RNA and DNA base editing via engineered ADAR recruitment

Abstract

Disclosed herein are systems for recruiting ADAR to catalyze therapeutic editing of point mutations via the use of an engineered RNA scaffold, an engineered DNA scaffold, or a DNA-RNA hybridization scaffold. The system includes an engineered ADAR2 guide RNA (adRNA) that carries a complementarity to 20-100 bp of the target RNA, and an ADAR2 recruitment domain from GluR2 mRNA at either or both of the 5'end and the 3 'end.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201980072798.1, application date September 6, 2019, and invention name “RNA and DNA base editing recruited by modified ADAR” after the PCT international application with PCT application number PCT / US2019 / 050095 entered the Chinese national phase.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to polynucleotide editing and vectors related thereto. Summary of the invention

[0005] One aspect of the present disclosure provides a vector. In some cases, the vector may include a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity recruitment domain, wherein: (a) the polynucleotide sequence encoding at least one RNA editing entity recruitment domain does not form a secondary structure containing a stem loop, or (b) wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain includes at least about 80% sequence identity with at least one sequence selected from the following items: Alu domain coding sequence, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain coding sequence, and any combination thereof. In some cases, the polynucleotide sequence encoding the RNA editing entity recruitment domain may include at least about 80% sequence identity with the Alu domain sequence. In some cases, the polynucleotide sequence encoding the RNA editing entity recruitment domain may include at least about 80% sequence identity with the APOBEC recruitment domain coding sequence. In some cases, the vector may 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 RNA editing entity recruitment domain may be configured to recruit ADAR proteins. 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 RNA editing entity recruitment domain may be configured to recruit 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 recruitment domain may not form a secondary structure containing a stem loop. In some cases, the polynucleotide sequence may encode at least two RNA editing recruitment domains. In some cases, at least one of the at least two RNA editing recruitment domains can be an Alu domain. In some cases, the Alu domain sequence can form a secondary structure including at least one stem loop. In some cases, the Alu domain encoding sequence can include multiple Alu repeats. In some cases, the Alu domain encoding sequence can be at least partially single-stranded.In some cases, at least one of the at least two RNA editing recruitment domains may be an APOBEC recruitment domain. In some cases, at least one of the at least two RNA editing recruitment domain coding sequences may include at least about 80% sequence identity with the GluR2 domain coding sequence. In some cases, at least one of the at least two RNA editing recruitment domains may be a GluR2 domain. In some cases, at least one of the at least two RNA editing recruitment domains may be a Cas13 domain. In some cases, the at least two RNA editing recruitment domains may be an Alu domain and an APOBEC recruitment domain. In some cases, the vector may further include a nucleic acid encoding an RNA that can be complementary to at least a portion of the target RNA. In some cases, the nucleic acid encoding the RNA that can be complementary to at least a portion of the target RNA can be about 10 base pairs (bp) to about 1000bp in length. In some cases, the nucleic acid encoding at least one RNA editing entity recruitment domain and the nucleic acid encoding the RNA that can be complementary to at least a portion of the target RNA may include a continuous nucleic acid of at least about 200bp 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 include 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 include RNA. In some cases, the RNA may include base modifications. 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 derivatives thereof, and any combination thereof. In some cases, the AAV may be an AAV5 serotype or a derivative thereof. In some cases, the derivative of the AAV may include a modified VP1 protein. In some cases, the APOBEC recruitment domain can be selected from the group consisting of an APOBEC1 recruitment domain, an APOBEC2 recruitment domain, an APOBEC3A recruitment domain, an APOBEC3B recruitment domain, an APOBEC3C recruitment domain, an APOBEC3E recruitment domain, an APOBEC3F recruitment domain, an APOBEC3G recruitment domain, an APOBEC3H recruitment domain, an APOBEC4 recruitment domain, and any combination thereof.In some cases, the RNA editing entity recruitment domain can recruit at least two RNA editing entities, and at least one of the at least two polynucleotide sequences encoding the RNA editing entity includes at least about 80% identity with the APOBEC protein coding sequence. In some cases, the RNA editing entity recruitment domain can recruit at least two RNA editing entities, and at least one of the at least two polynucleotide sequences encoding the RNA editing entity includes at least about 80% identity with the ADAR protein coding sequence. In some cases, the RNA recruitment domain encoded by the nucleic acid may include at least one stem loop. In some cases, the polynucleotide sequence encoding the RNA editing recruitment domain may include a secondary structure that may be substantially cross-shaped. In some cases, the polynucleotide sequence encoding the RNA editing recruitment domain may include at least two substantially cross-shaped secondary structures. In some cases, the polynucleotide sequence encoding the RNA editing entity recruitment domain may be located between the polynucleotide sequences forming the at least two substantially cross-shaped secondary structures. In some cases, the cross-shaped secondary structure may include a stem loop adjacent to at least one pair of at least partially complementary chains of the cross-shaped secondary structure. In some cases, the polynucleotide sequence encoding the RNA editing recruitment domain may include a secondary structure that is substantially a foothold. In some cases, the non-naturally occurring RNA may be encoded by the vector. In some cases, the kit may include the vector in a container. In some cases, the kit may further include a syringe. In some cases, the container may be the syringe. In some cases, the separated cells may include the vector. In some cases, a pharmaceutical composition may include the vector in a unit dosage form. In some cases, the pharmaceutical composition may further include a pharmaceutically acceptable excipient, diluent or carrier. In some cases, the pharmaceutical composition may include a second active ingredient. In some cases, the 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 administering a second therapy to the subject. In some cases, the disease or condition may be selected from a group consisting of neurodegenerative disorders, muscle disorders, metabolic disorders, ocular disorders, cell proliferation 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-Tooth Disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce the expression of a gene compared to before the 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 a read-through 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 a vector. In some cases, the vector may include a nucleic acid encoding RNA having a two-dimensional shape that can be substantially a cross, wherein the RNA includes at least one sequence encoding an RNA editing entity recruitment domain. In some cases, the vector may further include a nucleic acid encoding RNA having at least one targeting domain encoding sequence that can be complementary to at least a portion of a target RNA sequence. In some cases, the nucleic acid encoding RNA may further include a substantially linear two-dimensional structure, wherein the RNA has at least one targeting domain that can be complementary to at least a portion of a target RNA sequence. In some cases, the vector may encode non-naturally occurring RNA. In some cases, the kit may include the vector in a container. In some cases, the kit may further include a syringe. In some cases, the container may be the syringe. In some cases, the separated cells may include the vector. In some cases, a pharmaceutical composition may include the vector in a unit dosage form. In some cases, the pharmaceutical composition may 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 performed by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further include administering a second therapy 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 ocular disorder, a cell proliferation 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-Tooth disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce the expression of a gene compared to before the 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 a read-through of the stop codon. In certain cases, the administering may be sufficient to produce exon skipping in the subject.

[0007] Another aspect of the present disclosure provides non-natural RNA. In some cases, the non-natural RNA may include: a first domain sequence, including a substantially cross-shaped two-dimensional shape; and, a second domain sequence, having a substantially linear two-dimensional structure connected to the first domain sequence, wherein the first domain sequence encodes an RNA editing entity recruitment domain and the second domain sequence encodes a targeting domain, wherein the second domain sequence may be complementary to at least a portion of the target RNA. In some cases, the non-natural RNA may further include a third domain sequence attached to the second domain sequence. In some cases, the third domain sequence may include an RNA editing entity recruitment domain encoding sequence, and the RNA editing entity recruitment domain encoding sequence forms a secondary structure having a two-dimensional shape that is substantially cross-shaped. In some cases, at least one base in the non-natural RNA may include a chemical modification. In some cases, at least one sugar in the non-natural RNA may include a chemical modification. In some cases, the kit may include the non-natural RNA in a container. In some cases, the kit may further include a syringe. In some cases, the container may be the syringe. In some cases, the isolated cell may include the non-natural RNA. In some cases, a pharmaceutical composition may include the non-naturally occurring RNA in a unit dosage form. In some cases, the pharmaceutical composition may further include a pharmaceutically acceptable excipient, diluent or carrier. In some cases, the pharmaceutical composition may include a second active ingredient. In some cases, the method for treating a disease or condition in a subject comprises administering the non-naturally occurring RNA to the subject. In some cases, the administration may be performed by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further include administering a second therapy to the subject. In some cases, the disease or condition may be selected from a group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferation 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-Tooth Disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce the expression of a gene compared to before the 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 read-through 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 provides nucleic acids. In some cases, the nucleic acid may include an RNA editing entity recruitment domain and an antisense domain sequence, wherein as determined by Sanger Method sequencing of the target nucleic acid, when the nucleic acid contacts the RNA editing entity and the target nucleic acid complementary to at least a portion of the antisense domain, at least one base pair of the target nucleic acid is modified with an efficiency at least about 4 times higher than that of a comparable nucleic acid compounded with a Cas13b protein or its active fragment.

[0009] Another aspect of the present disclosure provides nucleic acids. In some cases, the nucleic acid may include an RNA editing entity recruitment domain and an antisense domain, wherein, as determined by Sanger sequencing of the target nucleic acid, the nucleic acid, when contacted with an RNA editing entity and 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 an efficiency at least about 4 times higher than that of a comparable nucleic acid complexed with a GluR2 domain and the antisense domain. In some cases, the nucleic acid may include RNA. In some cases, the target nucleic acid may include 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, the dysfunction of the protein or a portion thereof may be associated with a disease or condition. In some cases, the disease or condition may be selected from a group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an ocular disorder, a cell proliferation 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 RNA editing entity recruitment domain may include at least about 80% identity to the GluR2 domain. In some cases, the RNA editing entity recruitment domain may include at least about 80% identity with an Alu domain. In some cases, the RNA editing entity recruitment domain may be at least about 80% identical to an APOBEC recruitment domain. In some cases, the RNA editing entity recruitment domain may be configured to recruit ADAR proteins. 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 RNA editing entity recruitment domain may be configured to recruit APOBEC proteins. 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 the syringe.In some cases, the isolated cells may include the nucleic acid. In some cases, the pharmaceutical composition may include the nucleic acid in a unit dosage form. In some cases, the pharmaceutical composition may further include a pharmaceutically acceptable excipient, diluent or carrier. In some cases, the pharmaceutical composition may include a second active ingredient. In some cases, the 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 performed by intravenous injection, intramuscular injection, intrathecal injection, intraorbital injection, subcutaneous injection, or any combination thereof. In some cases, the method may further include administering a second therapy 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 ocular disorder, a cell proliferation 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-Tooth Disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce the expression of a gene compared to before the 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 a read-through of the stop codon. In some cases, the administration may be sufficient to produce exon skipping in the subject.

[0010] Another aspect of the present disclosure may provide a nucleic acid. In some cases, the nucleic acid may include a sequence including an antisense domain, a first stem-loop forming sequence, and a second stem-loop forming sequence, wherein the nucleic acid modifies at least one base pair of a target nucleic acid when in contact with: (a) a first polypeptide including a first portion of an RNA editing entity and a first polynucleotide binding domain configured to bind to the first stem-loop forming sequence; and, (b) a second polypeptide including a second portion of an RNA editing entity and a second polynucleotide binding domain configured to bind to the second stem-loop forming sequence; and, (c) a target nucleic acid complementary to at least a portion of the antisense domain. In some cases, the first stem-loop or the second stem-loop may be an MS2 stem-loop. In some cases, the first stem-loop or the second stem-loop may be a BoxB stem-loop. In some cases, the first stem-loop or the second stem-loop may be a U1A stem-loop. In some cases, the first portion of the RNA editing entity or the second portion of the RNA editing entity may include an N-terminal fragment of an ADAR deaminase domain encoding sequence. In some cases, the first portion of the RNA editing entity or the second portion of the RNA editing entity may include a C-terminal fragment of an ADAR deaminase domain encoding sequence. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain may include an MS2 coat protein. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain may include a λN peptide. In some cases, the first polynucleotide binding domain or the second polynucleotide binding domain may include a human nucleic acid binding protein. In some cases, the human nucleic acid binding protein may be a U1A 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 may be capable of performing a mutation from adenosine to inosine on the target nucleic acid. In some cases, the RNA editing entity may be capable of performing a mutation from cytosine to thymine on the target nucleic acid. 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 the syringe. In some cases, the isolated cells may include the nucleic acid. In some cases, the pharmaceutical composition may include the nucleic acid in a unit dosage form. In some cases, the pharmaceutical composition may further include a pharmaceutically acceptable excipient, diluent or carrier. In some cases, the pharmaceutical composition may include a second active ingredient. In some cases, the 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 include administering a second therapy 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 ocular disorder, a cell proliferation 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-Tooth disease. In some cases, the disease or condition may be Rett syndrome. In some cases, the administration may be sufficient to reduce the expression of a gene compared to before the 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 a read-through of the stop codon. In some cases, the administration may be sufficient to produce exon skipping in the subject.

[0011] Another aspect of the present disclosure provides a method for treating muscular dystrophy in a subject. In some cases, the method may include: administering to the subject a pharmaceutical composition including an adeno-associated virus (AAV) vector, the adeno-associated virus (AAV) vector including a first nucleic acid encoding a second nucleic acid, wherein the second nucleic acid includes (a) an antisense region that can be at least partially complementary to an RNA sequence associated with muscular dystrophy, and (b) at least one RNA editing entity recruitment domain, wherein the at least one RNA editing entity recruitment domain does not include a stem loop, or wherein the at least one RNA editing entity recruitment domain includes at least about 80% sequence identity with at least one of the following items: Alu domain, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain, and any combination thereof. In some cases, the pharmaceutical composition may be a unit dosage form. In some cases, the administration may be at least once a week. In some cases, the administration may be at least once a month. In some cases, the administration may be by injection. In some cases, the injection may be subcutaneous, intravenous, infused, intramuscular, intrathecal, or intraperitoneal. In some cases, the administration may be transdermal, transmucosal, oral, or pulmonary. In some cases, the method may further comprise administering to the subject a second therapy.

[0012] Another aspect of the present disclosure may provide a method for making 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 at least one RNA editing entity recruitment domain, and wherein the sequence encoding the at least one RNA editing entity recruitment domain does not form a secondary structure including a stem loop, or wherein the nucleic acid encoding the at least one RNA editing entity recruitment domain includes at least about 80% sequence identity with a sequence selected from the following items: Alu domain encoding sequence, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain encoding sequence 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 include 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 further includes transfecting or transducing the vector into isolated human cells.

[0013] Aspects of the present disclosure relate to modified ADAR1 or ADAR2 guide RNA ("adRNA") comprising a sequence complementary to a target RNA, or alternatively consisting essentially of it, or further consisting essentially of it, the sequence optionally comprising a modified ADAR2 and an ADAR2 recruitment domain derived from GluR2mRNA, or alternatively consisting essentially of it, or further consisting essentially of it. In some embodiments, the sequence complementary to the target RNA comprises about 15 to 200 or optionally 20 to 100 base pairs, or alternatively consisting essentially of it, or further consisting of it. In one aspect, the modified adRNA does not include the ADAR recruitment domain, or does not consist essentially of it, or further does not consist of it. In some embodiments, the ADAR recruitment domain comprises GluR2mRNA, Alu repeat sequence elements, or other RNA motifs that ADAR binds, or alternatively consists essentially of it, or further consists of it. In one aspect, the modified adRNA includes about 1 to 10 ADAR recruitment domains, is essentially composed of it, or is further composed of it. On the other hand, the ADAR2 recruitment domain can be derived from GluR2mRNA, and can be located at the 5' end or 3' end of the modified adRNA. In another embodiment, the modified adRNA includes GluR2mRNA at both the 5' end and the 3' end, or is alternatively essentially composed of it, or is further composed of it. In a further aspect, the modified adRNA of the present disclosure further includes two MS2 hairpins on both sides of the sequence complementary to the target RNA, or is alternatively essentially composed of it, or is further composed of it.

[0014] In some embodiments, the target RNA can be ornithine transcarbamylase. A complex is also provided herein, comprising an adRNA disclosed herein that hybridizes to a complementary polynucleotide under high stringency conditions, or alternatively substantially consisting of it, or further consisting of it. In one aspect, the polynucleotide can be DNA. On the other hand, the polynucleotide can be RNA.

[0015] In one aspect, the engineered adRNA of the present disclosure further comprises, or alternatively consists essentially of, or further consists of an editing-inducing element.

[0016] A further aspect relates to an engineered ADAR2 guide RNA ("adRNA") selected from Table 1 or Figure 2 The adRNA can be combined with a carrier, such as a pharmaceutically acceptable carrier, and such an embodiment is provided herein.

[0017] A modified adRNA-snRNA (small nuclear RNA) fusion is also disclosed herein. In one aspect, the modified adRNA further includes an N-terminal mitochondrial targeting sequence (MTS), or alternatively is essentially composed of it, or further consists of it, to promote the modified adRNA to be positioned to mitochondria. On the other hand, provided herein may be a modified zipper code (zipcode) further including a cis-acting one, or alternatively is essentially composed of it, or further consists of it, to promote the modified adRNA to be positioned to peroxisomes, endosomes and exosomes.

[0018] Further provided herein are modified adRNAs that are small molecule-regulatable. In one aspect, disclosed herein are modified adRNA-aptamer fusions. Non-limiting examples of aptamers that can be used for this purpose include aptamers that bind to flavin mononucleotides, guanine, other natural metabolites, or sugars. Also disclosed herein are U1A-ADAR fusions that are completely derived from humans.

[0019] Also disclosed herein is a complex comprising, or alternatively consisting essentially of, or further consisting of, the engineered adRNA of the present disclosure hybridized to a complementary polynucleotide under high stringency conditions.

[0020] Also provided herein is a vector comprising one or more isolated polynucleotide sequences and optionally a regulatory sequence operably linked to the isolated polynucleotide, or alternatively consisting essentially of, or further consisting of, wherein the isolated polynucleotide sequence encodes the modified adRNA of the present disclosure. Non-limiting examples of vectors include plasmids or viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

[0021] Further disclosed herein is a recombinant cell, further comprising or the above-mentioned vector, or alternatively essentially consisting of it, or further consisting of it, wherein the modified adRNA can be recombinantly expressed.

[0022] Provided are compositions comprising one or more of the above compounds and a carrier. In one embodiment, the composition can be a pharmaceutical composition, and therefore further comprises at least one pharmaceutically acceptable carrier or pharmaceutically acceptable excipient. The composition is formulated for various modes of delivery, such as systemic (oral) or local delivery.

[0023] Also provided herein is a method of modifying protein expression, comprising contacting a polynucleotide encoding the protein with the engineered adRNA of the present disclosure, or alternatively consisting essentially of it, or further consisting of it, wherein the expression of the protein is to be modified.

[0024] Other aspects relate to methods for treating diseases or disorders associated with abnormal protein expression, comprising, or alternatively consisting essentially of, or further consisting of, an effective amount of any one or more modified adRNAs disclosed herein, and / or an effective amount of any one or more modified adRNAs disclosed herein are used for subjects in need thereof, and for treating diseases or disorders associated with abnormal protein expression. In a specific aspect, a method for treating Duchenne muscular dystrophy is provided herein, comprising administering an effective amount of one or more modified adRNAs disclosed herein to a subject in need of such treatment, or alternatively consisting essentially of, or further consisting of.

[0025] The present disclosure demonstrates in vivo validation of this approach in an spf-ash mouse model of ornithine transcarbamylase deficiency, which carries a G->A point mutation in the last nucleotide of exon 4. After delivery of adRNA alone via AAV, correction of point mutations up to 1% was observed in the absence of overexpression of ADAR enzymes.

[0026] Other aspects relate to the same or similar structures, which include DNA or a combination of DNA and RNA, or alternatively consist essentially of it, or further consist of it. Other aspects relate to kits, which include any one or more of the above embodiments, and instructions for in vitro and / or in vivo use.

[0027] Aspects of the present disclosure may relate to ADAR and APOBEC systems for gene editing. Some aspects relate to ADAR systems for exon skipping, including adRNAs targeting splicing acceptors and / or branch points in introns, and optionally ADAR enzymes. In some embodiments, the ADAR enzyme may be ADAR1, ADAR2, or a respective 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 included in a gene selected from dystrophin, SCN9A or ornithine transcarbamylase. In some cases, the adRNA may be selected from sequence set 1. Further aspects may relate 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.

[0028] Other aspects relate to an APOBEC system for cytosine to thymine editing, comprising a pair of gRNAs that create a lipoprotein B mRNA-like structure, and optionally an APOBEC enzyme. In some embodiments, the pair of gRNAs can be a pair of sequences provided in sequence set 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram showing the endogenous recruitment of ADARs.

[0030] Figure 2 A list of stable scaffolds that were evaluated for (1) improved efficiency and (2) ability to recruit ADAR1 is provided (SEQ ID NOs: 2-9).

[0031] Figure 3 Results of in vitro and in vivo screens of exemplary adRNAs are shown.

[0032] Figure 4Schematic diagram of how to recruit MCP-APOBEC fusion or MCP-ACF-APOBEC fusion via MS2-RNA carrying two MS2 stem loops. As shown in the figure, the target cytosine can remain single-stranded so that it can be used for APOBEC-mediated editing via the generation of a bulge. The bulge is created by using the precise target sequence (20-30 base pairs) or the sequence ACATATATGATACAATTTGATCAGTATATT (SEQ ID NO: 175) (blue) between the MS2 stem loops in the MS2-RNA, and the complementary sequence (20-30 base pairs) (green) that binds to the target mRNA on either side of the MS2 stem loop. The sequence ACATATATGATACAATTTGATCAGTATATT (SEQ ID NO: 176) is obtained from the naturally occurring apoB substrate edited by APOBEC. The MS2 stem loop sequence used in this design is aACATGAGGATCACCCATGTc (SEQ ID NO: 177).

[0033] Figure 5 Shown are the percentage mRNA editing by constructs containing the Alu domain compared to editing by constructs containing the GluR2 domain.

[0034] Figure 6 Shown is the method used to generate Figure 5 Example of an Alu domain-containing construct (SEQ ID NO: 10) based on data from the invention.

[0035] Figure 7 An example of a construct is shown that includes two crosses connected by an antisense domain and includes an Alu domain. An exemplary structure (SEQ ID NO: 11) and an exemplary sequence (SEQ ID NO: 1) are provided.

[0036] Figure 8 Various construct designs, including those containing the GluR2 domain (SEQ ID NOs: 12-15, 252, and 16-34) and the respective percent mRNA editing are shown.

[0037] Fig. 9 Enzyme targeting preferences for localizing ADAR1 and ADAR2 for each base (eg, A, C, G, and T) are shown.

[0038] Fig.10The next generation adRNAs that have enhanced ADAR1 and ADAR2 recruitment potential are shown. The first series of columns show the relative activity without adRNA. The second series of columns show the relative activity of constructs containing the GluR2 domain. The third series of columns show the relative activity of constructs containing the Alu domain, wherein the Alu domain is associated with two cruciform structures. Fig.10 SEQ ID NO: 11 is disclosed.

[0039] FIG. 11A to FIG. 11D Different construct designs are shown. Fig.11A An antisense domain linked to two GluR2 domains is illustrated. Fig. 11B Only the antisense domain is exemplified. Fig. 11C The antisense domain linked to two crosses is illustrated. Fig.11D A toe-hold is illustrated.

[0040] Fig.12 The percent editing yields between different construct designs are shown.

[0041] Fig.13 Shown is a comparison of a short antisense oligonucleotide (AON) with mismatch bulges and a longer construct containing a hairpin structure.

[0042] Fig.14 Exemplary adRNA designs are shown (SEQ ID NOs: 250 and 251).

[0043] Fig.15 Exemplary adRNA structures (SEQ ID NOs: 35-37) are shown with parameters (d, l, m), where d = number of GluR2 domains, l = length of the antisense domain, and m = position of the mismatch.

[0044] Fig.16 Schematic diagram showing RNA editing via recruitment of endogenous ADARs in the presence of adRNA.

[0045] Fig.17 AdRNAs with increasing antisense domain lengths transcribed from the U6 promoter are shown, binding to 0, 1 or 2 GluR2 domains, and their ability to induce targeted RNA editing with or without exogenous ADAR2 expression is assessed. Values ​​represent mean + / - SEM (n = 3). Long adRNAs can recruit endogenous ADARs for RNA editing.

[0046] Fig.18Shown are chemically synthesized adRNA patterns tested against a panel of mRNAs with or without exogenous ADAR2 expression. Chemical modifications and sources of adRNAs were identified. Values ​​represent mean + / - SEM (n=3).

[0047] Fig.19 Shown in adults treated with SPF ash In vivo RNA proofreading efficiency in correctly spliced ​​OTC mRNA in mouse liver (retro-orbital injection) A level of 0.6% RNA editing was seen in mice injected with U6-transcribed short adRNA.

[0048] Fig. 20 Design of Alu adRNA is shown. Left: Structure of the Alu element. Middle: Design as described herein, including a locus-specific antisense sequence with a C mismatch relative to the target A. Right: Recruitment of the RNA editing enzyme ADAR to the target.

[0049] Fig.21 Exemplary Alu guide sequences are shown (SEQ ID NOs: 38-41).

[0050] Fig. 22 A schematic diagram of a split ADAR2DD system is shown.

[0051] Fig.23 An exemplary sequence of a split ADAR2DD is shown (SEQ ID NO:42), with the split ADAR2DD having potential sites for splitting highlighted.

[0052] Fig.24 Pairs of fragments 1 to 16 assayed via a cypridina luciferase reporter (Cluc W85X) are shown.

[0053] Fig.25 Fragment 9 and fragment 10 assayed against the Cluc reporter are shown.

[0054] Fig.26 Exemplary sequences are shown (SEQ ID NOs: 43-55).

[0055] Fig. 27 A schematic diagram of ADAR recruitment via U1A (SEQ ID NO: 56) is shown.

[0056] Fig.28 Exemplary sequences of several fusion constructs (SEQ ID NOs: 57-68) or exemplary sequences of one or more APOBEC family members (SEQ ID NOs: 57-68) are shown.

[0057] Fig.29 Exemplary sequences of engineered apRNAs for recruiting APOBEC3A are shown (SEQ ID NOs: 69-73).

[0058] Fig.30 Exemplary sequences of engineered MS2-apRNAs for recruiting MCP-APOBEC3A are shown (SEQ ID NOs: 74-78).

[0059] Fig.31 Two different scenarios are shown, with no ADAR recruitment and ADAR recruitment allowing ribosome readthrough leading to normal luciferase expression.

[0060] Figures 32A to 32C Demonstrated engineering of programmable RNA editing and characterization of specificity profiles: Fig.32A ) Schematic diagram of RNA editing via constructs utilizing full-length ADAR2 and engineered adRNA derived from GluR2 transcripts or MS2 coat protein (MCP) fusions with the ADAR1 / 2 deaminase domain and the corresponding MS2 hairpin with adRNA. ( Fig.32B ) Comparison of RNA editing efficiency of different RNA editing constructs on endogenous RAB7A transcripts quantified by Sanger sequencing (efficiency is calculated as the ratio of Sanger peak height G / (A+G)). Experiments were performed in HEK 293T cells. Values ​​represent mean + / - SEM (n = 3). ( Fig.32C ) represents a violin plot of the A->G editing yield distribution observed at the reference site, where the editing yield of at least one treated sample was found to have a significant change relative to the control sample (Fisher's exact test, FDR=1%). The blue circles represent the editing yield at the target A site within the RAB7A transcript. The black dots represent the median off-target editing yield. In order to better visualize the shape of the distribution, their maximum range along the y-axis was equalized on all graphs and truncated at 60% of the yield.

[0061] Figures 33A to 33E In vivo RNA editing in a mouse model of human disease is shown. Fig.33A ) Schematic representation of DNA- and RNA-targeted 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. Fig.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 was also seen in treated samples (scale bar: 250 μm). ( Fig.33C ) RNA editing efficiency of TAA->TGG / TAG / TGA in vivo in corresponding treated adult mdx mice. Values ​​represent mean + / - SEM (n = 4, 3, 7, 3, 3, 10, 3, 4 independent TA muscles, respectively). ( Fig.33D ) in ornithine transcarbamylase deficiency spf ash Schematic representation of the OTC locus in a mouse model of ornithine transcarbamylase deficiency with a G->A point mutation at the donor splice site of the last nucleotide of exon 4 and the method for correction of mutant OTC mRNA via ADAR2-mediated RNA editing. Fig.33E ) in treated adult spf ash In vivo RNA correction efficiency in correctly spliced ​​OTC mRNA in liver of mice injected retro-orbitally with AAV8-ADAR2 and AAV8-ADAR2(E488Q). Values ​​represent mean + / - SEM (n=4, 4, 3, 3, 4, 5 independent animals, respectively).

[0062] Figure 34A to Figure 34C Antisense domain engineering is shown. ( Fig.34A ) Optimization of adRNA antisense regions using adRNA scaffold 2: length and distance to the ADAR2 recruitment region were systematically varied. Values ​​represent mean + / - SEM (n=3) (SEQ ID NOs: 253 and 79-102). ( Fig.34B ) The ability of adRNAs with increasing antisense domain lengths transcribed from the U6 promoter, combined with 0, 1, or 2 GluR2 domains, to induce targeted RNA editing in the presence or absence of exogenous ADAR2 expression was evaluated. Values ​​represent mean + / - SEM (n = 3). All experiments above were performed in HEK 293T cells. ( Fig.34C ) experiments confirmed the expression of endogenous ADAR1 and ADAR2 (relative to GAPDH) in HEK 293T and HeLa cell lines. The levels observed were similar to those documented in the Human Protein Atlas (see the global webpage at proteinatlas.org (www)).

[0063] Figure 35A to Figure 35B Modified MS2 adRNAs are shown. Fig.35A) The antisense RNA targeting domain of MS2 adRNA (SEQ ID NO: 103-110) was systematically evaluated. Values ​​represent mean + / - SEM (n = 3). ( Fig.35B ) On-target RNA editing by MCP-ADAR2DD-NLS requires co-expression of MS2adRNA. Values ​​represent mean + / - SEM (n=3). All experiments were performed in HEK 293T cells.

[0064] Figure 36A-C shows an analysis of the RNA editing yield of a group of targets. (Figure 36A) Comparison of the efficiency of RNA editing by OTC reporter transcripts through GluR2adRNA and MS2adRNA guide RNA editing constructs, and Cas13b-based REPAIR constructs. Numerical values ​​represent mean values ​​+ / - SEM (for reporter factors and Cas13b-based constructs, n = 6, for other constructs, n = 3). (Figure 36B) For a group of mRNAs, chemically synthesized adRNA patterns were tested with or without exogenous ADAR2 expression. The exact chemical modification and the source of adRNA are illustrated in the figure. Numerical values ​​represent mean values ​​+ / - SEM (n = 3). (Figure 36C) The RNA editing yield of a series of selected endogenous targets is analyzed to cover a range of expression levels. Also for multiple endogenous mRNA targets, with or without exogenous ADAR2 expression, long adRNAs transcribed by U6 without or with two GluR2 domains were evaluated. Editing was observed at the tested loci even in the absence of exogenous ADAR2 expression. Values ​​represent mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0065] Figure 37A-D shows ADAR2 variants and their effects on editing and specificity. (Figure 37A) Comparison of RAB7A transcripts by GluR2adRNA and MS2adRNA guide RNA editing constructs, as well as Cas13b-based REPAIR constructs, for editing of the upper target RNA of RAB7A transcripts and editing in the flanking adenosine. Depicted are the average (n=3) editing yields (SEQID NO: 111). All experiments were performed in HEK 293T cells, and the editing efficiency was calculated as the ratio of Sanger peak height G / (A+G). (Figure 37B) In spf ashADAR2(E488Q) showed higher efficiency than ADAR2 in in vitro editing of OTC reporter transcripts (p=0.037, unpaired t-test, two-tailed; values ​​represent mean + / - SEM (n=3)) and (Figure 37C) mdx DMD reporter transcripts (p=0.048, p=0.012, unpaired t-test, two-tailed; values ​​represent mean + / - SEM (n=3)). (Figure 37D) ADAR2, ADAR2(E488Q) and ADAR2(Δ1-138) were compared for the editing efficiency and specificity profiles of OTC reporter transcripts (upper panel) and endogenous RAB7A transcripts (lower panel). The heat map indicates A->G editing near the target (red arrow). Values ​​represent mean + / - SEM (n=3). All experiments were performed in HEK 293T cells, and editing efficiency was calculated as the ratio of Sanger peak height G / (A+G). Figure 37D discloses SEQ ID NOs: 254-255, respectively, in order of appearance.

[0066] Fig.38 A transcriptome-scale specificity profile of the RNA editing approach (Cas13b-ADAR REPAIR+ / -gRNA) is shown.

[0067] Fig.39 The transcriptome-scale specificity profile of the RNA editing approach (ADAR2+ / -adRNA) is shown. The format used in these studies was GluR2adRNA (1, 20, 6).

[0068] Fig.40 A transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR1DD+ / -adRNA) is shown.

[0069] Fig.41 A transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR2DD+ / -adRNA) is shown.

[0070] Figure 42A~B shows the change of transcriptome scale editing with construct features.(Figure 42A) Each point in the box plot corresponds to the score of the edited site of one of the MCP-ADAR constructs listed in Figure 32.The score of the edited site of each construct is calculated by dividing the number of reference sites where A- to -G editing output (see Table 3) changes significantly by the total number of reference sites considered 8,729,464.The construct features indicated on the horizontal axis were compared using the Mann-Whitney U test, and the p value of NLS vs.NES was 0.16, the p value of ADAR1 vs.ADAR2 was 0.0070, the p value of "-adRNA" vs. "+adRNA" was 0.72, and the p value of "ADAR WT" vs. "ADAR E>Q" was 0.038 (n=8 under all conditions). (Figure 42B) 2D histograms compare the A->G editing yield (y-axis) of the transcriptome range observed using each construct with the yield (x-axis) observed using the control sample. The illustration shows a violin plot, which represents the distribution of the A->G editing yield observed at the reference site, wherein relative to the control sample, it is found that the editing yield of at least one treated sample has a significant change (Fisher's exact test, FDR=1%). The blue circle represents the editing yield at the target A-site within the RAB7A transcript. In order to better visualize the shape of the distribution, they are balanced on the graph along the maximum range of the y-axis and truncated at 60% of the yield. The sample here corresponds to 293T transfected with a long antisense domain carrying adRNA, which can be recruited via exogenous and / or endogenous ADARs for RNA editing.

[0071] Figure 43A-E shows the optimization and evaluation of in vitro and in vivo dystrophin RNA editing experiments in mdx mice. (Figure 43A) Schematic diagram of RNA editing using full-length ADAR2 and modified adRNA or reverse adRNA (radRNA); (ii) RNA editing efficiency of amber and ochre stop codons in one-step and two-step manner. The experiment was performed in HEK 293T cells. The values ​​represent the mean + / - SEM (n = 3). (Figure 43B) RNA editing of ochre codons requires two cytosine mismatches in the antisense RNA targeting domain of adRNA or radRNA (SEQ ID NO: 112-116) to restore GFP expression. The experiment was performed in HEK 293T cells. The values ​​represent the mean + / - SEM (n = 3). (FIG. 43C) Schematic diagram of AAV vectors for in vivo delivery of adRNA and ADAR2, and in vitro optimization of RNA editing of amber and ochre stop codons delivered via AAV vectors in the presence of 1 or 2 copies of adRNA (p=0.0003, p=0.0001, p=0.0015, unpaired t-test, two-tailed, respectively). Experiments were performed in HEK 293T cells. Values ​​represent mean + / - SEM (n=3 for reporter, n=6 for other conditions). (FIG. 43D) Representative Sanger sequencing plots showing editing of ochre stop codons (TAA->TGG) in mdx DMD reporter transcripts (quantified by NGS) (SEQ ID NO: 117-118). Experiments were performed in HEK 293T cells (n=3). ( FIG. 43E ) Representative example of in vivo RNA editing analysis of treated mdx mice (quantified using NGS) (SEQ ID NOs: 119-130).

[0072] Figure 44A-C shows the analysis of immunofluorescence and immunoblotting of in vivo dystrophin RNA editing experiments in mdx mice. (Figure 44A) Immunofluorescence staining of dystrophin in TA muscle shows partial recovery of expression in treated samples (intramuscular injection of AAV8-ADAR2, AAV8-ADAR2 (E488Q), AAV8-MCP-ADAR1 (E1008Q) NLS). Partial recovery of nNOS localization was also seen in the treated samples (scale bar: 250 μm). (Figure 44B) Immunoblotting shows partial recovery of dystrophin expression (1-2.5%) in TA muscles of mdx mice (which were injected with two components of the editing tool, enzyme and adRNA), as well as stable ADAR2 expression for up to 8 weeks after injection in injected TA muscles. (Figure 44C) Immunoblotting shows partial recovery of dystrophin expression (10%) using AAV8-CRISPR.

[0073] Figures 45A to 45E show the spf ash Optimization and evaluation of OTC RNA editing experiments in vitro and in vivo in mice. (Figure 45A) Representative Sanger sequencing profiles show that spf ash Correction of point mutations in OTC reporter transcripts (quantified using NGS) (SEQ ID NOs: 131-132). Experiments were performed in HEK 293T cells (n=3). (Figure 45B) Spf treated ash Representative examples of in vivo RNA editing analysis in mice show correction of point mutations in correctly spliced ​​OTC mRNA (quantified using NGS) (SEQ ID NOs: 133-139). (FIG. 45C) In treated adult spf ash In vivo RNA correction efficiency of OTC pre-mRNA in the liver of mice (retro-orbitally injected with A488-ADAR2 and AAV8-ADAR2(E488Q)). Values ​​represent mean + / - SEM (n=4, 4, 3, 3, 4, 5 independent animals, respectively). (FIG. 45D) PCR products show correctly and incorrectly spliced ​​OTC mRNA. The incorrectly spliced ​​mRNA was extended by 48 base pairs. The fraction of incorrectly spliced ​​mRNA was reduced in mice treated with adRNA+ADAR2(E488Q). (FIG. 45E) Immunoblot for OTC shows that in treated adult spf ash Partial restoration of expression in mice (2.5% to 5%), and stable ADAR2(E488Q) expression three weeks after injection.

[0074] Fig.46 Toxicity analysis of in vivo RNA editing experiments is shown.

[0075] Fig.47 is a schematic diagram showing exon skipping via creation of splice acceptor and / or branch point mutations.

[0076] Fig.48 Schematic diagram of C→T editing via APOBEC.

[0077] Figures 49A to 49D Schematic diagram showing editing of DNA and both strands of a DNA / RNA hybrid.

[0078] Figures 50A to 50D show the results of the study on the ornithine transcarbamylase deficiency model. Figure 50A depicts the results of the study on the ornithine transcarbamylase deficiency model in adult spf treated with ash In vivo RNA correction efficiency in the liver of mice (retro-orbitally injected with AAV8-ADAR2 and AAV8-ADAR2(E488Q)). Each data point represents an independent animal. Editing efficiency measured in spliced ​​OTC mRNA. Error bars represent + / - SEM. FIG50B depicts the effect of AAV8-ADAR2 on the editing efficiency of spliced ​​OTC mRNA in adult spf treated mice. ash In vivo RNA correction efficiency of OTC pre-mRNA in the liver of mice (retro-orbital injection of A488-ADAR2 and AAV8-ADAR2 (E488Q)). Each data point represents an independent animal. Figure 50C shows PCR products showing correctly and incorrectly spliced ​​OTC mRNA. The incorrectly spliced ​​mRNA was extended by 48 base pairs. In mice treated with adRNA + ADAR2 (E488Q), the fraction of incorrectly spliced ​​mRNA was reduced. Figure 50D is an immunoblot of OTC, which shows that in treated adult spf ash Partial restoration of expression in mice (2.5% to 5%).

[0079] Figures 51A to 51B show the results of the study in the Duchenne muscular dystrophy model. Figure 51A depicts the RNA editing efficiency of TAA->TGG / TAG / TGA in vivo in the corresponding treated adult mdx mice. Each data point represents an independent TA muscle. Error bars represent + / -SEM. Figure 51B is an immunoblot against dystrophin, showing partial restoration of expression (1-2.5%) in the corresponding treated adult mdx mice.

[0080] Fig.52 Provides more information about the location of potential branch points. DETAILED DESCRIPTION

[0081] As one aspect of the present disclosure, nucleic acids, non-naturally occurring RNAs, vectors including nucleic acids, compositions, and pharmaceutical compositions for RNA editing are provided. Any of the above or as described herein can be configured for A (adenosine) to I (inosine) editing, C (cytosine) to T (thymine) editing, or a combination thereof. In some cases, A to I editing can be interpreted or read as a C to U mutation. In some cases, A to I editing can be interpreted or read as an A to G mutation. Compared to natural systems, nucleic acids, non-naturally occurring RNAs, vectors including nucleic acids, compositions, and pharmaceutical compositions described herein can provide enhanced editing efficiency, reduced off-target editing, enhanced stability or in vivo half-life, or any combination thereof.

[0082] One aspect of the present disclosure provides a vector. The vector may include a nucleic acid having a polynucleotide sequence encoding (i) an RNA editing entity recruitment domain, (ii) a targeting domain complementary to at least a portion of a target RNA, (iii) any one or more domains, or (iv) any combination thereof. In some cases, the vector may be administered to a subject, such as a subject in need thereof. In some cases, the vector may be administered to a subject, such as a subject in need thereof, as part of a pharmaceutical composition.

[0083] One aspect of the present disclosure provides non-natural RNA. Non-natural RNA may include (i) an RNA editing entity recruitment domain, (ii) a targeting domain complementary to at least a portion of a target RNA, (iii) any one or more domains, or (iv) any combination thereof. In some cases, the non-natural RNA may be administered to a subject, such as a subject in need thereof. In some cases, the non-natural RNA may be administered to a subject as part of a pharmaceutical composition, such as a subject in need thereof. In some cases, the non-natural RNA may be formulated in a carrier for administration. The carrier may include a viral vector, a liposome, a nanoparticle, or any combination thereof. In some cases, the non-natural RNA may include at least one base, at least one sugar, any one or more of them, with modification (e.g., chemical modification).

[0084] One aspect of the present disclosure provides nucleic acids. The nucleic acid may include (i) an RNA editing entity recruitment domain, (ii) a targeting domain complementary to at least a portion of a target RNA, (iii) any one or more 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 as part of a pharmaceutical composition, such as a subject in need thereof. 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.

[0085] The nucleic acid may include one or more domains, such as 1, 2, 3, 4, 5 or more domains. In some cases, the nucleic acid may include a recruitment domain, a targeting domain, any domain or a combination thereof. In some cases, the nucleic acid may include a targeting domain and a recruitment domain. In some cases, the nucleic acid may include a targeting domain and two recruitment domains.

[0086] The domain can form a two-dimensional shape or a 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 cross or a portion thereof, a toehold shape, 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 cross. 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 shape.

[0087] In some cases, the targeting domain can be located near the recruitment domain, including directly adjacent, or adjacent but separated by multiple nucleotides. In some cases, the two sides of the targeting domain can be two recruitment domains. In some cases, two or more recruitment domains can be adjacent to each other.

[0088] One aspect of the present disclosure includes reducing off-target editing. A method described herein includes limiting the catalytic activity of ADAR or APOBEC by a split recombination method. In such a design, the first domain (e.g., a recruitment domain) itself can be catalytically inactive, and the second domain itself can be catalytically inactive, but when reassembled together, the two domains provide catalytic activity together to recruit ADAR or APOBEC. A nucleic acid comprising two domains can be split at any number of positions, such as splitting at a position between the two domains. In some cases, the first domain or the second domain may include an MS2 stem loop, a BoxB stem loop, a U1A stem loop, a modified form of any of these, or any combination thereof.

[0089] Compared with nucleic acids that can form different two-dimensional shapes or secondary structures, the two-dimensional shape or secondary structure of the domain can affect editing efficiency, off-target effects, or a combination thereof. Therefore, one aspect of the present disclosure includes modified nucleic acids so that a two-dimensional shape can be advantageously designed to enhance editing efficiency and reduce off-target effects. Modification of sequences including naturally occurring recruitment domains can also enhance editing efficiency and reduce off-target effects. Therefore, one aspect of the present disclosure includes modified nucleic acids so that sequences (e.g., synthetic sequences) can be advantageously designed to enhance editing efficiency and reduce off-target effects. Modification can include changing the length of the domain (e.g., extending the length), changing the native sequence to cause changes in the secondary structure, adding chemical modifications, or any combination thereof. Nucleic acids as described herein can provide these advantages.

[0090] In some cases, nucleic acid as described herein can be compared with comparable nucleic acids, high at least about 3,4 or 5 times the efficiency, modify at least one base pair of target nucleic acid, wherein comparable nucleic acid and natural recruitment domain and antisense domain (complementary to target nucleic acid) compound.In some cases, nucleic acid as described herein can be compared with comparable nucleic acids, high at least about 3,4 or 5 times the efficiency, modify at least one base pair of target nucleic acid, wherein comparable nucleic acid and GluR2 domain and antisense domain (complementary to target nucleic acid) compound.In some cases, nucleic acid as described herein can be compared with comparable nucleic acids, high at least about 3,4 or 5 times the efficiency, modify at least one base pair of target nucleic acid, wherein comparable nucleic acid and Cas13b albumen or its active fragment and antisense domain (complementary to target nucleic acid) compound.The improvement in efficiency can be measured by sequencing method, such as Sanger method.

[0091] One aspect of the present disclosure provides a vector. The vector may include a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity recruitment domain. In some cases, the polynucleotide sequence may not form a secondary structure containing a stem loop. In some cases, the polynucleotide sequence may form one or more stem loops. In some cases, the polynucleotide sequence may form a secondary structure including a cross. In some cases, the polynucleotide sequence may form a secondary structure that may be substantially linear. In some cases, the polynucleotide sequence may include at least about 80% sequence identity with one or more sequences including the following: Alu domain encoding sequence, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain encoding sequence and any combination thereof. In some cases, the nucleic acid may be genetically encoded. In some cases, the nucleic acid may be chemically synthesized.

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

[0093] In some cases, the polynucleotide sequence may include at least about 80% sequence identity to an APOBEC domain coding sequence. In some cases, the polynucleotide sequence may include at least about 85% sequence identity to an APOBEC domain coding sequence. In some cases, the polynucleotide sequence may include at least about 90% sequence identity to an APOBEC domain coding sequence. In some cases, the polynucleotide sequence may include at least about 95% sequence identity to an APOBEC domain coding sequence. In some cases, the APOBEC domain coding sequence may be a non-naturally occurring sequence. In some cases, the APOBEC domain coding sequence may include a modified portion. In some cases, the APOBEC domain coding sequence may include a portion of a naturally occurring APOBEC domain sequence.

[0094] In some cases, the polynucleotide sequence may include at least about 80% sequence identity with the GluR2 domain encoding sequence. In some cases, the polynucleotide sequence may include at least about 85% sequence identity with the GluR2 domain encoding sequence. In some cases, the polynucleotide sequence may include at least about 90% sequence identity with the GluR2 domain encoding sequence. In some cases, the polynucleotide sequence may include at least about 95% sequence identity with the GluR2 domain encoding sequence. In some cases, the GluR2 domain encoding sequence may be a non-naturally occurring sequence. In some cases, the GluR2 domain encoding sequence may include a modified portion. In some cases, the GluR2 domain encoding sequence may include a portion of a naturally occurring GluR2 domain sequence.

[0095] In some cases, the polynucleotide sequence may include at least about 80% sequence identity with the coding sequence for raising ADAR. It is possible to separate and purify, or it is possible to synthesize, a polynucleotide sequence encoding at least one RNA editing entity recruitment domain. Such a polynucleotide sequence can be specifically configured to recruit ADAR to a target site. The recruitment may include exogenous ADAR recruitment (which may be co-delivered or delivered alone), endogenous ADAR recruitment, or a combination thereof. In some cases, compared with a naturally occurring recruitment domain, a polynucleotide sequence may be specifically configured to enhance the recruitment of ADAR or enhance the specificity of ADAR recruitment to a specific site. In some cases, the coding sequence may be a non-naturally occurring sequence. In some cases, the coding sequence may include a modified portion. In some cases, the coding sequence may include a portion of a naturally occurring ADAR recruitment domain sequence. It is conceivable that any sequence (natural or synthetic) for raising ADAR is included in the polynucleotide sequence. In some cases, a polynucleotide sequence may include an exemplary sequence as described herein. Figure 2 , Figure 6 , Figure 7 , Figure 8 , Fig.14 , Fig.15 , Fig.21 , Fig.26 , Fig.28 , Fig.29 , Fig.30 and Table 1 include exemplary sequences. The sequences provided herein include sequences having at least a portion that can encode at least one RNA editing entity recruitment domain.

[0096] Table 1: DNA encoding adRNA sequences. The adRNA sequences generated from these sequences are identical to the DNA sequences, but T is replaced by U:

[0097]

[0098]

[0099]

[0100]

[0101] The polynucleotide sequence of the RNA editing entity recruitment domain can include recruiting 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., exogenously delivered ADAR or APOBEC), modified (e.g., exogenously delivered ADAR or APOBEC), endogenous (e.g., endogenous ADAR or APOBEC), or any combination thereof.

[0102] In some cases, the at least one RNA editing entity recruitment domain does not form a secondary structure including a stem loop. In some cases, the at least one RNA editing entity recruitment domain forms a secondary structure including a stem loop. In some cases, the at least one RNA editing entity recruitment domain forms a secondary structure without a stem loop. In some cases, the at least one RNA editing entity recruitment domain forms a secondary structure including a linear portion. In some cases, the at least one RNA editing entity recruitment domain forms a secondary structure including a cross or a portion thereof.

[0103] The polynucleotide sequence may encode more than one RNA editing recruitment domain. In some cases, the polynucleotide sequence may encode multiple recruitment domains. In some cases, the polynucleotide sequence may encode 2, 3, 4, 5, 6 or more recruitment domains. Multiple recruitment domains may include Alu domains, APOBEC domains, GluR2 domains, Cas13 domains or any combination thereof. In some cases, the Alu domain, APOBEC domain, Cas13 domain or GluR2 domain may be a naturally occurring recruitment domain. In some cases, the Alu domain, APOBEC domain, Cas13 domain or GluR2 domain may be non-naturally occurring, may be modified from a natural sequence, or may be recombinant. At least one of the multiple recruitment domains may include a single-stranded sequence. At least one of the multiple recruitment domains may include multiple Alu repeats. At least one of the multiple recruitment domains may form a secondary structure containing a stem loop. At least one of the multiple recruitment domains may form a secondary structure without a stem loop. At least one of the multiple recruitment domains may form a secondary structure including a cross or part thereof. At least one of the plurality of recruitment domains can form a secondary structure comprising a toehold.

[0104] In some cases, the nucleic acid may encode at least one RNA editing entity recruitment domain. In some cases, the nucleic acid may encode an RNA complementary to at least a portion of the target RNA. In some cases, the nucleic acid may encode a recruitment domain and a targeting domain. In some cases, the nucleic acid may encode a recruitment domain, and the nucleic acid may encode a targeting domain. A portion of the target RNA may include a single base. A portion of the target RNA may include multiple bases. A portion of the target RNA may include 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, 100 base pairs or more. In some cases, the target RNA may include about 1bp to about 10bp. In some cases, the target RNA may include about 10bp to about 100bp. In some cases, the target RNA may include about 10bp to about 500bp. In some cases, the target RNA may include about 10bp to about 1000bp. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 200bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 150bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 250bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 275bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 300bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 400bp in length. The nucleic acid comprising the targeting domain and the recruiting domain may include a continuous sequence of at least about 500bp in length.

[0105] A vector may be used to deliver nucleic acid. The vector may include DNA, such as double-stranded DNA or single-stranded DNA. The vector may include RNA. In some cases, the RNA may include base modifications. The vector may include a recombinant vector. The vector may be a vector modified from a naturally occurring vector. The vector may include at least a portion of a non-naturally occurring vector. Any vector may be used. In some cases, the vector may include a viral vector, a liposome, a nanoparticle, an exosome, an extracellular vesicle, or any combination thereof. In some cases, the viral vector may include an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a retroviral vector, a portion of any of these vectors, or any combination thereof. In some cases, a nanoparticle vector may include polymer-based nanoparticles, amino lipid-based nanoparticles, metal nanoparticles (e.g., gold-based nanoparticles), a portion of any of these particles, or any combination thereof. In some cases, the vector may include an AAV vector. The vector may be modified to include a modified VP1 protein (e.g., an AAV vector modified to include a VP1 protein). AAV may include serotypes—e.g., AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, derivatives of any of these, or any combination thereof.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Unless otherwise noted, all nucleotide sequences provided herein are presented in a 5' to 3' direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods, devices and materials are now described. All technologies and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein shall be construed as admitting that the present disclosure is not entitled to be disclosed earlier than the disclosure by virtue of prior disclosure.

[0107] The practice of the techniques will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook, eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al., eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press of Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield et al. (2003) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield et al. (2003) PCR: The Basics (Garland Science); ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 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, ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins, eds. (1984) Transcription and Translation; Buzdin and Lukyanov, eds. (2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells (enzyme and cell fixation); Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos, eds., (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides, ed. (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 most recent editions.

[0108] The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present disclosure.

[0109] All numerical indications, such as pH values, temperatures, times, concentrations and molecular weights (including ranges) are approximate and vary by (+) or (-) increments of 1.0 or 0.1, or by + / -15% or 10% or 5% or 2%, as appropriate. It is to be understood that all numerical indications are preceded by the term "about", although not always expressly stated. It is also to be understood that the agents described herein are exemplary only and that equivalents thereof are known in the art, although not always expressly stated.

[0110] Unless the context indicates otherwise, the various features of the disclosure specifically intended to be described herein may be used in any combination. In addition, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification indicates that a composite comprises components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted and abandoned individually or in any combination.

[0111] Unless expressly stated otherwise, all specified embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and its biological equivalents.

[0112] definition

[0113] 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 "polypeptide" includes a plurality of polypeptides, including mixtures thereof. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this application are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0114] As used herein, the term "about" may mean that a particular value is within an acceptable error range, as determined by a person of ordinary skill in the art, which may depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, according to the practice of the art, "about" may mean ±10%. Alternatively, "about" may represent a range of ±20%, ±10%, ±5%, or ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, within 5 times or within 2 times. Where a particular value is described in the present application and claims, unless otherwise stated, it may be assumed that the term "about" means that the particular value is within an acceptable error range. Similarly, where a range and / or subrange of a value is provided, the range and / or subrange may include the endpoint values ​​of the range and / or subrange. In some cases, a change may include an amount or concentration of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of a specified amount.

[0115] For the numerical ranges recited herein, each intervening number with the same degree of precision is expressly contemplated. For example, for the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated; for the range of 6.0 to 7.0, the numbers 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.

[0116] As used herein, the terms "adenine," "guanine," "cytosine," "thymine," "uracil," and "hypoxanthine" (the nucleobase in inosine) refer to nucleobases as such.

[0117] The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to a nucleobase linked to a (deoxy)ribosyl sugar.

[0118] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the virus class associated with the name and belonging to the Parvoviridae family of the genus Dependoparvovirus. Various serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. In the prior art, at least 11 (sequential numbering) are disclosed. Non-limiting exemplary serotypes for the purposes disclosed herein include any of the 11 serotypes, for example, AAV2 and AAV8. The term "lentivirus" as used herein refers to a member of the virus class associated with the name and belonging to the genus Dependoparvovirus, the family Retroviridae. Although some lentiviruses are known to cause disease, other lentiviruses are also known to be suitable for gene delivery. See, e.g., Tomás et al. (2013) Biochemistry, Genetics and Molecular Biology: “Gene Therapy–Tools and Potential Applications” ISBN 978-953-51-1014-9, DOI: 10.5772 / 52534.

[0119] As used herein, the term "adenosine deaminase acting on RNA" or "ADAR" may refer to an adenosine deaminase that can convert adenosine (A) in an RNA sequence into inosine (I). ADAR1 and ADAR2 are two exemplary ADARs involved in mRNA editing in vivo. Non-limiting exemplary sequences of ADAR1 can be found under the following reference numbers: HGNC: 225; Entrez Gene: 103; Ensembl: ENSG 00000160710; OMIM: 146920; UniProtKB: P55265; and, GeneCards: GC01M154554, and their bioequivalents. Non-limiting exemplary sequences of ADAR2 can be found under the following reference numbers: HGNC: 226; Entrez gene: 104; Ensembl: ENSG00000197381; OMIM: 601218; UniProtKB: P78563; and, GeneCards: GC21P045073, and their biological equivalents. Other non-limiting exemplary sequences of catalytic domains are provided above. The forward and reverse RNAs for guiding site-specific ADAR editing are referred to as "adRNA" and "radRNA", respectively. The catalytic domains of ADAR1 and ADAR2 are included in the sequences provided below.

[0120] ADAR1 catalytic domain:

[0121] KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV(SEQ ID NO:140)

[0122] ADAR2 catalytic domain:

[0123] QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT(SEQ ID NO:141)

[0124] The double-stranded RNA-binding domain (dsRBD) of ADAR is included in the sequence provided below.

[0125] ADAR dsRBD:

[0126] MDIEDEENMSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEA HLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN(SEQ ID NO:142)

[0127] It should be understood that further mutations can be performed to the sequence of ADAR and / or its various domains. For example, the disclosure provides E488Q and E1008Q mutants of both ADAR1 and ADAR2, and a "mixed" variant of ADAR2-caused by C-terminal deletion. This "mixed" variant is referred to as a "variant" because it shows promiscuity in the edited reads, with several A's approaching the target sequence (verified on 2 different loci) showing A to G conversion. The sequence of the variant is provided below.

[0128] "Promiscuous" ADAR2 variants:

[0129] *(SEQ ID NO:143)

[0130] Without being bound by theory, C-terminal deletions in ADAR1 may produce the same or similar effects.

[0131] The term "Alu domain" may refer to a sequence obtained from an Alu transposable element ("Alu element"). Typically, the length of an Alu element is about 300 base pairs. An Alu element typically includes the following structure: a cross-poly (poly) A5-TAC-polyA6-cross-polyA tail, wherein two cross-shaped domains are similar in nucleotide sequence. An "Alu domain" may include a cross-shaped portion of an Alu element. In some embodiments, two Alu domains including a cross-shaped structure are connected by a sequence complementary to a target RNA sequence.

[0132] As used herein, the term "APOBEC" may refer to any protein and its equivalent falling into the evolutionarily conservative cytidine deaminase family (which catalyzes the editing of C to T, which can be interpreted as the conversion of C to U) involved in mRNA editing. In some aspects, the term APOBEC may refer to any one of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4 or its equivalent. Provided herein are non-limiting exemplary sequences of fusion proteins including one or more APOBEC domains, which are fused to the ADAR domain or to an alternative domain so that they are suitable for RNA editing systems. For this reason, APOBEC can be regarded as an equivalent of ADAR, but it can be catalyzed and edited by different conversions. Therefore, without being bound by theory, all embodiments of the editing system based on ADAR considered herein are believed to be suitable for use in the RNA editing system based on APOBEC. In some cases, the use of APOBECs may involve certain modifications, such as, but not limited to, the use of specific guide RNAs or "gRNAs" to recruit the enzyme.

[0133] "Aptamer" may refer to a short single-stranded oligonucleotide that can bind to 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.

[0134] As used herein, the term "comprising" is intended to indicate that compositions and methods include the listed elements, but do not exclude other elements. Unless otherwise indicated, open-ended terms such as "comprising", "containing", "containing", "having", etc., represent inclusion. When "consisting essentially of..." is used to define compositions and methods, it should be indicated that other elements that are of any importance to the combination for the intended use are excluded. Therefore, a composition consisting essentially of the elements defined herein may not exclude trace contaminants from separation and purification methods, and pharmaceutically acceptable carriers (e.g., phosphate buffered saline, preservatives, etc.). "Consisting of..." refers to trace elements excluding other ingredients, and substantial method steps for applying the compositions of the present disclosure. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0135] "Classical amino acids" refer to the 20 amino acids naturally occurring in humans, shown in the following table, each with a three-letter abbreviation, a one-letter abbreviation, a structure, and a corresponding codon:

[0136] Nonpolar aliphatic residues

[0137]

[0138] Aromatic residues

[0139]

[0140] Polar uncharged residues

[0141]

[0142] Positively charged residues

[0143]

[0144] Negatively charged residues

[0145]

[0146] The term "Cas9" may refer to the CRISPR-associated nuclease referred to by the name. Non-limiting exemplary Cas9s include Staphylococcus aureus Cas9, nuclease-dead Cas9, and their respective orthologs and biological equivalents. Orthologs include, but are not limited to, Streptococcus pyogenes Cas9 ("spCas9"), Cas 9 from Streptococcus thermophiles, Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida; and Cpf1 from various bacterial species including Acidaminococcus spp. and Francisella novicida U112 (which performs a cleavage function similar to Cas9). For example, UniProtKB G3ECR1 (CAS9_STRTR) and dead Cas9 or dCas9 lacking endonuclease activity (e.g., having mutations in both RuvC and HNH domains) can be used. The term "Cas9" may further refer to an equivalent of a reference 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, Cas9 is derived from Campylobacter jejuni, or another Cas9 ortholog having a length of 1000 amino acids or less.

[0147] The term "CRISPR" as used herein may refer to a sequence-specific genetic manipulation technique that relies on a clustered regularly interspaced short palindromic repeat pathway. CRISPR can be used to perform gene editing and / or gene regulation, as well as simply targeting proteins to specific genomic locations. "Gene editing" may refer to a type of genetic modification in which the nucleotide sequence of a target polynucleotide is changed by introducing deletions, insertions, single-strand or double-strand breaks, or base substitutions into a polynucleotide sequence. In certain aspects, CRISPR-mediated gene editing utilizes a pathway of non-homologous end joining (NHEJ) or homologous recombination for editing. Gene regulation may refer to increasing or decreasing the production of a specific gene product (eg, protein or RNA).

[0148] As used herein, the term "absence" may refer to below normal (physiologically acceptable) levels of a particular agent. With respect to a protein, absence may refer to below normal levels of a full-length protein.

[0149] As used herein, the term "detectable label" may refer to at least one label that is capable of directly or indirectly generating a detectable signal. A non-exhaustive list of such labels includes enzymes that generate a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups with electron density detectable by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltammetry, impedance detectable groups, such as the size of the molecule is sufficient to induce a detectable modification of its physical and / or chemical properties, such detection can be by optical methods (e.g., diffraction, surface plasmon resonance, surface changes, contact angle changes) or physical methods (e.g., atomic force spectroscopy, tunneling or radioactive molecules, such as 32 P. 35 S or 125 I) to complete.

[0150] As used herein, the term "domain" may refer to a specific region of a protein or polypeptide and is associated with a specific function. For example, a "domain associated with an RNA hairpin motif" may refer to a domain of a protein that binds one or more RNA hairpins. The binding may optionally be specific for a particular hairpin.

[0151] The term "dystrophin" as used herein refers to the protein corresponding to this name and encoded by the gene Dmd; non-limiting examples of which are found under the UniProt references P11532 (for human) and P11531 (for mouse).

[0152] "Editing-inducing element" may refer to a structure that is primarily double-stranded RNA, which is necessary for efficient RNA editing. Non-limiting examples of editing-inducing elements are described in Daniel, C. et al. (2017) Genome Biol. 18, 195. The following structure (SEQ ID NO: 15) provides another non-limiting example of an editing-inducing element:

[0153]

[0154] ADAR is a naturally occurring RNA editing enzyme that catalyzes the hydrolytic deamination of adenosine to inosine, which is biochemically known as guanosine. APOBEC is an enzyme described above that performs a similar function but for the deamination of cytosine to thymine.

[0155] The term "encoding" as applied to a polynucleotide may refer to a polynucleotide that is said to "encode" a polypeptide if, in its native state or by manipulations well known to those skilled in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragments. The antisense strand is the complement of such nucleic acid, and the coding sequence can be deduced therefrom.

[0156] The terms "equivalent" or "bioequivalent" are used interchangeably when referring to specific molecular, biological, or cellular materials and refer to those that have minimal homology but still retain the desired structure or function.

[0157] "Eukaryotic cells" include all kingdoms of life except prokaryotes. They can be easily identified by the membrane surrounding the nucleus. Animals, plants, fungi and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by inner membranes and cytoskeleton. The most characteristic membrane-enclosed structure is the nucleus. Unless otherwise specified, the term "host" includes 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.

[0158] As used herein, "expression" may 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 may include splicing of mRNA in eukaryotic cells.

[0159] The term "functional" as used herein may be applied to the modification of any molecular, biological or cellular material so that it performs a specific, particular role.

[0160] As used herein, the term "Glur2 mRNA" may refer to an mRNA encoding the ionotropic AMPA glutamate receptor 2 ("Glur2"), which undergoes adenosine to inosine (A->I) editing. This mRNA recruits ADARs in a site-specific manner.

[0161] As used herein, the term "gRNA" or "guide RNA" may refer to a guide RNA sequence for targeting a specific polynucleotide sequence for gene editing using CRISPR technology. The techniques for designing gRNA and donor therapeutic polynucleotides for target specificity are well known in the art. 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. The gRNA includes, or is essentially composed of, or is further composed of: a fusion polynucleotide comprising CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA); or, a polynucleotide comprising CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA). In some aspects, the gRNA is synthetic (Elley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83).

[0162] The terms "hairpin", "hairpin loop", "stem-loop" and / or "loop" used alone or in combination with "motif" in the context of oligonucleotides refer to a structure formed in a single-stranded oligonucleotide that, when the sequences are within a single strand, are complementary when base pairs are read in opposite directions to form a region whose conformation resembles a hairpin or loop.

[0163] "Homology" or "identity" or "similarity" can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then 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. "Unrelated" or "non-homologous" sequences share less than 40% identity or less than 25% identity with one of the sequences of the present disclosure.

[0164] Homology refers to the % identity of a sequence to a reference sequence. In fact, whether any particular sequence can have at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with any sequence described herein (which can correspond to a specific nucleic acid sequence described herein), this specific polypeptide sequence can routinely use known computer programs, such as Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin, University Research Park, Postal Code 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference sequence, parameters can be set so that the identity percentage is calculated over the full length of the reference sequence, and the homology gap is allowed to be no more than 5% of the total reference sequence.

[0165] For example, in a specific embodiment, the FASTDB computer program can be used to determine the identity between a reference sequence (query sequence, i.e., sequence of the present disclosure) and a subject sequence (also referred to as a global sequence alignment) based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6: 237-245 (1990)). In some cases, in a specific embodiment in which identity is narrowly interpreted, the parameters for FASTDB amino acid alignments can include: scoring scheme = PAM (percentage of mutations accepted) 0, k-tuple = 2, mismatch penalty = 1, contiguous penalty = 20, random group length = 0, cutoff score = 1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the subject sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions (not due to internal deletions), the result can be manually corrected to take into account the fact that the N- and C-terminal truncations of the subject sequence are not taken into account when the FASTDB program calculates the global identity percentage. For the subject sequence truncated at the N- and C-terminal ends relative to the query sequence, the identity percentage can be corrected by calculating the number of residues on both sides of the subject sequence N- and C-terminal ends of the query sequence, which residues do not match / align with the corresponding subject residues, and are expressed as a percentage of the total bases of the query sequence. Determine whether the residue is matched / aligned, and can be determined by the result of the FASTDB sequence alignment. Then the percentage can be subtracted from the identity percentage calculated using specific parameters by the FASTDB program to obtain a final identity percentage score. This final identity percentage score can be used for the purpose of this embodiment. In some cases, for the purpose of manually adjusting the identity percentage score, only the residues that the N- and C-terminal ends of the subject sequence do not match / align with the query sequence are considered. That is to say, for this manual correction, only the query residue position outside the farthest N-terminal and C-terminal residues of the subject sequence is considered.For example, the subject sequence of 90 residues can be compared with the query sequence of 100 residues to determine the identity percentage.The deletion occurs at the N-terminal of the subject sequence, so FASTDB compares the matching / comparison of the first 10 residues that are not obvious at the N-terminal.10 unpaired residues represent 10% of the sequence (the total number of residues in the number of residues / query sequence that do not match at the N-terminal and C-terminal), so 10% is deducted from the identity percentage score calculated by the FASTDB program.If the remaining 90 residues are fully matched, the final identity percentage can be 90%.In another embodiment, the subject sequence of 90 residues is compared with the query sequence of 100 residues.This deletion is an internal deletion, so there is no residue that does not match / comparison with the query at the N-terminal or C-terminal of the subject sequence.In this case, the identity percentage calculated by FASTDB cannot be manually corrected.Again, only residue positions outside the N- and C-termini of the subject sequence that were not matched / aligned with the query sequence (as displayed in the FASTDB alignment) were manually corrected.

[0166] "Hybridization" may refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding may occur by Watson-Crick base pairing, Hogstein binding, or in any other sequence-specific manner. The complex may include a double strand forming a double helix structure, three or more strands forming a multi-stranded complex, a self-hybridizing single strand, or any combination thereof. A hybridization reaction may constitute a step in a broader process, such as the initiation of a PC reaction, or enzymatic cleavage of a polynucleotide by a ribozyme.

[0167] 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 washing solution of about 4x SSC to about 8x SSC. Examples of mild 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 washing solution of about 5x SSC to about 2x SSC. Examples of high stringency 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 washing solution of about 1x SSC, 0.1x SSC or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the wash incubation time is about 1, 2 or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that the equivalent of SSC using other buffer systems may be employed.

[0168] As used herein, "inhibit" refers to the ability to substantially antagonize, inhibit, prevent, control, slow, destroy, alter, eliminate, stop or reverse the development or severity of the activity of a particular agent (e.g., infectious agent) or disease.

[0169] As used herein, the term "interferon" may refer to a group of signal transduction proteins known to be associated with an immune response. In the context of the present application, the interferons of interest are those that cause enhanced 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 interferon alpha include, but are not limited to, Sigma-Aldrich, R&D Systems, Abcam, and Thermo Fisher Scientific. Alternatively, known vectors and given protein sequences, such as Q6QNB6 (human IFNA), can be used to produce interferon alpha.

[0170] As used herein, the term "separation" may refer to molecules or biological or cellular materials that are substantially free of other materials. On the one hand, the term "separation" may refer to nucleic acids (such as DNA or RNA), or proteins or polypeptides (such as antibodies or derivatives thereof), or cells or organelles, or tissues or organs separated from other DNA or RNA present in natural sources, or proteins or polypeptides, or cells or organelles, or tissues or organs, respectively. The term "separation" may also refer to nucleic acids or peptides that are substantially free of cell materials, viral materials or culture media when produced by recombinant DNA technology, or nucleic acids or peptides that are substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acids" refers to, including nucleic acid fragments that are not naturally present as fragments and may not be found in the natural state. The term "separation" is also used herein to refer to polypeptides separated from other cell proteins, and is intended to cover purified and recombinant polypeptides. The term "separation" is also used herein to refer to cells or tissues separated from other cells or tissues, and is intended to cover cultured and transformed cells or tissues.

[0171] "Messenger RNA" or "mRNA" is a nucleic acid molecule that is transcribed from DNA and then processed to remove non-coding portions called introns. The resulting mRNA is exported from the cell nucleus (or another location where DNA is present) and translated into protein. The term "pre-mRNA" may refer to the strand before processing to remove non-coding portions.

[0172] The term "mutation" as used herein may refer to a change in the nucleic acid sequence encoding the protein relative to the consensus sequence of the protein. A "missense" mutation causes a codon to be replaced by another codon; a "nonsense" mutation changes a codon from a codon encoding a specific amino acid to a stop codon. Nonsense mutations usually result in truncated translation of the protein. "Silent" mutations are those mutations that have no effect on the resulting protein. The term "point mutation" as used herein may refer to a mutation that affects only one nucleotide in a gene sequence. "Splice site mutations" refer to those mutations present in pre-mRNA (before processing to remove introns), which result in protein translation errors due to incorrect delineation of splice sites, often truncating the protein. Mutations may include single nucleotide variations (SNVs). Mutations may include sequence variants, sequence changes, sequence changes, or allelic variants. Reference DNA sequences may be obtained from a reference database. Mutations may affect function. Mutations may not affect function. Mutations may occur at one or more nucleotides at the DNA level, at one or more nucleotides at the ribonucleic acid (RNA) level, at one or more amino acids at the protein level, or in any combination thereof. Reference sequences can be obtained from databases such as the NCBI Reference Sequence Database (RefSeq) database. Specific changes that may constitute mutations may include substitutions, deletions, insertions, inversions or conversions of one or more nucleotides, or one or more amino acids. Mutations may be point mutations. Mutations may be fusion genes. Fusion pairs or fusion genes may be produced by mutations such as translocations, interstitial deletions, chromosomal inversions, or any combination thereof. Mutations may constitute the variability of the number of repeated sequences (e.g., triplicates, quadruples, or others). For example, a mutation may be an increase or decrease in the number of copies associated with a given sequence (i.e., copy number variation or CNV). Mutations may include two or more sequence changes in different alleles, or two or more sequence changes in one allele. Mutations may include two different nucleotides at one position of one allele, such as mosaic mutations. Mutations may include two different nucleotides at one position of one allele, such as chimeric mutations. Mutations may be present in malignant tissues. The presence or absence of mutations may indicate an increased risk of developing a disease or condition. The presence or absence of mutations may indicate the presence of a disease or condition. Mutations may be present in benign tissues. The absence of a mutation may indicate that the tissue or sample is benign. Alternatively, the absence of a mutation may not indicate that the tissue or sample is benign. The methods described herein may include identifying the presence of a mutation in a sample.

[0173] The term "non-classical amino acid" may refer to those synthetic or other modified amino acids outside of this group, typically produced by chemical synthesis or modification of classical amino acids (e.g., amino acid analogs). The present disclosure employs proteinogenic non-classical amino acids in some of the methods and vectors disclosed herein. A non-limiting exemplary non-classical amino acid is pyrrolysine (Pyl or O), the chemical structure of which is provided below:

[0174]

[0175] Inosine (I) is another exemplary non-classical amino acid that can be found in tRNA and is essential for correct translation according to "wobble base pairing." The structure of inosine is provided above.

[0176] The term "ornithine transcarbamylase" or "OTC" as used herein may refer to the protein corresponding to the name and encoded by the gene Otc; non-limiting examples of which are found under UniProt reference numbers P00480 (for humans) and P11725 (for mice). OTC deficiency is an X-linked genetic disease that results in higher ammonia concentrations in the blood. In some cases, OTC deficiency is caused by a G->A splice site mutation at the exon 4 donor splice site, which results in mis-splicing of the pre-mRNA. This mutation results in the formation of proteins that are elongated or carry point mutations. OTC protein levels are reduced 15 to 20 times. See, e.g., Hodges, PE & Rosenberg, LE The spf ash Mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc. Natl. Acad. Sci. USA 86, 4142–4146 (1989)) (showing an alternative form of the generated OTC). Its sequence is provided below:

[0177] OTC pre-mRNA (wild type):

[0178] .....CTCACAGACACCGCTC G GTTTGTAAAACTTTTCTTC.....(SEQ ID NO:144)

[0179] OTC pre-mRNA (mutant):

[0180] .....CTCACAGACACCGCTC AGTTTGTAAAACTTTTCTTC.....(SEQ ID NO:145)

[0181] OTC mRNA (mis-spliced, mutant):

[0182] .....CTCACAGACACCGCTC A GTTTGTAAAACTTTTCTTC.....(SEQ ID NO:146)

[0183] OTC mRNA (correctly spliced, mutant):

[0184] .....CTCACAGACACCGCTC A TGTCTTATCTAGCATGACA....(SEQ ID NO:147)

[0185] OTC mRNA (correctly spliced, wild type):

[0186] .....CTCACAGACACCGCTC G TGTCTTATCTAGCATGACA....(SEQ ID NO:148)

[0187] As shown above, when a mutation is present, the correct splice variant can be produced; however, such production results in a missense mutation, which may also result in OTC deficiency.

[0188] The terms "protein", "peptide" and "polypeptide" are used interchangeably and refer to compounds of two or more subunit amino acids, amino acid analogs or peptidomimetics in their broadest sense. These subunits can be connected by peptide bonds. In another embodiment, the subunits can be connected by other bonds, such as esters, ethers, etc. A protein or peptide can contain at least two amino acids, and there is no restriction on the maximum number of amino acids that can constitute a protein or peptide sequence. The term "amino acid" as used herein can refer to natural and / or non-natural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs and peptidomimetics. The term "fusion protein" as used herein can refer to a protein composed of domains from more than one naturally occurring or recombinantly produced protein, wherein each domain generally performs a different function. In this regard, the term "joint" can refer to a protein fragment used to connect these domains together-optionally retaining the conformation of the fused protein domains, and / or preventing unfavorable interactions between the fused protein domains, which may impair their respective functions.

[0189] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. 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, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polynucleotide assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term can also refer to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure encompasses a double-stranded form and each of two complementary single-stranded forms (known or expected to constitute the double-stranded form).

[0190] A polynucleotide is composed of a specific sequence of the following four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, when the polynucleotide is RNA, thymine is uracil (U). In some embodiments, a polynucleotide may include one or more other nucleotide bases, such as inosine (I), a nucleoside formed when hypoxanthine is linked to a ribofuranosyl sugar via a β-N9-glycosidic bond, resulting in the following chemical structure:

[0191]

[0192] Inosine is read by the translation machinery as guanine (G).

[0193] The term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database of a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searches.

[0194] As used herein, the term "purification marker" may refer to at least one marker that can be used 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, soft tag (Softag) 1, Softag 3, streptomycin (Strep) or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, biotin, digitoxin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

[0195] As used herein, the term "recombinant expression system" refers to one or more gene constructs used to express certain genetic materials formed by recombination; in this regard, the term "construct" is interchangeable with the term "vector" as defined herein.

[0196] The term "recombinant protein" as used herein may refer to a polypeptide produced by recombinant DNA technology, wherein typically, 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.

[0197] As used herein, the term "restoration" with respect to protein expression may refer to the ability to establish full-length protein expression where protein expression was previously truncated due to a mutation. In the context of "restoration of activity," the term includes bringing the expression of a protein to its normal, unmutated level where a mutation results in abnormal expression (e.g., too low or too high).

[0198] The term "sample" as used herein generally refers to any sample (e.g., a blood sample or a tissue sample) of a subject. A sample or a portion thereof may include stem cells. A portion of a sample may be enriched with stem cells. Stem cells may be isolated from a sample. A sample may include tissues, cells, serum, plasma, exosomes, body fluids, or any combination thereof. Body fluids may include urine, blood, serum, plasma, saliva, mucus, spinal fluid, tears, semen, bile, amniotic fluid, or any combination thereof. A sample or a portion thereof may include extracellular fluid obtained from a subject. A sample or a portion thereof may include cell-free nucleic acids, DNA, or RNA. A sample or a portion thereof may be analyzed for the presence or absence of one or more mutations. Genomic data may be obtained from a sample or a portion thereof. A sample may be a sample suspected or confirmed to have a disease or condition. A sample may be a sample removed from a subject via non-invasive, minimally invasive, or invasive techniques. A sample or a portion thereof may be obtained by a tissue brush, a swab, a tissue biopsy, excised tissue, a fine needle aspiration, a tissue wash, a cytological specimen, a surgical resection, or any combination thereof. A sample or a portion thereof may include tissue or cells from a 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, kidney tissue, uterine tissue, ovarian tissue, endometrial tissue, vaginal tissue, vulva tissue, uterine 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.

[0199] The term "sequencing" as used herein may include bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore sequencing, shotgun sequencing, RNA sequencing, Enigma sequencing, or any combination thereof.

[0200] The term "stop codon" means a continuous sequence of three nucleotides in a messenger RNA indicating the termination of translation. Non-limiting examples include UAG, UAA, UGA in RNA, and TAG, TAA or TGA in DNA. Unless otherwise indicated, the term also includes nonsense mutations that introduce premature stop codons in DNA or RNA, which cause any protein produced to be abnormally shortened. The specific names of the tRNA corresponding to each stop codon are: amber (UAG), ochre (ochre) (UAA), and opal (opal) (UGA).

[0201] "Transfer RNA" or "tRNA" is a nucleic acid molecule that helps translate mRNA into protein. tRNA has a unique folded structure, including three hairpin loops; one of these loops includes a "stem" portion that encodes an anticodon. The anticodon recognizes the corresponding codon on the mRNA. Each tRNA is "charged with" an amino acid corresponding to the mRNA codon; this "charging" is achieved by the enzyme tRNA synthetase. When the tRNA recognizes the codon corresponding to its anticodon, the tRNA transfers its charged amino acid to the growing amino acid chain to form a polypeptide or protein. Endogenous tRNAs can be charged by endogenous tRNA synthetases. Therefore, endogenous tRNAs are usually charged with classical amino acids. Orthogonal tRNAs from external sources require corresponding orthogonal tRNA synthetases. Such orthogonal tRNAs can be charged with classical amino acids and non-classical amino acids. In some embodiments, the amino acid charged by the tRNA can be detectably labeled to enable detection in vivo. Labeling techniques are known in the art and include, but are not limited to, click chemistry, where an azide / alkyne containing an unnatural amino acid is added by an orthogonal tRNA / synthetase pair and can therefore be detected using an alkyne / azide containing a fluorophore or other such molecule.

[0202] As used herein, the terms "treat," "treating," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease, disorder or condition, or a sign or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disorder and / or an adverse effect attributable to the disorder.

[0203] The term "vector" as used herein may refer to a nucleic acid construct designed to be transferred between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, etc. "Viral vector" is defined as a recombinant virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, in vitro or in vitro. In some embodiments, a plasmid vector may be prepared by a commercially available vector. In other embodiments, a viral vector may be prepared by baculovirus, retrovirus, adenovirus, 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, alpha viral vectors, etc. Vectors based on infectious tobacco mosaic virus (TMV) may be used to manufacture proteins, and it has been reported that Griffithsin has been expressed in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106 (15): 6099-6104). Alphavirus vectors, such as those based on Semliki Forest virus and those based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al. (1999) Nat. Med. 5 (7): 823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct may refer to a polynucleotide comprising a retroviral genome or a portion thereof and a gene of interest. More details on modern methods for vectors used for gene transfer can be found, for example, in Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors comprising 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, California) and Promega Biotech (Madison, Wisconsin). In one aspect, the promoter is a polIII promoter.

[0204] The pharmaceutical composition for administering AdRNA can be conventionally present in dosage unit form and can be prepared by any method known in the pharmaceutical field. The pharmaceutical composition can be prepared, for example, by the following manner: the compound provided herein is uniformly and closely combined with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, the product is formed into a desired preparation. In the pharmaceutical composition, the compound provided herein is included in an amount sufficient to produce the desired therapeutic effect. For example, the pharmaceutical composition of the technology can take a form suitable for almost any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal and vaginal administration, or a form suitable for inhalation or insufflation.

[0205] For topical administration, the compounds can be formulated into solutions, gels, ointments, creams, suspensions, etc., as is known in the art.

[0206] Systemic formulations include those designed for administration by injection (eg, subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0207] Useful injectable formulations include sterile suspensions, solutions or emulsions of compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulation agents, such as suspending agents, stabilizers and / or dispersants. Injectable formulations may exist in unit dosage form, such as in ampoules or multidose containers, and may include added preservatives.

[0208] Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle prior to use, including but not limited to sterile, pyrogen-free water, buffer and dextrose solution. To this end, the compounds provided herein can be dried by any technique known in the art (e.g., lyophilization) and reconstituted prior to use.

[0209] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0210] For oral administration, the pharmaceutical composition can be in the form of, for example, lozenges, tablets or capsules, which are prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon oxide); disintegrants (e.g., potato starch or sodium starch glycolate); or, wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated with, for example, sugar, film or enteric coatings by methods well known in the art.

[0211] Compositions intended for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions can include one or more agents selected from the group consisting of sweeteners, flavoring agents, colorants and preservatives to provide pharmaceutically exquisite and palatable preparations. Tablets contain a mixture of compounds provided herein and non-toxic, pharmaceutically acceptable excipients suitable for making tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants (e.g., corn starch or alginic acid); binders (e.g., starch, gelatin or gum arabic); and lubricants (e.g., magnesium stearate, stearic acid or talc). Tablets may not be coated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period of time. For example, delay materials such as monostearate or distearate can be used. They can also be coated by techniques well known to technicians. The pharmaceutical composition of this technology can also be in the form of an oil-in-water emulsion.

[0212] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be in the form of dry products for reconstitution with water or other suitable vehicles before use. Such liquid preparations may be prepared in a conventional manner with the following pharmaceutically acceptable additives: for example, suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol, cremophore TM or fractionated vegetable oil); and, preservatives (e.g., methyl or propyl paraben or sorbic acid). The preparation may also contain buffer salts, preservatives, flavoring agents, coloring agents and sweeteners as appropriate.

[0213] "Administration" can be performed in one dose, continuously or intermittently, throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and may vary with the composition used for treatment, the purpose of treatment, the target cell treated, and the subject treated. Single or multiple administrations may be performed according to the dosage level and mode selected by the treating physician. Suitable dosage formulations and methods for administering agents are known in the art. The route of administration may also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and may vary with the composition used for treatment, the purpose of treatment, the health condition or disease stage of the subject treated, and the target cell or tissue. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical application.

[0214] Application can refer to a method that can be used to enable a compound or composition (e.g., a DNA construct, a viral vector or other) to be delivered to a desired biological site of action. These methods may include topical application (e.g., lotion, cream, ointment) to the outer surface of a surface (e.g., skin). These methods may include parenteral administration (including intravenous, subcutaneous, intrathecal, abdominal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration. In some cases, the subject may apply the composition without supervision. In some cases, the subject may apply the composition under the supervision of a medical professional (e.g., a doctor, a nurse, a physician's assistant, a caregiver, a hospice worker, etc.). In some cases, a medical professional may apply the composition. In some cases, a cosmetic professional may apply the composition.

[0215] The administration or use of the compositions disclosed herein can be performed continuously or discontinuously for 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. In some cases, the treatment period can be 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, 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.

[0216] Administration or application of the compositions disclosed herein may 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 a day. In some cases, administration or application of the compositions disclosed herein may 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 a week. In some cases, the administration or application of the compositions disclosed herein can be performed for 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 , 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.

[0217] In some cases, the composition can be used as a single dose or a separate dose. In some cases, the composition described herein can be used at a first time point and a second time point. In some cases, the composition can be used so that the first application precedes another application, and the application time differs by 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.

[0218] The term "effective amount" may refer to an amount sufficient to achieve the desired effect. In the context of therapeutic or preventive applications, the effective amount will depend on the type and severity of the disorder in question, as well as the characteristics of the individual subject, such as overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of immunogenic compositions, in some embodiments, an effective amount is an amount sufficient to cause a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is an amount sufficient to cause antibody production against an antigen. In some embodiments, an effective amount is the amount required to confer passive immunity to a subject in need. With regard to immunogenic compositions, in some embodiments, in addition to the above factors, the effective amount may also depend on the intended use, the degree of immunogenicity of a specific antigenic compound, and the health / responsiveness of the subject's immune system. A technician can determine the appropriate amount based on these and other factors.

[0219] In the case of in vitro applications, in some embodiments, the effective amount may depend on the size and nature of the application in question. It may also depend on the nature and sensitivity of the in vitro target, as well as the method used. The technician can determine the effective amount based on these and other considerations. According to the embodiment, the effective amount may include one or more administrations of the composition.

[0220] It can be inferred without explicit citation that, unless otherwise specified, when the present disclosure relates to polypeptides, proteins, polynucleotides or antibodies, their equivalents or biological equivalents are intended to be within the scope of the present disclosure. As used herein, the term "biological equivalents thereof" is intended to be synonymous with "equivalents thereof" when referring to reference proteins, antibodies, polypeptides or nucleic acids, meaning those with minimal homology while still maintaining the desired structure or function. Unless specifically described herein, it is expected that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, equivalents mean at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% homology or identity, and show biological activity substantially equivalent to reference proteins, polypeptides or nucleic acids. Alternatively, when referring to polynucleotides, their equivalents are polynucleotides that hybridize with reference polynucleotides or their complementary sequences under stringent conditions.

[0221] The present disclosure provides polypeptide and / or polynucleotide sequences for gene and protein editing techniques described below. It should be understood that, although not always explicitly stated, the sequences provided herein can be used to provide expression products, as well as substantially identical sequences of proteins having the same biological properties. These "bioequivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides described herein. When compared using a sequence identity method run under default conditions, they may have a primary amino acid 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. The amino acids of the sequence are modified with spare amino acids having similar charges. In addition, an equivalent polynucleotide is a polynucleotide that hybridizes with a reference polynucleotide or its complementary sequence under stringent conditions, or, with respect to a polypeptide, refers to a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide encoding a reference or its complementary chain under stringent conditions. Alternatively, an equivalent polypeptide or protein is a polypeptide or protein expressed by an equivalent polynucleotide.

[0222] "Composition" generally refers to an active agent, such as an adRNA, compound or composition of the present disclosure, and a naturally occurring or non-naturally occurring carrier, an inert (e.g., a detectable agent or marker) or active substance, such as a combination of adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc., and includes a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acid lipids and carbohydrates (e.g., sugars, including monosaccharides, di-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, by weight or volume, alone or in combination accounting for 1 to 99.99%. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / antibody components that can also play a buffering role include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also intended to be included in the scope of this technology, and examples include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and inositol.

[0223] Compositions used according to the present disclosure, including cells, treatments, therapies, medicaments, drugs and pharmaceutical preparations, can be packaged in the form of dosage units for ease of administration and uniform dosage. The term "unit dose" or "dose" may refer to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of compositions, which is calculated to produce a desired response in relation to its administration (i.e., appropriate routes and schemes). The amount administered according to the number of treatments and the unit dose depends on the desired result and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique for each person. Factors affecting dosage include the subject's physical and clinical state, route of administration, expected therapeutic goals (symptom relief and cure), and the efficacy, stability and toxicity of a particular composition. After preparation, the solution can be administered in a manner compatible with the dosage formulation, and the amount administered is an effective amount for treatment or prevention. The preparation can be easily administered in a variety of dosage forms, such as the type of injectable solution described herein.

[0224] As used herein, the term "reduce or eliminate its expression and / or function" may refer to reducing or eliminating the transcription of the polynucleotide to mRNA, or alternatively reducing or eliminating the translation of the mRNA to a peptide, polypeptide or protein, or reducing or eliminating the function of the peptide, polypeptide or protein. In a non-limiting embodiment, the transcription of the polynucleotide to mRNA is reduced to at least half of its normal level found in wild-type cells.

[0225] The phrases "first-line" or "second-line" or "third-line" can refer to the order in which a patient receives treatment. A first-line therapy regimen is the first treatment given, while a second-line or third-line therapy is given after the first-line therapy or after the 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 primary treatment may be surgery, chemotherapy, radiation therapy, or a combination of these therapies. Those skilled in the art also refer to first-line therapy as "primary therapy and primary treatment." See the National Cancer Institute's website at cancer.gov, last accessed November 15, 2017. Typically, a subsequent chemotherapy regimen is given to a patient because the patient did not show a positive clinical or subclinical response to the first-line therapy, or the first-line therapy has been stopped.

[0226] The term "contacting" refers to the direct or indirect binding or interaction between two or more entities. A specific example of direct interaction is binding. A specific example of indirect interaction is where one entity acts on an intermediate molecule, which in turn acts on a second mentioned entity. As used herein, contacting includes in solution, in solid phase, in vitro, ex vivo, in cells, and in vivo. Contacting in vivo can be referred to as administration or dosing.

[0227] "Cryoprotectants" are known in the art and include, but are not limited to, for example, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are generally used.

[0228] Disclosed herein are adRNAs for site-specific editing of RNA in the absence of overexpression of ADAR enzymes. Further provided herein are modifications of A->G editing of DNA. Additionally, provided herein are screening for ADAR2 mutants capable of site-specific C->T editing of RNA and DNA. Further provided herein are modifications of C->T editing of RNA via the use of APOBEC1 expressed with ACF.

[0229] Compared to other ADAR2 systems, the present disclosure is unique because it proposes a new method to recruit endogenous ADARs to catalyze therapeutic RNA editing. In addition, none of the prior art systems provide a means to use ADAR enzymes to modify C->T editing. Finally, they do not disclose programmable site-specific RNA editing using APOBEC.

[0230] Disclosed herein is an exemplary adRNA comprising an RNA targeting domain complementary to a target RNA, and one or more ADAR recruitment domains. When bound to its target, the adRNA is capable of recruiting the ADAR enzyme to the target RNA. The ADAR enzyme is then capable of catalyzing the conversion of the target adenosine to inosine. Without being bound by theory, it is believed that adRNA can be similarly used to recruit one of the ADAR2 mutants or APOBEC1 to affect the editing of C->T RNA.

[0231] Also disclosed herein are in vitro and in vivo experiments using engineered adRNAs to recruit endogenous ADAR enzymes. Also disclosed herein are experiments showing the C->T editing efficiency of ADAR mutants and APOBEC1 / ACF constructs.

[0232] The viral vector as described herein may include a nucleic acid sequence encoding at least one RNA editing entity recruitment domain. In some cases, the nucleic acid sequence may encode more than one RNA editing entity recruitment domain, such as 2, 3, 4 or more. The RNA editing entity recruitment domain may include at least about 80% sequence identity with at least one of the following items: Alu domain, apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain, Cas13 domain, GluR2 domain, or any combination thereof. The recruitment domain may include one or more GluR2 domains, one or more Alu domains, one or more APOBEC domains, or any combination thereof. The recruitment domain may include more than one GluR2 domain, more than one Alu domain, more than one APOBEC domain, Cas13 domain, or any combination thereof. The recruitment domain may not include an Alu domain. The recruitment domain may not include a GluR2 domain. The recruitment domain may not include an APOBEC domain. The recruitment domain may not include a Cas13 domain.

[0233] The APOBEC recruitment domain can include an APOBEC1 recruitment domain, an APOBEC2 recruitment domain, an APOBEC3A recruitment domain, an APOBEC3B recruitment domain, an APOBEC3C recruitment domain, an APOBEC3D recruitment domain, an APOBEC3E recruitment domain, an APOBEC3F recruitment domain, an APOBEC3G recruitment domain, an APOBEC3H recruitment domain, an APOBEC4 recruitment domain, a derivative of any of these domains, or any combination thereof.

[0234] The raising domain can include at least about 80% sequence identity with any of the Alu domains as described herein. In some cases, the raising domain can include at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of the Alu domains as described herein.

[0235] The recruitment domain may comprise at least about 80% sequence identity with any of the APOBEC domains as described herein. In some cases, the recruitment domain may comprise at least about 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any of the APOBEC domains as described herein.

[0236] The recruitment domain may comprise at least about 80% sequence identity with any of the GluR2 domains as described herein. In some cases, the recruitment domain may comprise at least about 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any of the GluR2 domains as described herein.

[0237] The recruitment domain may include at least about 80% sequence identity with any of the Cas13 domains as described herein. In some cases, the recruitment domain may include at least about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of the Cas13 domains as described herein.

[0238] The nucleic acid sequence may encode at least 1, 2, 3, 4, or 5 RNA editing recruitment domains. The nucleic acid sequence may encode at least 2 RNA editing recruitment domains, one of which is an Alu domain. The nucleic acid sequence may encode at least 2 RNA editing recruitment domains, one of which is an APOBEC domain. The nucleic acid sequence may encode at least 2 RNA editing recruitment domains, one of which is a GluR2 domain. The nucleic acid sequence may encode at least 2 RNA editing recruitment domains, one of which is a Cas13 domain.

[0239] The recruitment domain may include one or more stem-loop structures. The recruitment domain may include at least 2 stem-loop structures. The recruitment domain may include at least 3 stem-loop structures. The recruitment domain may not include a stem-loop structure. The recruitment domain including at least one stem-loop structure may be an Alu domain, an APOBEC domain, a GluR2 domain, a Cas13 domain, or any combination thereof.

[0240] At least a portion of the recruitment domain can be single-stranded. In some cases, the Alu domain can be at least partially single-stranded. In some cases, the APOBEC domain can be at least partially single-stranded. In some cases, the GluR2 domain can be at least partially single-stranded. In some cases, the Cas13 domain can be at least partially single-stranded.

[0241] The recruitment domain may include multiple repeat sequences. The recruitment domain may include multiple Alu repeat sequences.

[0242] In some cases, the viral vector may include one or more RNA editing recruitment domains. In some cases, the viral vector may include more than one RNA editing recruitment domain. In some cases, the viral vector may include 2, 3, 4, 5 or more RNA editing recruitment domains. The nucleic acid sequence may encode one or more RNA editing recruitment domains. The nucleic acid sequence may encode more than one RNA editing recruitment domain. The nucleic acid sequence may encode 2, 3, 4, 5 or more RNA editing recruitment domains. The nucleic acid sequence may encode at least an Alu domain and a GluR2 domain. The nucleic acid sequence may encode at least an Alu domain and a Cas13 domain. The nucleic acid sequence may encode at least an Alu domain and an APOBEC domain. The nucleic acid sequence may encode at least a GluR2 domain and an APOBEC domain. The nucleic acid sequence may encode at least a GluR2 domain and a Cas13 domain. The nucleic acid sequence may encode at least a Cas13 domain and an APOBEC domain.

[0243] The nucleic acid sequence can encode a target RNA that can be complementary to at least a portion of a target RNA. It can be complementary to at least a portion of the target RNA. The length of the complementary portion can be about 50 base pairs (bp) to about 200bp. The length of the complementary portion can be about 20bp to about 100bp. The length of the complementary portion can be about 10bp to about 50bp. The length of the complementary portion can be about 50bp to about 300bp. Modifying the length of the complementary portion can improve editing efficiency. In some cases, the longer length of the portion can improve editing efficiency compared to a shorter length.

[0244] The nucleic acid sequence can encode at least one RNA editing entity recruitment domain, and a nucleic acid sequence encoding an RNA that can be complementary to at least a portion of a target RNA, and includes a continuous nucleic acid sequence of at least about 200bp in length. The continuous nucleic acid sequence can include a length of about 100bp to about 300bp in length. The continuous nucleic acid sequence can include a length of about 150bp to about 400bp in length. The continuous nucleic acid sequence can include a length of about 200bp to about 500bp in length. The continuous nucleic acid sequence can include a length of about 50bp to about 300bp in length. Modifying the length of the continuous nucleic acid sequence can improve editing efficiency. In some cases, the longer length of the continuous sequence can improve editing efficiency compared to a shorter length.

[0245] The nucleic acid may include a linker sequence, such as a linker sequence located between a targeting domain and a recruitment domain. In some cases, the nucleic acid may include a sequence such as 5'-X-(Y-X')nLZ-3', wherein X is complementary to a target RNA sequence downstream of a specific position, X' is complementary to a 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 may be an integer from 1 to 10, L may be a linker sequence including any number of nucleotides (including 0), and Z may be a sequence that is recognized and bound to an RNA editing entity. L may also be composed of different chemical bonds, such as (oligo)peptide linkages or PEG linkages.

[0246] Nucleic acids can include 20 to hundreds of nucleotides. In some cases, longer targeting moieties provide higher specificity for the target site of the RNA sequence to be edited, less off-target effects due to unintentional (off-target) binding, and more space to create secondary structures (e.g., stem-loop structures, cross-shaped, foothold-shaped structures) within the targeting moiety itself, mismatches or wobble bases (due to mismatches of one or more complementary bases in the target RNA sequence, at or near the site to be edited), etc. In some cases, in addition to mismatches relative to the nucleotide to be edited, the targeting moiety can be complementary to the target RNA sequence over the entire length of the targeting moiety, and optionally has 1 or 2 wobble bases.

[0247] Nucleic acids can be modified using various chemical methods and modifications. In some cases, the regular internucleoside connections between nucleotides can be changed by monothio or dithio to the phosphodiester bond to produce thiophosphate or dithiophosphate, respectively. Other modifications to the internucleoside connection can include amidation or peptide linkers. Ribose can be modified by replacing the 2'-O part with a lower alkyl (C1-4, such as 2'-O-Me), alkenyl (C2-4), alkynyl (C2-4), methoxyethyl (2'-MOE) or other substituents. In some cases, the substituent of the 2'OH group can include methyl, methoxyethyl or 3,3'-dimethylpropylene. In some cases, a locked nucleic acid sequence (LNA) can be applied, which contains a 2'-4' intramolecular bridge (e.g., a methylene bridge between 2' oxygen and 4' carbon) bond inside the ribose ring. Purine nucleobases and / or pyrimidine nucleobases can be modified, for example, by amination or deamination of a heterocycle, to change their properties.

[0248] The viral vector can be an adeno-associated virus (AAV) vector. AAV can be a recombinant AAV. AAV can include AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, any of these derivatives, or any combination thereof. AAV can 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 of these derivatives, and any combination thereof. The viral vector can be a modified viral vector. The viral vector can be modified to include a modified protein. In some cases, the viral vector can include a modified VP1 protein.

[0249] The nucleic acid sequence encoding the RNA editing entity recruitment domain, the targeting domain or a combination thereof may include a substantially cruciform structure (e.g., a secondary structure). The nucleic acid sequence may include at least two substantially cruciform structures. The recruitment domain may include a substantially cruciform structure. The recruitment domain may include at least two substantially cruciform structures. The secondary structure of the nucleic acid sequence (e.g., a portion encoding the recruitment domain) may be modified to enhance the recruitment or binding of ADARs. The structure may be modified to enhance the recruitment or binding of ADARs, and may include forming a cruciform structure.

[0250] The RNA editing entity recruitment domain may be located between at least two structures that may be substantially cross-shaped. The targeting domain may be located between at least two structures that may be substantially cross-shaped. The RNA editing entity recruitment domain may be flanked by at least one structure that may be substantially cross-shaped. The targeting domain may be flanked by at least one structure that may be substantially cross-shaped.

[0251] The cruciform structure can include a stem loop adjacent to at least one pair of at least partially complementary strands of the cruciform structure. The cruciform structure can be substantially cruciform. The cruciform structure can include substantially less than a cruciform, such as 3 of 4 stem loops, or 2 of 4 stem loops. The one or more stem loops that can form a cruciform can include different lengths. The one or more stem loops that can form a cruciform can include the same length. The one or more stem loops that can form a cruciform can include one or more mismatched protrusions.

[0252] The RNA editing entity recruitment domain may include a structure that is substantially a toehold-shaped. The RNA editing entity may include one or more mismatch protrusions. The RNA editing entity may not include a mismatch protrusion. The RNA editing entity recruitment domain may include a substantially toehold-shaped structure, a substantially cruciform structure, a substantially linear structure, a stem-loop structure, a double stem-loop structure, or a combination thereof.

[0253] The viral vector may include a nucleic acid sequence encoding an RNA having a two-dimensional shape. The two-dimensional shape may show excellent recruitment or binding of ADARs compared to RNAs having different two-dimensional shapes. The sequence of the nucleic acid sequence encoding the RNA may be modified so that the RNA includes a two-dimensional shape that shows excellent recruitment or binding of ADARs. The two-dimensional shape may be substantially cross-shaped, foothold-shaped, stem-loop, or any combination thereof. The two-dimensional shape may include substantially cross-shaped. The two-dimensional shape may include foothold-shaped. The two-dimensional shape may include stem-loop. The two-dimensional shape may be linear.

[0254] The RNA encoded by the nucleic acid sequence may include a first domain and a second domain. The first domain may include a cross, and the second domain may include a linear structure. The first domain and the second domain may be directly or indirectly connected. The first domain may be a recruitment domain, and the second domain may be a targeting domain. The RNA may include a third domain. The third domain may be directly or indirectly connected to the first domain or the second domain. The third domain may be a recruitment domain. The third domain may include a cross structure.

[0255] The RNA encoded by the nucleic acid sequence can be a non-naturally occurring RNA. The RNA encoded by the nucleic acid sequence can include at least one base or at least one sugar including chemical modifications. The RNA can include two or more chemical modifications. Chemical modifications can increase the stability of the RNA, for example, have a biologically active half-life of the RNA in vivo.

[0256] Nucleic acid can include one or more recruitment domains and one or more antisense domains.As determined by sequence methods (such as Sanger method), when the nucleic acid contacts the target nucleic acid complementary to at least a portion of the antisense domain with the RNA editing entity, it can be compared with the comparable nucleic acid compounded with Cas13b protein or its active fragment, at least about 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times or 6 times higher efficiency, to modify at least one base pair of the target nucleic acid.The efficiency can be at least about 3 times higher.The efficiency can be at least about 4 times higher.The efficiency can be at least about 5 times higher.

[0257] Nucleic acid can include one or more recruitment domains and one or more antisense domains. As determined by sequence methods (e.g., Sanger methods), when the nucleic acid contacts a target nucleic acid that is complementary to at least a portion of an antisense domain and an RNA editing entity, it can be at least about 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times or 6 times higher than a comparable nucleic acid compounded with a GluR2 protein or its active fragment, to modify at least one base pair of the target nucleic acid. The efficiency can be at least about 3 times higher. The efficiency can be at least about 4 times higher. The efficiency can be at least about 5 times higher.

[0258] Compared to at least a portion of a native recruitment domain (e.g., a GluR2 domain), nucleic acids as described herein can provide higher editing efficiencies.Compared to at least a portion of a modified recruitment domain (e.g., a modified GluR2 domain), nucleic acids can provide higher editing efficiencies.

[0259] The target nucleic acid may include RNA. The RNA may be mRNA. The RNA may encode a protein or a portion thereof. The dysfunction of the protein or a portion thereof may be associated with a disease or condition. The administration of the compositions, vectors, nucleic acids, non-naturally occurring RNA as described herein may treat, eliminate, cure or alleviate one or more symptoms of a disease or condition.

[0260] The disease or condition may include a neurodegenerative disease, a muscle disorder, a metabolic disorder, an eye disorder, or any combination thereof. The disease or condition may include cystic fibrosis, albinism, alpha 1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, beta-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Beck muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, factor V Leiden-related disease, familial adenoma, polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Hunter syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, Leber congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy , myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-related cancers, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary disease, diseases associated with prothrombin mutations, such as 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-linked breast and ovarian cancer). The disease or condition can include muscular dystrophy, ornithine transcarbamylase deficiency, retinitis pigmentosa, breast cancer, ovarian cancer, Alzheimer's disease, pain, Stargardt macular muscular dystrophy, Charcot-Marie-Tooth disease, Rett syndrome, or any combination thereof. Administration of the composition can be sufficient to: (a) reduce gene expression relative to gene expression prior to administration; (b) edit at least one point mutation in a subject (e.g., a subject in need thereof); (c) edit at least one stop codon in a subject to produce read-through of a stop codon; (d) produce exon skipping in a subject; or, (e) any combination thereof.

[0261] The pharmaceutical composition may include a first active ingredient. The first active ingredient may include a viral vector as described herein, a non-naturally occurring RNA as described herein, or a nucleic acid as described herein. The pharmaceutical composition may be formulated into a unit dosage form. The pharmaceutical composition may include a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition may include a second, third, or fourth active ingredient.

[0262] The compositions described herein may include excipients. Excipients may be added to stem cells or may be co-isolated from their source together with stem cells. Excipients may include cryopreservatives such as DMSO, glycerol, polyvinylpyrrolidone (PVP) or any combination thereof. Excipients may include cryopreservatives such as sucrose, trehalose, starch, any salt of these, any derivative of these, or any combination thereof. Excipients may include pH agents (to minimize oxidation or degradation of composition components), stabilizers (to prevent modification or degradation of composition components), buffers (to enhance temperature stability), solubilizers (to increase protein solubility), or any combination thereof. Excipients may include surfactants, sugars, amino acids, antioxidants, salts, nonionic surfactants, solubilizers, triglycerides, alcohols or any combination thereof. Excipients can 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, HCl, disodium edetate, lecithin, glycerol, xanthan gum, soy isoflavones, polysorbate 80, ethanol, water, teprenone or any combination thereof. Excipients can be excipients described in Handbook of Pharmaceutical Excipients (1986) of the American Pharmaceutical Association.

[0263] Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compacting agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, coloring agents.

[0264] In some cases, the excipient can be a buffer. The non-limiting examples of suitable buffers can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate. As buffers, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen 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 can be used in pharmaceutical preparations.

[0265] In some cases, the excipient may include a preservative. Non-limiting examples of suitable preservatives may include antioxidants, such as alpha-tocopherol and ascorbic acid esters, and antimicrobials, such as parabens, chlorobutanol and phenol. Antioxidants may further 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 cases, the preservative may include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethyl ketone, Na-tosyl-Lys-chloromethyl ketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, 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, antimicrobial agents, oxidase inhibitors or other inhibitors.

[0266] In some cases, the pharmaceutical preparation can include a binder as an excipient. Non-limiting examples of suitable binders can include starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyl oxazolidinone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, sugar, oligosaccharide, and combinations thereof.

[0267] Binders that can be used in pharmaceutical preparations can be selected from: starches, such as potato starch, corn starch, wheat starch; sugars, such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives, such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinyl pyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols, such as sorbitol, xylitol, mannitol and water, or combinations thereof.

[0268] In some cases, pharmaceutical preparations can include lubricants as excipients. The non-limiting examples of suitable lubricants can include magnesium stearate calcium stearate, zinc stearate, hydrogenated vegetable oil, hydrogenated vegetable oil (sterotex), polyoxyethylene monostearate, talcum powder, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate and light mineral oil. The lubricant that can be used in pharmaceutical preparations can be selected from metal stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), aliphatic alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), lauryl metal sulfate (such as sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate and talcum powder, or their combination.

[0269] In some cases, the pharmaceutical formulation may include a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants may include starch, alginic acid, polyvinyl pyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate and microcrystalline cellulose as high HLB emulsifier surfactants.

[0270] In some cases, pharmaceutical preparations can include disintegrants as excipients. In some cases, disintegrants can be non-effervescent disintegrants. The non-limiting examples of suitable non-effervescent disintegrants can include starch such as corn starch, potato starch, its pregelatinization and modified starch, sweeteners, clay such as bentonite, microcrystalline cellulose, alginate, sodium starch glycolate, gums such as agar, guar bean, locust bean, karaya gum, pectin and tragacanth. In some cases, disintegrants can be effervescent disintegrants. The non-limiting examples of suitable effervescent disintegrants can include sodium bicarbonate combined with citric acid, and sodium bicarbonate combined with tartaric acid.

[0271] In some cases, the excipient may include a flavoring agent. The flavoring agent incorporated into the outer layer may be selected from synthetic flavoring 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; wintergreen 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 flavors, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot.

[0272] In some cases, the excipient may include a sweetener. Non-limiting examples of suitable sweeteners may 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); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, sylitol, etc.

[0273] In one aspect, the present disclosure, adRNA, helps to recruit endogenous ADAR enzymes to target mRNA and achieve site-specific A to G editing. This has great potential for gene therapy, where delivery of a single adRNA can potentially correct G to A point mutations. It can also achieve target RNA editing without having to overexpress RNA editing enzymes (such as ADAR2). The present disclosure demonstrates the applicability of the technology in vitro and in vivo. The present disclosure also demonstrates that by creating long double-stranded RNA, endogenous ADARs can be recruited even without an ADAR recruitment domain. In addition, using these modified adRNAs, multiple A to G editing can be created in the mRNA of the target region. In one aspect, the system uses adRNA transcribed by U6 promoter (pol III), as well as chemically synthesized adRNA, and shows effective RNA editing. Therefore, in one aspect, the construct further includes a promoter, such as a pol II promoter, to transcribe adRNA. Transcription from a promoter (such as a pol III promoter) can improve the editing efficiency of the target RNA. Alu transcripts from pol III promoters are preferentially edited. Also provided herein are engineered adRNAs derived from Alu repeat sequence structures that serve as endogenous ADAR targets.

[0274] The construct disclosed herein can be used to localize adRNA to a specific cell compartment. For example, for nuclear localization, adRNA-snRNA fusions can be used. Similarly, by adding an N-terminal mitochondrial targeting sequence (MTS), adRNA can be localized to mitochondria. Therefore, in one aspect, the construct further includes an N-terminal mitochondrial targeting sequence (MTS). By adding appropriate cis-acting zipper coding, adRNA can be localized in peroxisomes, endosomes, and exosomes. Therefore, on the other hand, the construct further includes appropriate cis-acting zipper coding for localizing adRNA in peroxisomes, endosomes, and exosomes. In the case of neurons, localizing adRNA to endosomes can potentially enable them to be transported over long distances. Localization to exosomes can potentially help propagate adRNA to neighboring cells. Tethering parts such as cholesterol to adRNA can help cells to take up. Therefore, in one aspect, the construct further includes a targeting moiety, such as cholesterol.

[0275] In one aspect, the present disclosure demonstrates that, in order to create small molecule regulatable adRNA, the use of adRNA-aptamers is disclosed in one aspect to achieve temporal control of RNA editing, such as aptamers that bind to flavin mononucleotides, guanine, and other natural metabolites. Aptamers that bind to sugars can also be used for this purpose.

[0276] In one aspect, the creation of U1A-ADAR fusions is entirely of human origin. The N-terminal RNA recognition motif of the spliceosomal U1A protein binds to its cognate U1 hairpin II RNA with a dissociation constant of 63 nM.

[0277] The present disclosure also provides constructs further comprising a toehold shape.

[0278] In one aspect, the constructs of the disclosure can be used without overexpression of an ADAR enzyme.

[0279] Therefore, in some aspects, the adRNA of the present disclosure has certain components: an RNA targeting domain of about 15 to about 200 base pairs in length, which is complementary to the target RNA; 0 to 10 ADAR recruitment domains, which can be derived from GluR2mRNA, Alu repeat elements, or other RNA motifs bound by ADAR; and, cytosine mismatches required to direct ADAR to target adenosine, which may be present at any position in the targeting domain. When this adRNA binds to its target RNA, it recruits the ADAR enzyme to the target RNA. Now, the ADAR enzyme can catalyze the conversion of target adenosine to inosine. For adRNAs with a length of more than 50 base pairs, when expressed in HEK 293T and HeLa, adRNA cells can recruit ADARs even in the absence of ADAR recruitment domains, and can achieve significant levels of target RNA editing. A single adRNA can also be used to create multiple A to G edits in the target mRNA. In addition, by utilizing multiple adRNAs, multiple different adRNAs can be edited in the same cell. For example, in the mdxmdx mouse model of Duchenne muscular dystrophy, not only could a mutation in dystrophin be corrected, but also the mRNA sequence of a gene encoding a protein involved in nonsense-mediated degradation could be disrupted. Another application is to use this technology to create loss-of-function, gain-of-function, and dominant-negative mutations, and on the one hand, it could be used for cancer screening, tumor progression, and immunoediting studies.

[0280] Modified adRNA

[0281] Provided herein is a modified ADAR1 or ADAR2 guide RNA ("adRNA") comprising, or alternatively consisting essentially of, or further consisting of: a sequence complementary to a target RNA. In a specific aspect, the modified adRNA of the present disclosure further comprises, or alternatively consists essentially of, or further consists of a sequence complementary to ornithine transcarbamylase.

[0282] In one aspect, the modified adRNA of the present disclosure further includes the ADAR2 recruitment domain derived from GluR2mRNA, or alternatively is substantially composed of it, or further consists of it. On the other hand, the modified adRNA of the present disclosure further includes the ADAR1 recruitment domain derived from Alu repetitive sequence, or alternatively is substantially composed of it, or further consists of it. On the other hand, the modified adRNA of the present disclosure further includes two MS2 hairpins on both sides of the sequence complementary to the target RNA, or alternatively is substantially composed of it, or further consists of it. In some embodiments, the sequence complementary to the target RNA in the modified adRNA of the present disclosure comprises, or alternatively consists essentially of, or further consists of: about 15 to 30 base pairs, or about 30 to 45 base pairs, or about 45 to 60 base pairs, or about 60 to 75 base pairs, or about 75 to 90 base pairs, or about 90 to 105 base pairs, or about 105 to 120 base pairs, or about 120 to 135 base pairs, or about 135 to 150 base pairs, or about 150 to 165 base pairs, or about 165 to 180 base pairs, or about 180 to 200 base pairs. In another aspect, it is about 40 to about 200, or about 50 to about 200, or about 60 to about 200, or about 70 to about 200, or about 80 to about 200, or about 90 to about 200, or about 100 to about 200 base pairs.

[0283] Disclosed herein is a modified adRNA, which includes the following items, or alternatively is substantially composed of the following items, or is further composed of the following items: no ADAR recruitment domain, or about 1-2 ADAR recruitment domains, or about 2-3 ADAR recruitment domains, or about 3-4 ADAR recruitment domains, or about 4-5 ADAR recruitment domains, or about 5-6 ADAR recruitment domains, or about 6-7 ADAR recruitment domains, or about 7-8 ADAR recruitment domains, or about 8-9 ADAR recruitment domains, or about 9-10 ADAR recruitment domains. In some embodiments, the ADAR recruitment domain includes the following items, or alternatively is substantially composed of the following items, or is further composed of the following items: GluR2mRNA, Alu repeat sequence elements, or other RNA motifs that ADAR binds. In addition, provided herein is a modified adRNA, wherein the ADAR2 recruitment domain of the modified adRNA derived from GluR2mRNA is located at the 5' end or 3' end of the modified adRNA. In some embodiments, GluR2 mRNA is located at the 5' end and the 3' end of the engineered adRNA.

[0284] In one aspect, the modified adRNA of the present disclosure further includes an editing inducing element, or alternatively is essentially composed of it, or is further composed of it. "Editing inducing element" can refer to a structure of a mainly double-stranded RNA that is necessary for effective RNA editing. Non-limiting examples of editing inducing elements are described in Daniel, C. et al. (2017) Genome Biol. 18, 195.

[0285] In a specific aspect, the modified adRNA of the present disclosure is selected from Table 1 or Figure 2 The polynucleotide sequences encoding a set of sequences provided in or their respective equivalents.

[0286] Also disclosed herein is a complex comprising, or alternatively consisting essentially of, or further consisting of, the engineered adRNA of the present disclosure hybridized to a complementary polynucleotide under high stringency conditions.

[0287] The present disclosure also provides polypeptide and / or polynucleotide sequences for gene and protein editing techniques described below. It should be understood that, although not always explicitly stated, the sequences provided herein can be used to provide expression products, as well as substantially identical sequences of proteins having the same biological properties. These "bioequivalent" or "biologically active" polypeptides are encoded by equivalent polynucleotides described herein. When compared using a sequence identity method run under default conditions, they may have a primary amino acid 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. The amino acids of the sequence are modified with spare amino acids having similar charges. In addition, an equivalent polynucleotide is a polynucleotide that hybridizes with a reference polynucleotide or its complementary sequence under stringent conditions, or, with respect to a polypeptide, refers to a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide encoding a reference or its complementary chain under stringent conditions. Alternatively, an equivalent polypeptide or protein is a polypeptide or protein expressed by an equivalent polynucleotide.

[0288] Also disclosed herein is a modified adRNA-snRNA (small nuclear RNA) fusion. In one aspect, the modified adRNA further includes an N-terminal mitochondrial targeting sequence (MTS), or alternatively is substantially composed of it, or is further composed of it, to promote the modified adRNA to be positioned to mitochondria. On the other hand, provided herein is a modified zipper code (zipcode) further including a cis-acting, or alternatively is substantially composed of it, or is further composed of it, to promote the modified adRNA to be positioned in peroxisomes, endosomes and exosomes. In the case of neurons, positioning adRNA in endosomes can potentially enable them to be transported over long distances. Positioning in exosomes can potentially help propagate adRNA to neighboring cells. Tethering parts such as cholesterol to adRNA can help cells to take in.

[0289] Further provided herein are modified adRNAs that are regulatable by small molecules. In one aspect, disclosed herein are modified adRNA-aptamer fusions. Non-limiting examples of aptamers that can be used for this purpose include aptamers that bind to flavin mononucleotides, guanine, other natural metabolites, or sugars. "Aptamers" may refer to short single-stranded oligonucleotides that can bind to various molecules with high affinity and specificity. Non-limiting examples of aptamers are described in Lakhin, AVet al. (2013). Acta naturae, 5 (4), 34-43.

[0290] Also disclosed herein is a fully human derived U1A-ADAR fusion. N-terminal RNA recognition of the spliceosomal U1A protein motif, binding to its cognate U1 hairpin II RNA with a dissociation constant of 63 nM.

[0291] Vectors and recombinant cells expressing modified adRNA

[0292] Also provided herein is a vector comprising one or more isolated polynucleotide sequences and a regulatory sequence optionally operably connected to the isolated polynucleotide, or alternatively consisting essentially of, or further consisting of, wherein the isolated polynucleotide sequence encodes the modified adRNA of the present disclosure. Non-limiting embodiments of the vector include plasmids or viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors. The vector may further include targeting sequences, zipper coding, or toeholds known in the art.

[0293] In one aspect, the regulatory sequence comprises, or alternatively consists essentially of, or further consists of, a promoter, an enhancer element, and / or a reporter factor. In some embodiments, the promoter is human U6, mouse U6 promoter, CMV promoter, or pol III promoter or pol II promoter. In one aspect, the vector further comprises, or alternatively consists essentially of, or further consists of, a detectable marker or a purification marker.

[0294] Further disclosed herein is a recombinant cell, which further comprises the above-mentioned vector, or alternatively consists essentially of it, or further consists of it, wherein the modified adRNA is recombinantly expressed.

[0295] Composition of modified adRNA

[0296] A composition is disclosed herein, comprising, or alternatively consisting essentially of, or further consisting of: a carrier and one or more modified adRNAs of the present disclosure, a polynucleotide encoding the isolated modified adRNAs of the present disclosure, a vector expressing the modified adRNAs of the present disclosure, or a recombinant cell expressing the modified adRNAs of the present disclosure. In one aspect, the carrier is a pharmaceutically acceptable carrier or solid support. In another aspect, the composition further comprises a chemotherapeutic agent or drug, or alternatively consists essentially of, or further consists of.

[0297] Methods of using engineered adRNA

[0298] Provided herein is a method for modifying protein expression, the method comprising contacting a polynucleotide encoding the protein with an engineered adRNA of the present disclosure, or alternatively consisting essentially of it, or further consisting of it, wherein the expression of the protein will be modified.

[0299] Also provided herein is a method for treating a disease or disorder associated with abnormal protein expression, the method comprising administering an effective amount of one or more modified adRNAs of the present disclosure to a subject in need of such treatment, or alternatively consisting essentially of it, or further consisting of it. In a specific aspect, provided herein is a method for treating Duchenne muscular dystrophy, comprising administering an effective amount of one or more modified adRNAs of the present disclosure to a subject in need of such treatment, or alternatively consisting essentially of it, or further consisting of it.

[0300] In the case of in vitro applications, in some embodiments, the effective amount may depend on the size and nature of the application in question. It may also depend on the nature and sensitivity of the in vitro target, and the method used. The technician can determine the effective amount based on these and other considerations. According to the embodiment, the effective amount may include one or more administrations of the composition.

[0301] The terms "subject", "host", "individual" and "patient" are used interchangeably herein and refer to animals, typically mammals. Any suitable mammal can be treated by 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, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, adolescent, child, infant, or intrauterine mammal). The mammal can be male or female. The mammal can be a pregnant female. In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is suspected of having cancer or a neoplastic disorder. In other embodiments, the subject suffers from or is suspected of having a disease or disorder associated with abnormal protein expression.

[0302] refer to Fig.13 , and antisense oligonucleotide (AON) design that may include short ssRNA (e.g., about 35 nt in length) ( Fig.13 ) compared to the exemplary constructs of the present disclosure ( Fig.13 The right side of the ) can be a long ssRNA of about 60 bp to about 100 bp in length with excellent target specificity, with a total sequence length 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 can be added to create a hairpin-like RNA bulge, such as Fig.13 The AON design is shown on the left. Advantages of the true hairpin design of the construct include optimal and superior ADAR recruitment efficiency compared to AON constructs, resulting in higher target editing yields. The hairpin design can be completely independent of the mRNA target sequence and can be easily used for any new mRNA target. The target site for deamination can be unique and precise, without the need for undesirable chemical modifications and without the risk of undesirable deamination at other locations. In contrast, the use of mismatched hairpin-like RNA protrusions (such as Fig.13AON designs (shown on the left) can: (a) have limited efficacy in recruiting ADARs (not a true hairpin, structure may be too short); (b) prevent reuse of sequences and bulges (need unique design for each new target mRNA); (c) have reduced specificity for cellular mRNA target sites due to mismatches (increased risk of target damage), and (d) require 2'OMe modification in the bulge to protect other adenines from ADAR deamination (potentially causing undesirable mutations at the wrong ribonucleotide).

[0303] Recruitment of exogenous and endogenous ADARs via long antisense adRNA

[0304] The CRISPR / Cas9 system is widely used in research, but there are still concerns about its in vivo application. The two main concerns are permanent editing of the genome and the immune response that may be caused by the introduction of a bacterial-derived system. As a result, RNA editing has attracted interest as a potential solution to both challenges. The first challenge can be easily overcome by applying RNA editing; RNA transcribed from genes can be transient, and editing will not permanently change the cell. However, because many RNAs may be edited to achieve phenotypic effects, this may create new problems that reduce editing efficiency. In addition, the second challenge of immune response may not necessarily be overcome. The enzymes of the ADAR family that edit RNA are of human origin. In particular, ADAR1 is almost universally expressed in many cell types; by taking advantage of its natural preference for RNA A-to-I (A-to-G) editing, there may be many potential therapeutic applications. This strategy also overcomes the major obstacle of delivery, because the transportation of small guide RNAs is much easier than that of bulky enzymes. Therefore, the goal of the methods and compositions described herein may include transforming guides (referred to as adRNAs) to recruit endogenous ADARs. ADARs may prefer to edit regions of double-stranded RNA, especially near sequences with secondary structures. Therefore, it may be advantageous to engineer a guide with increased antisense domain length. The adRNA may have three parts: (1) an RNA targeting domain (e.g., about 15 base pairs to about 200 base pairs in length), which may be complementary to the target RNA; (2) about 0 to about 10 ADAR recruitment domains to which the ADAR binds (which may be derived from GluR2 mRNA, Alu repeat elements, or other RNA motifs); and, (3) a cytosine mismatch, which may be necessary to direct the ADAR to the target adenosine, wherein the target adenosine may be present at any position within the targeting domain.

[0305] When the adRNA binds to its target RNA, it can recruit the ADAR enzyme to the target RNA. The ADAR enzyme can catalyze the conversion of target adenosine to inosine. Interestingly, when expressed in HEK 293T and HeLa cells, long adRNAs with a length of more than 50 base pairs can even recruit ADARs without an ADAR recruitment domain, and can achieve significant levels of target RNA editing. A single adRNA can also be used to create multiple A-to-G edits in a target mRNA. In addition, 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 mutations in dystrophin can be corrected, but also mRNA sequences of genes encoding proteins involved in nonsense-mediated degradation can be destroyed. Another application can be to use this technology to create loss of function, gain of function, dominant negative mutations, or any combination thereof. This is crucial in cancer screening (tumor progression and immunoediting research).

[0306] refer to Fig.16 , schematic diagram showing RNA editing via recruitment of endogenous ADARs in the presence of adRNA. These adRNAs can be provided as chemically modified RNA or as U6 transcribed RNA. Reference Fig.17 , adRNAs with increasing antisense domain lengths transcribed from the U6 promoter, combined with 0, 1, or 2 GluR2 domains, were evaluated for their ability to induce targeted RNA editing in the presence or absence of exogenous ADAR2 expression. Values ​​represent mean + / - SEM (n = 3). Long adRNAs can recruit endogenous ADARs for RNA editing. Reference Fig.18 Chemically synthesized adRNA formats were tested against a panel of mRNAs in the presence or absence of exogenous ADAR2 expression. The exact chemical modifications and sources of adRNA are described in the figure. Values ​​represent mean + / - SEM (n = 3). Fig.19 , treated adult spf ash In vivo RNA proofreading efficiency of correctly spliced ​​OTC mRNA in mouse liver (retro-orbital injection). In mice injected with short adRNA transcribed from U6, RNA editing levels were 0.6%.

[0307] Recruitment of exogenous and endogenous ADARs via Alu-adRNA

[0308] Alu genes are transposable elements in the genome and are natural targets for RNA editing by ADARs. AdRNAs can be designed based on the Alu element structure to enable editing by endogenously recruited ADARs. Various positions on the Alu element structure can be tested where the native sequence can be replaced with an antisense sequence that is complementary to the target. Single-stranded linker regions can be selected, such as Fig. 20 As shown. The length of the antisense guide can be optimized to change the length from about 20 bases to about 100 bases, targeting the RAB7A locus. Each guide can be designed to include one or more mismatches. The mismatch can be located between the cytosine of the antisense sequence and the target adenosine base to be edited. The mismatch can be located in the middle of each antisense sequence length. The editing efficiency can be compared with the 100-50 antisense guide that can recruit both ADAR1 and ADAR2, and the GluR220-6 guide that can only recruit ADAR2.

[0309] refer to Fig. 20 , showing the design of Alu adRNA. Left: Structure of the Alu element. Middle: Design incorporating a locus-specific antisense sequence with a C mismatch relative to the target A. Right: Recruitment of the RNA editing enzyme ADAR to the target.

[0310] These guides were tested in 293FT cells by transfection with lipofectamine. Each guide was tested in cells overexpressing ADAR1p110, ADAR1p150, ADAR2, or in cells without enzyme overexpression to test the ability of adRNA to recruit endogenous ADARs. 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 ratio of peak heights. Figure 5 Results of an Alu guide length experiment are shown.

[0311] refer to Figure 5 , the long Alu-v2-100-50 guide showed improved editing in cells that did not overexpress the ADAR enzymes. Overexpression of ADAR1p150 resulted in significantly higher editing rates for the Alu construct, while the linear 100-50 guide had similar editing rates. ADAR1p150 may preferentially bind to Z-RNA due to its additional ds-RNA binding domain compared to the shorter isoform ADAR1p110. Alu elements with high GC content are known to form Z-DNA and Z-RNA, which helps recruit ADAR1p150.

[0312] Split ADAR2 deaminase domain (DD)

[0313] Overexpression of ADARs may result in multiple transcriptome-wide off-target editing. The ability to restrict the catalytic activity of ADAR2DD only to the target mRNA may reduce the number of off-targets. Creating a split ADAR2DD may be a potential approach to reduce the number of off-targets. Split protein reassembly or protein fragment complementation may be a widely used approach to study protein-protein interactions. Split ADAR2DD may be designed so that each fragment of the split ADAR2DD may be catalytically inactive by itself. However, in the presence of adRNA, the split halves may dimerize to form an enzyme that is catalytically active against the intended mRNA target.

[0314] The region for splitting the protein can be identified by studying the crystal structure of the complex of ADAR2DD and its naturally occurring substrate, inferring the solvent accessibility score, using prediction software or any combination thereof. The MS2-MCP system and the box B-λN system (which can effectively recruit ADARs) can be used alone or in combination to recruit the N- and C-termini of the split ADAR2DD, respectively. The adRNA can include an MS2 stem loop and a boxB hairpin, as well as an antisense domain complementary to the target. This can recruit the N- and C-termini of the split ADAR2DD at the target, thereby constituting a catalytically active DD.

[0315] refer to Fig. 22 and Fig.23 , a schematic diagram of the split ADAR2DD system is shown, and an exemplary sequence of ADAR2DD with sites for splitting is highlighted.

[0316] refer to Fig.24 , the paired fragments 1 to 16 can be assayed via a cypridina luciferase reporter (cluc W85X). Fragments 9 and 10 showed the highest activity. The split position corresponding to fragments 9 and 10 is Fig.23 Circled in blue.

[0317] refer to Fig.25 , fragments 9 and 10 were assayed against the Cluc reporter. In addition, the NES-MCP-AD2-CU variant can also be tested for AG editing. N: N-terminal fragment, C: C-terminal fragment, MM: MS2-MS2adRNA, MB: MS2-BoxBadRNA, BB: BoxB-BoxB adRNA.

[0318] Further fully humanized versions of these constructs can be created by utilizing human RNA binding proteins, such as (a) U1A, or (b) its evolved variant TBP6.7 (which has no known endogenous human hairpin targets), 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 create a fully human programmable RNA editing tool set that can edit adenosine with extremely high specificity. In addition, chimeric RNAs with two corresponding RNA hairpins can be used to recruit ADAR2 fragments. Sequences of RNA hairpins are provided herein.

[0319] A CU RNA editing enzyme can be created by making one or more mutations (e.g., 16 mutations) in the ADAR2 deaminase domain. Even with one or more mutations, the editing enzyme can still display several transcriptome-wide AG and To improve the specificity of this enzyme, it can be split at certain residues (such as those identified in previous screens), thereby developing a split ADAR system for CU editing.

[0320] Recruitment of exogenous and endogenous APOBECs for CU editing

[0321] APOBEC (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) is an RNA editing enzyme that converts cytidine to uracil and creates diversity at the mRNA level. These entities can be recruited for CU editing of RNA by guide RNAs, such as modified guide RNAs (apRNAs) that recruit APOBEC. In some cases, fusion constructs can be created that include 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) for MCP (MS2 coat protein). The modified MS2-apRNA can be used to recruit one or more MCP fusions. The protein sequences used for this construct and the MS2-apRNA sequences are shown in Figure 28 to Figure 30 The protein sequence used in the fusion construct, or in any of the methods described herein, may include Fig.28 have at least about: 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to at least a portion of any sequence of.

[0322] In some cases, the method may include a construct configured to recruit APOBEC (e.g., APOBEC3A). Recruitment may be endogenous recruitment. A construct configured to recruit APOBEC may be designed by targeting preference of primary sequence, secondary structure, or a combination thereof. The design of one or more apRNAs may include, for example, Fig.29 In some cases, sequences that can recruit APOBEC (e.g., APOBEC3A), or sequences used in any of the methods described herein, can include sequences that are related to Fig.29 have at least about: 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to at least a portion of any sequence of.

[0323] To recruit MCP-APOBEC3A, MS2-apRNAs can be designed and their sequences can include Fig.30 In some cases, the sequence that can recruit MCP-APOBEC3A, or the sequence used in any of the methods described herein, can include any one or more sequences of Fig.30 have at least about: 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to at least a portion of any sequence of.

[0324] Luciferase assay

[0325] When ADAR converts the target A in TAG to I (read as G), the ribosome is able to fully translate the protein, e.g. Fig.31 Schematic diagram of the transfection. Cells can be transfected and visualized using a luciferase reporter assay 48 hours later. Light readout from the cells can indicate restoration of luciferase activity.

[0326] refer to Fig.31 , the first scheme proves an embodiment, wherein ribosome can reach the early TAG stop codon in the luciferase gene, and can stop translation, produce truncated non-functional luciferase.In the second scheme, ADAR can be recruited to a site by adRNA, at which it can edit the TAG stop codon into the TGG codon of tryptophan, which can allow ribosome to read through, and normal luciferase expression can be produced.Such a system can allow assessment of the appropriate ADAR recruitment carried out by adRNA.When luciferase expression is detected, ADAR recruitment by adRNA may have occurred.When luciferase expression is not detected, ADAR recruitment may not occur.

[0327] Reagent test kit

[0328] Also disclosed herein is a kit, which includes the following items, or alternatively consists essentially of the following items, or further consists of the following items: the modified adRNA of the present disclosure, the isolated polynucleotide encoding the modified adRNA of the present disclosure, the vector expressing the modified adRNA of the present disclosure, the recombinant cell expressing the modified adRNA of the present disclosure, or the composition disclosed herein, and instructions for use. In one aspect, the instructions record the method of using the modified adRNA disclosed herein.

[0329] The kit may include a vector. The vector may be packaged in a container. The kit may include a 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 a vector, nucleic acid, or non-naturally occurring RNA may be packaged. The kit may include a pharmaceutical composition as described herein. The kit may include instructions for administering a viral vector, non-naturally occurring RNA, or pharmaceutical composition as described herein to a subject in need thereof.

[0330] Example

[0331] The following examples are non-limiting and illustrate procedures that can be used in various situations to practice the present disclosure.In addition, all references disclosed herein are incorporated herein by reference in their entirety.

[0332] Example 1

[0333] Without being bound by theory, it is believed that ADARs found in mammals can be recruited to catalyze therapeutic editing of point mutations. ADAR1 or ADAR2 can be recruited to target RNA or potential DNA using a modified RNA scaffold, a modified DNA scaffold, or a DNA-RNA hybrid scaffold. Tissues that can be potentially targeted by this method include, but are not limited to, the central and peripheral nervous system, lungs, liver, gastrointestinal tract, pancreas, myocardium, kidneys, and skin.

[0334] An exemplary embodiment proposed herein is a modified ADAR2 guide RNA (adRNA) carrying 20-100 bp complementarity with the target RNA. The modified adRNA contains the ADAR2 recruitment domain from GluR2 mRNA at the 5' end or 3' end, or at both ends.

[0335] SPF in ornithine transcarbamylase deficiency ashIt was tested in vivo in a mouse model. This model carries a G->A point mutation in the last nucleotide of exon 4. After delivery of adRNA alone via AAV, correction of up to 1% of the point mutation was observed in the absence of overexpression of the ADAR enzyme. The present disclosure also shows this efficacy in HeLa cells known to express ADAR in vitro. RNA editing was observed in these cells after delivery of adRNA alone.

[0336] In addition, this efficacy can be applied to other recruitment domains. Therefore, further aspects relate to engineered single-stranded ADAR2 guide DNA (adDNA) and adDNA-RNA hybrids that have greater potential stability than adRNA. Methods of using the enzymes disclosed herein are also provided.

[0337] Without being bound by theory, since enzymes of the ADAR family catalyze the hydrolytic deamination of adenosine to inosine, it is believed that these enzymes can be used to catalyze the hydrolytic deamination of cytosine to thymine by mutating three specific residues of ADAR2, V351, E396, and C451, which interact with the target adenosine. Therefore, by mutation of these sites, a method for providing this catalytic potential is provided.

[0338] All possible amino acid substitutions at the above three residues were created and screened to test this hypothesis.

[0339] In addition, experiments were performed to explore the role of other amino acids, such as S486, that might be able to abrogate the inherent preference of ADARs for the UAG editing site.

[0340] To modify C->T editing, the effects of overexpression of hAPOBEC1 and rAPOBEC1 and Apobec1 complementation factor (ACF) were determined. In addition, the present invention further provides MCP-(h / r)APOBEC1-ACF fusion protein.

[0341] Also provided are methods for editing adenine bases for Cas9 (or Cpf1)-ADAR deaminase domain fusions (ADAR1, ADAR2, and their catalytically active mutants E1008Q and E488Q), and further provided herein are compositions for targeting ssDNA replacement strands by current base editors. To this end, a gRNA binding strand having an AC bulge (ideally in the first 10 bp close to the 5' end of the gRNA) is targeted.

[0342] Additional embodiments are illustrated in the Supplementary Files, which are incorporated herein by reference.

[0343] Example 2

[0344] refer to Figure 6 , Alu elements can be the main target of RNA editing based on endogenous ADARs. Therefore, for the purpose of programmable RNA editing, the goal is to design ADAR-recruiting RNAs (adRNAs) based on Alu elements because they can potentially effectively recruit endogenous ADARs. Alu-adRNA is expressed from the human U6 promoter, and the linker sequence between the Alu repeats is replaced with antisense domains of various lengths, targeting RAB7A transcripts. Each antisense domain has a mismatched nucleotide in the middle of the antisense region, targeting the cytosine opposite the adenosine.

[0345] By transfecting 293FT cells, together with ADAR1p110, ADAR1p150, ADAR2, or in the absence of overexpressed enzymes, Alu-adRNA was tested in vitro to demonstrate the recruitment of endogenous ADARs. Cells were harvested 48 hours after transfection, RNA was extracted, and converted into cDNA using random hexamer or oligonucleotide (oligo)-dT primers. The RAB7A locus was then amplified and sent to Sanger for sequencing. Editing efficiency was calculated as the ratio of Sanger peak height G / (A+G).

[0346] Example 3

[0347] Vector design and construction

[0348] 0, 1 or 2 copies of GluR2adRNA were cloned into an AAV vector containing human U6 and mouse U6 promoters, as well as a CMV promoter, driving the expression of GFP or the full-length human ADAR2 enzyme or its hyperactive mutant ADAR2(E488Q). Similarly, 1 or 2 copies of MS2adRNA were cloned into an AAV vector carrying MCP-ADAR1 or MCP-ADAR2 deaminase domain fusions and their hyperactive mutants. To construct GFP reporters, GFP-Amber, GFP-Ochre and GFP-Opal, three gene blocks were synthesized, replacing the Y39 residue of wild-type GFP with 'TAG', 'TAA' and 'TGA', respectively, and cloned downstream of the CAG promoter. To construct OTC and DMD reporters, spf carrying the target adenosine to be edited was cloned into an AAV vector containing 100 copies of the target adenosine. ash A 200 bp fragment of the OTC and mdx DMD transcripts was cloned downstream of the CAG promoter.

[0349] Mammalian cell culture and transfection

[0350] 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 and 5% CO2 atmosphere. All in vitro transfection experiments were performed in HEK 293T cells using the commercial transfection reagent Lipofectamine 2000 (Thermo Fisher). All in vitro RNA editing experiments involving reporter factors were performed in 24-well plates using 400ng reporter plasmid and 800ng adRNA+enzyme plasmid. All in vitro RNA editing experiments targeting endogenous transcripts were performed in 24-well plates using 800ng adRNA / enzyme plasmid. RNA editing experiments based on dCas13b-ADAR2DDE488Q were performed using 800ng of enzyme plasmid (Addgene#103864) and 800ng of gRNA plasmid. Cells were transfected at 25-30% confluence and harvested 60 hours after transfection for quantification of editing. Chemically synthesized adRNA (by IDT or Synthego) was transfected at 20 pmol / well using Lipofectamine 3000 (Thermo Fisher).

[0351] Production of AAV vectors

[0352] AAV8 particles were produced by triple transfection using HEK 293T cells and purified by iodixanol gradient. The confluence during transfection was about 80%. Two hours before transfection, DMEM supplemented with 10% FBS was added to HEK293T cells. Each virus was produced in a 5x 15cm plate, each of which was transfected with 7.5μg pXR-8, 7.5μg recombinant transfer vector, 7.5μg pHelper vector, using PEI (1μg / μL linear PEI in 1xDPBS, pH 4.5, using HCl) at a PEI:DNA mass ratio of 4:1. The mixture was incubated at RT for 10 minutes and then added dropwise to the cell culture medium. The virus was harvested after 72 hours and purified using iodixanol density gradient ultracentrifugation. The virus was then dialyzed against 1x PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher) using a 50 kDA filter (Millipore) to a final volume of ~1 mL and quantified by qPCR using specific primers for the ITR region according to the standard (ATCC VR-1616).

[0353] AAV-ITR-F:5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO:149) and

[0354] AAV-ITR-R: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 150).

[0355] Example 4 - RNA editing of point mutations in vivo by RNA-guided adenosine deaminase

[0356] A system was designed to perform sequence-specific RNA base editing through the RNA-acting adenosine deaminase (ADAR) enzyme, in conjunction with the associated ADAR guide RNA (adRNA). The system was systematically engineered to utilize ADARs, and its specificity and activity were comprehensively evaluated in vitro and in vivo using mouse models of two human diseases. In some cases, the platform enables tunable and reversible modification of RNA for different applications.

[0357] Adenosine to inosine RNA editing, a post-transcriptional RNA modification, is catalyzed by adenosine deaminase (ADAR) enzymes acting on RNA. Inosine is a deaminated form of adenosine that is biochemically recognized as guanine. Recently, multiple studies have demonstrated ADAR-mediated targeted RNA editing. Building on these foundations, two orthogonal tool sets have been engineered for sequence-specific, programmable RNA base editing in vitro and in vivo. Specifically, a system was utilized that couples ADAR1 / 2 with the associated ADAR guide RNA (adRNA) for targeted RNA editing ( Fig.32A ). The adRNA partially includes a programmable antisense region that is complementary to the target RNA sequence and carries a mismatched cytidine relative to the target adenosine. In addition, in one version, they carry 0, 1 or 2 ADAR-recruiting domains modified from the naturally occurring ADAR substrate GluR2 pre-mRNA (hereinafter referred to as GluR2adRNA); in the second version, they carry 2 MS2 hairpins located on both sides of the antisense region (hereinafter referred to as MS2adRNA). By evaluating multiple scaffold variants, including mutagenesis scaffolds based on GC and AU pairing, addition of editing-inducing elements, and modification of antisense domain length and mismatch position, GluR2adRNA was systematically optimized to enhance the recruitment of exogenous and / or endogenous ADARs ( Figure 8 , Fig.34AThe latter MS2 adRNA format was in turn optimized to utilize synthetic proteins comprising the deaminase domain (DD) of ADAR1 or ADAR2 fused to the MS2 coat protein (MCP) via systematic modification of the antisense domain length and mismatch position, combined with the use of a hyperactive version of the deaminase domain, and a version carrying nuclear localization (NLS) and export (NES) signals ( Fig.32B , Fig.35A , 41B).

[0358] The activity of the two systems described above was thoroughly evaluated and benchmarked using a recently developed Cas13b-based RNA editing system. These in vitro experiments demonstrated that: (1) the engineered constructs were able to efficiently perform targeted RNA editing with yields comparable to those of the Cas13b-based system ( Fig.32B , Figure 36A, Table 2, Table 3), U6-transcribed adRNA and chemically synthesized adRNA are both effective forms (Figure 36B); (2) adRNA with a long antisense domain (with or without the GluR2 domain) is sufficient to recruit exogenously expressed ADARs, and to a certain extent, endogenous ADARs to achieve effective RNA editing ( Fig.32B , Fig.34B , 40C, 42C); (3) constructs based on MS2adRNA and the corresponding MCP-ADAR1 / 2 fusions showed the highest and most robust activity, including a large panel of endogenous genes selected across a range of different expression levels ( Fig.32B , Figure 36C); (4) the use of NES and / or hyperactive deaminase domains in MCP-ADAR1 / 2 fusions consistently resulted in higher RNA editing yields for the target adenosine, but also resulted in a higher propensity to edit non-target adenosines in flanking sequences ( Fig.32B , Figure 37A). To further verify this, similar promiscuity was generated by deleting the native NLS domain (Δ1-138) in ADAR2 (Figure 37B to Figure 37D); and, 5) the two tool sets are operably orthogonal: specifically, the editing efficiency of the MCP-ADAR2 deaminase domain fusion with co-expressed MS2adRNA or GluR2adRNA was evaluated, and on-target editing was shown only by the former. In contrast, full-length ADAR2 was observed to be recruited by GluR2adRNA rather than MS2adRNA ( Fig.35B ).

[0359] After demonstrating the robust activity of the toolset, we investigated its specificity profile by analyzing transcriptome-wide off-target A->G editing enabled by the system. Fig.32C). To this end, HEK 293T cells were transfected with each construct and analyzed by RNA-seq. Untransfected cells were used as controls. From each sample, approximately 40 million uniquely aligned sequencing reads were collected. Fisher's exact test was used to quantify significant changes in A->G editing yield relative to untransfected cells at each reference adenosine site with sufficient read coverage. The number of sites where at least one A->G editing event was detected in any sample was calculated. Among these, the number of sites with statistically significant A->G editing, a false discovery rate (FDR) of 1%, and a fold change of at least 1.1 was found to vary widely, from the lowest for the MCP-ADAR2DD-NLS construct, to the highest for the MCP-ADAR1DD(E1008Q)-NES( Figures 38 to 41 , Tables 4, 5). To investigate the distribution of editing yields, violin plots were generated considering A-sites whose editing yields varied significantly in at least one sample (Figure 32). In summary, RNA-seq experiments showed that transcriptome-wide off-target editing was: 1) less prevalent in MCP-ADAR constructs with NLS than in constructs with NES; 2) less prevalent in MCP-ADAR2 constructs than in MCP-ADAR1 constructs; 3) less prevalent in wild-type MCP-ADAR constructs than in E>Q hyperactive mutants (Figure 42A, Table 5); and, 4) off-targets were primarily attributed to ADAR overexpression, while the use of adRNA alone resulted in the least number of off-targets (Figure 42B).

[0360] Table 2: List of adRNA and gRNA antisense sequences:

[0361]

[0362] Table 3: List of primers used for next generation sequencing (NGS) analysis.

[0363]

[0364] Table 4: Record of RNA-seq reads from high-throughput sequencing experiments. Record of RNA-seq reads from high-throughput sequencing experiments. The counts given represent read mates for paired-end sequencing, not matepairs. The columns are: sn, sample name; nt, total number of raw reads after demultiplexing; nu, number of paired reads uniquely aligned to the reference genome; nd, number of reads in duplicate pairs; nr, number of remaining reads; df, downsampling fraction. The samples named "293T", "293T L2", "293T L8" and "293T L4" were all taken from the same control library but were sequenced on different lanes of the Illumina instrument:

[0365]

[0366] Table 5: Results of quantification of A->G editing yields from aligned RNA-seq reads. Results of quantification of A->G editing yields from aligned RNA-seq reads. Columns are: Sample name; Total sites, the total number of reference sites with significant changes in A->G editing yield in at least one comparison between treated and control samples; Changed sites, the number of reference A-sites found to have significant changes in A->G editing yield when comparing treated samples to control samples (which is the first sample in Table 4); On-target editing yield, the editing yield observed at the expected target A-site within RAB7A mRNA; Median editing yield, the median yield at all other sites considered except the target site:

[0367]

[0368] Following these in vitro studies, the system was evaluated for in vivo RNA targeting in gene therapy applications using adRNA and exogenous ADAR expression construct formats, as these consistently achieved the highest in vitro RNA editing yields. The mdx mouse model of Duchenne Muscular Dystrophy (DMD) was first evaluated, which carries an ochre stop site on exon 23 of the dystrophin gene. This choice was also inspired by the fact that nonsense mutations can often lead to nearly 11% of the above-mentioned genetic damage that causes heritable human diseases, and nearly 20% of disease-associated single base substitutions that affect gene coding regions. Therefore, the validation of the RNA editing strategy here can have a wide range of therapeutic applications. To this end, RNA editing of the stop codon was first optimized in vitro (Figure 43). It is worth noting that the addition of a second copy of adRNA was observed to significantly increase the targeting efficiency (Figure 43C), so a dual adRNA delivery method was used in the in vivo study. The construct was then packaged into AAV8 and 2E+12 vector genomes (vg) / muscle were injected into the tibialis anterior (TA) or gastrocnemius muscle of mdx mice. To further validate this approach, CRISPR-Cas9-based excision of exon 23 ( Fig.33A ), and mdx mice were targeted at the same time. Four or eight weeks after injection, TA and gastrocnemius muscles were collected from mdx mice, wild-type mice, adRNA-targeted and non-targeted controls, and CRISPR-Cas9-treated mice. Immunofluorescence staining showed that the expression of anti-dystrophin could be significantly restored by targeted RNA editing ( Fig.33B , Figure 44A). In addition, nNOS activity was also restored at the sarcolemma ( Fig.33B , Figure 44A). In treated mice, RNA editing yields up to 3.6% (TAA->TGG / TAG / TGA) and up to 2.4% (TAA->TGG) were observed. Fig.33C , Figure 43E). Immunoblotting of treated muscles confirmed the immunofluorescence observations, indicating 1-2.5% protein recovery (Figure 44B). As a benchmark, injection of muscles with a vector carrying CRISPR-Cas9 also expectedly resulted in restoration of dystrophin expression in a subset of muscle cells ( Fig.33B ), immunoblotting of treated muscles confirmed up to 10% protein recovery (Fig. 44C).

[0369] To further confirm the effectiveness of this approach, we next used an independent mouse model of human disease, ornithine transcarbamylase (OTC)-deficient male sparse fur ash (SPF). ash ) mouse model, ADAR-mediated RNA editing was evaluated. ash Mice carry a G->A point mutation in the last nucleotide of the fourth exon of the OTC gene, which results in a lack of OTC mRNA and production of a mutant protein. Recent studies have shown that this mutation can be robustly corrected in newborn mice using CRISPR-Cas9 and homologous recombination-based strategies. To test this system in spf ash Editing point mutations in OTC mRNA ( Fig.33D ) was evaluated in vitro (Figure 45A). The construct was packaged into AAV8 with high liver tropism and injected into 10-12 week old spf mice at 2.5E+12 vg / mouse. ash Three to four weeks after injection, the ash Mice, wild-type littermates, and SPF treated with ADAR2-targeting and non-targeting vectors ash Mice were transfected, liver samples were collected, and the corresponding editing efficiency was evaluated by NGS. Notably, after delivery of adRNA and ADAR2, 0.8-4.7% of edited mRNA was observed in the correctly spliced ​​OTC mRNA. Interestingly, only adRNA resulted in a low but significant RNA editing yield ( Fig.33E ). In addition, after delivery of a hyperactive ADAR2 mutant (E488Q), high editing fractions (4.6-33.8%) were observed in correctly spliced ​​OTC mRNA ( Fig.33E, Figure 45B), 4.6-8.2% editing fraction was observed in OTC pre-mRNA (Figure 45C), and the reduction of mis-splicing products was confirmed (Figure 45D). Immunoblotting of treated liver samples confirmed partial recovery of OTC protein (2.5-5%) (Figure 45E).

[0370] Collectively, the results establish the utility of RNA-guided ADARs for in vivo RNA editing of point mutations. In some cases, sequence preferences, RNA folding, intrinsic half-life, localization, translation machinery, and resident RNA-binding proteins of the ADAR enzyme may influence the accessibility and editability of target sites in RNA and may be important design parameters to consider for effective targeting. For example, in the mdx model, ADAR-based RNA editing may have to compete with nonsense-mediated decay of mutant dystrophin mRNA and also require achieving two A->I substitutions in the presence of nonideal flanking nucleotides to eliminate premature stop codons and potential effects on RNA stability and function. Furthermore, in the spf ash The need to target transient OTC pre-mRNAs in this model may require rapid target ligation and editing. Important limitations of this system may also need to be further addressed, such as off-target effects induced by inherent enzyme-RNA binding, processivity, promiscuity, stimulation of the interferon response by the delivery format itself (such as lipids, nanoparticles, or viruses), leading to increased endogenous ADAR expression, the potential for adRNA to induce RNAi, and off-target hybridization of the antisense domain of adRNA (which may have deleterious effects). In this regard, studies have shown toxicity in mice injected systemically with hyperactive ADAR mutants ( Fig.46 ). These studies are crucial to help improve the specificity and safety of this approach systematically. When considering RNA targeting for gene therapy, especially via the use of non-integrating vectors, another important consideration may be that due to the limited half-life of edited mRNA and effector factors, the effector construct needs to be re-administered regularly. In this regard, compared to CRISPR-based RNA editing methods, RNA-guided ADAR strategies can be directly relevant to human treatment because the same format only utilizes effector RNA and human proteins. In addition, with the widespread expression of ADARs, for example, ADAR1 in most human tissues and ADAR2 especially in the lungs and brain, these ADARs are endogenously recruited via adRNA with long antisense domains (as shown in Figure 32, Fig.33E 34, and 36), presents a very attractive strategy for efficient RNA editing. With continued improvement, this toolset could have a broad impact on a variety of basic science and therapeutic applications.

[0371] Figure 32: Remodeling programmable RNA editing and characterizing specific profiles: (A) Schematic diagram of RNA editing via constructs using full-length ADAR2 and modified adRNA derived from GluR2 transcripts, or MS2 coat protein (MCP) fusions with ADAR1 / 2 deaminase domains and corresponding MS2 hairpins with adRNA. (B) Comparison of RNA editing efficiencies of endogenous RAB7A transcripts by different RNA editing constructs quantified by Sanger sequencing (efficiency calculated as the ratio of Sanger peak height G / (A+G)). The experiment was performed in HEK 293T cells. Values ​​represent mean values ​​+ / - SEM (n = 3). (C) Violin plots representing the distribution of A->G editing yields observed at reference sites, in which it was found that the editing yield of at least one treated sample had a significant change relative to the control sample (Fisher's exact test, FDR = 1%). Blue circles represent the editing yield at the target 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 is equalized on the plot and truncated at 60% yield.

[0372] Figure 33: In vivo RNA editing in mouse models of human disease: (A) Schematic diagram of DNA and RNA targeting methods to restore dystrophin expression in the mdx mouse model of Duchenne muscular dystrophy: (i) a dual gRNA-CRISPR-based approach that results in in-frame excision of exon 23, and (ii) ADAR2- and MCP-ADAR1-based editing of ochre codons. (B) 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 was also seen in treated samples (scale bar: 250 μm). (C) RNA editing efficiency of TAA->TGG / TAG / TGA in vivo in corresponding treated adult mdx mice. Values ​​represent mean + / - SEM (n = 4, 3, 7, 3, 3, 10, 3, 4 independent TA muscles, respectively). (D) In ​​ornithine transcarbamylase deficiency, SPF ash Schematic representation of the OTC locus and the method for correction of mutant OTC mRNA via ADAR2-mediated RNA editing in a mouse model of ornithine transcarbamylase deficiency with a G->A point mutation at the donor splice site of the last nucleotide of exon 4. (E) ashIn vivo RNA correction efficiency in correctly spliced ​​OTC mRNA in liver of mice injected retro-orbitally with AAV8-ADAR2 and AAV8-ADAR2(E488Q). Values ​​represent mean + / - SEM (n=4, 4, 3, 3, 4, 5 independent animals, respectively).

[0373] Vector design and construction

[0374] One or two copies of the adRNA were cloned into an AAV vector containing the human U6 and mouse U6 promoters, as well as a CMV promoter driving enzyme expression. To construct the GFP reporters, GFP-Amber, GFP-Ochre, and GFP-Opal, three gene blocks were synthesized by replacing the Y39 residue of wild-type GFP with 'TAG', 'TAA', and 'TGA', respectively, and cloned downstream of the CAG promoter. To construct the OTC and DMD reporters, spf with the target adenosine to be edited was cloned into the AAV vector containing the human U6 and mouse U6 promoters, as well as a CMV promoter driving enzyme expression. ash A 200 bp fragment of the OTC and mdx DMD transcripts was cloned downstream of the CAG promoter.

[0375] Mammalian cell culture and transfection

[0376] All HEK 293T cells were grown in Dulbecco's modified Eagle's medium (Thermo Fisher) supplemented with 10% FBS and 1% antibiotic-antimycotic in an incubator at 37°C and 5% CO2 atmosphere. All in vitro transfection experiments were performed in HEK 293T cells using the commercial transfection reagent Lipofectamine 2000 (Thermo Fisher). All in vitro RNA editing experiments involving reporter factors were performed in 24-well plates using 400ng reporter plasmid and 800ng adRNA+enzyme plasmid. All in vitro RNA editing experiments targeting endogenous transcripts were performed in 24-well plates using 800ng adRNA / enzyme plasmid. RNA editing experiments based on dCas13b-ADAR2DDE488Q used 800ng enzyme plasmid (Addgene #103864) and 800ng gRNA plasmid, and cells were transfected when they were 25-30% confluent and harvested 60 hours after transfection to quantify editing. Chemically synthesized adRNA (synthesized by IDT or Synthego) was transfected using Lipofectamine3000 (Thermo Fisher) at 20pmol / well.

[0377] Production of AAV vectors

[0378] HEK 293T cells were used to produce AAV8 particles via a triple transfection method and purified via an iodixanol gradient. The confluence at the time of transfection was approximately 80%. Two hours before transfection, DMEM supplemented with 10% FBS was added to HEK293T cells. Each virus was produced in a 5x15cm plate, where each plate used PEI (1μg / uL linear PEI in 1xDPBS pH 4.5, using HCl), with a PEI:DNA mass ratio of 4:1, using 7.5μg pXR-8, 7.5μg recombinant transfer vector, and 7.5μg pHelper vector for transfection. The mixture was incubated at RT for 10 minutes and then added dropwise to the cell culture medium. The virus was harvested after 72 hours and purified using iodixanol density gradient ultracentrifugation. The virus was then dialyzed against 1x PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% Pluronic F68 (Thermo Fisher) using a 50 kDA filter (Millipore) to a final volume of ~1 mL and quantified by qPCR using specific primers for the ITR region according to the standard (ATCC VR-1616).

[0379] AAV-ITR-F: 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 149), and

[0380] AAV-ITR-R: 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 150).

[0381] RNA isolation and next-generation sequencing library preparation

[0382] According to the manufacturer's protocol, RNA was extracted from animal tissues using RNeasy+ Universal Mini Kit (RNeasy Plus Universal MiniKit, Qiagen). RNA was extracted from cells using RNeasy Mini Kit (Qiagen). cDNA was synthesized from 500ng RNA using Protoscript II First Chain cDNA Synthesis Kit (NEB). The preparation of the next generation sequencing library is as follows. In brief, 1 μl of the cDNA prepared above was amplified by PCR using KAPA Hifi HotStart PCR mixture (Kapa Biosystems), wherein PCR used about 150bp around the amplification target site. PCR products were purified (Qiagen PCR Purification Kit / Gel Extraction Kit) to eliminate by-products. NEB Next Multiplex Oligos for Illumina Kit (NEB) was used to construct the library. 10ng of the DNA input was amplified using index primers. The samples were then merged and loaded onto Illumina Miseq (150bp single-end run) or HiSeq (100bp double-end run). Data analysis was performed using CRISPResso (Pinello, L. et al. 2016). For all in vivo experiments, at least 100,000 reads were analyzed. RNA-seq libraries were prepared from 300 ng RNA using the NEBNext Poly(A) mRNA Magnetic Separation Module and the NEBNext Ultra RNA Library Prep Kit for Illumina. Samples were pooled and loaded onto an Illumina Hiseq (100 bp double-end run).

[0383] Spf ash Quantification of OTC mRNA editing yield in mice

[0384] spf ash Mice carry three forms of OTC RNA: pre-mRNA, correctly spliced ​​mRNA, and an extended mRNA that is mis-spliced ​​due to the use of a cryptic splice site 48 base pairs in intron 4. Let the total number of correctly spliced ​​mRNA be X, the total number of mis-spliced ​​variants be Y, and the pre-mRNA be Z. Xe, Ye, and Ze represent the three forms of A->G edited mRNA. Ideally, the editing yield of mRNA can be calculated as (Xe+Ye+Ze) / (X+Y+Z). However, because it is not possible to use the same primers to amplify spliced ​​variants and pre-mRNA variants, Fig.33EThe fraction (Xe / X) of the edited transcript in the correctly spliced ​​mRNA is shown, which in turn will be translated to produce the OTC protein. In addition, Figure 45 C shows the fraction (Ze / Z) of the edited transcript in the pre-mRNA. This fraction will contribute to the formation of the OTC protein after the correct splicing. Finally, the mRNA of the wrong splicing leads to the production of a protein that extends 16 amino acids, which is selectively degraded. In Figure 45 D, the bands corresponding to X and Y are shown.

[0385] Animal experiments

[0386] All animal procedures 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 Laboratory. AAV was injected into mdx mice (C57BL / 10ScSn-Dmd mdx / J) in the gastrocnemius or TA muscle. Using 2.5E+12 vg / mouse, AAV was injected into the spf via retro-orbital injection. ash Mouse (B6EiC3Sn a / A-Otc spf-ash Mice that showed rough fur, slow movement, and slightly hunched backs were designated as sick mice and euthanized.

[0387] Immunofluorescence

[0388] Harvested gastrocnemius or TA muscles were placed in molds containing OCT compound (VWR) and snap frozen in liquid nitrogen. 10 μm sections were cut on pretreated histological slides. The slides were fixed with 4% paraformaldehyde. Dystrophin and nNOS were detected using 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, ThermoFisher).

[0389] Immunoblotting

[0390] Muscle biopsies from mdx mice and ashLiver biopsies from mice were fragmented in RIPA buffer (Sigma) containing protease inhibitor cocktail (Roche) and incubated on ice for 1 hour with intermittent vortexing. Samples were centrifuged at 15,500 x g for 30 minutes at 4°C, and the supernatant was separated 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 of total protein from liver biopsies were loaded into each well of a 4-15% Mini Protean TGX gel (Biorad) with Tris-Glycine-SDS buffer (Biorad) and electrophoresed at 100 V for 60 minutes. Proteins from muscle biopsies were transferred to nitrocellulose membranes at 34V overnight, while proteins from liver biopsies were transferred in 1X tris-glycine transfer buffer at 4°C, 65V for 1 hour and 30 minutes. The blots were blocked in 5% milk-TBST for 1 hour. The blots were probed 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 at 4°C overnight. 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 SuperSignal West FemtoChemiluminescent Substrate (Thermo Fisher) and X-ray film.

[0391] Statistics and reproducibility

[0392] In vitro experiments: Each in vitro experiment was repeated at least three times independently. In vivo experiments: For the mdx mouse model, two experiments based on ADAR2 and MCP-ADAR1(E1008Q) NLS were performed. Both rounds of experiments yielded consistent RNA editing efficiency, dystrophin immunofluorescence, and restoration of dystrophin (as shown by immunoblot). One experiment based on ADAR2(E488Q) and CRISPR-Cas9 was performed. For spfash Mouse Model. Two experiments were performed based on the availability of mice. RNA editing efficiency was consistent for OTC transcripts, spliced ​​mRNA, and pre-mRNA in both rounds of experiments. In experimental group 1, animals were subjected to RT-PCR and immunoblotting.

[0393] 1. Quantification of RNA A->G Editing

[0394] (a) RNA-seq read alignment

[0395] RNA-seq read pairs with 100 bases per read mate were aligned to the GRCh38 reference genome using STAR aligner version 2.6.0c (Dobin A et al. 2013). The genome index was constructed using the primary assembly annotation from GENCODE Release 28 (GRCh38.p12). STAR was run using default parameters except for the following related settings: readMapNumber = -1, alignSJoverhangMin = 5, alignSJDBoverhangMin = 1, alignEndsType = EndToEnd, outFilterMismatchNmax = 10, outFilterMultimapNmax = 1, outSAMunmapped = None, outSAMmultNmax = 1. Samtools sorting was used to sort the reads of the resulting unique alignment pairs by genomic coordinates (Li H. et al., 2009). Duplicate read pairs were marked using Samtools markup and removed from subsequent analysis. The records of total, aligned, duplicate and remaining reads (unpaired) for each sample are reported in Table 4.

[0396] (b) Selection of reference sites for quantification of editing yield

[0397] The evaluation of sites with significant changes in A- to -G editing yield (see below) is sensitive to the number of unique alignment reads available for each sample. In order to minimize potential errors in terms of significant editing sites when comparing different samples, Samtools view (option -s) was used to downsample the unique alignment reads of each HEK293T sample and report the downsampling scores in Table 4. These scores are calculated by dividing the number of unique alignment reads available for the sampled sample with the minimum number of unique alignment reads in the sample. The readings of the control sample (the first in Table 4) were not downsampled. Then, Samtools mpileup was used to process the downsampled readings. The output of the tool was parsed to extract the counts of each base found in the alignment reads at each A-site and T-site in the GRCh38 reference genome sequence. Insertions and deletions are ignored. Reference sites with read coverage less than 10 are omitted in downstream analysis. In the samples listed in Table 4, the number of reference A-sites and T-sites with a remaining read coverage of at least 10 varied by 15%. Without downsampling, the variation in this number was found to be 50%. In the aligned reads of at least one sample, a final list of total sites (A-sites and T-sites) was selected from reference A-sites and T-sites with a read coverage of at least 10 by selecting those sites that were common to all samples and at least one G or C was observed at the reference A-site or T-site, respectively. Those sites that were not common to all samples or had zero editing events observed in all samples were discarded.

[0398] (c) Assessment of significant changes in A-to-G editing yields

[0399] To reveal significant changes in the amount of A-to-G editing, 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 compared samples, and for each reference A-site selected as described above, a Fisher's exact test was performed using a 2x2 weight matrix C with entries defined as follows: C1,1 = counts of bases other than G observed in the control sample, C2,1 = counts of G bases observed in the control sample, C1,2 = counts of bases other than G observed in the test sample, C2,2 = counts of G bases observed in the test sample. For each selected reference T-site, a similar weight matrix was used, except that G was replaced by C in the above definition. The p-values ​​calculated for comparisons of all selected reference sites and a given sample were adjusted for multiple testing using the Benjamini-Hochberg method. A-sites and T-sites with an adjusted p-value of less than 1% false discovery rate (FDR) and a fold change in editing yield of at least 1.1 were considered to have significant changes in A-to-G editing yield on the forward and reverse transcripts, respectively. The counts of these sites were calculated for each sample comparison. Figure 38 to Figure 4 2 is shown as N sig , and reported in the "Changed Sites" column of Table 5. The total number of reference sites with significant changes in A-to-G editing yields was calculated. The editing yields at these sites were used to construct the distribution shown in Figure 32. The on-target A-to-G editing yields of each sample (as shown in Figures 32 and Figure 38 to Figure 4 The blue circle in 2 is estimated to be C 2,2 / (C 1,2 +C 2,2 ). These values ​​are reported in the “Editing Yield” column in Table 5. After selecting the reference assembly hg38, the 1-based genomic coordinates of the expected target A-site were found to be chr3:128814202 by submitting the following sequence to BLAT:

[0400] AGCGGCAGTATTCTGTACAGTAGACACAAGAATTATGTACGCCTTTTATCA (SEQ ID NO: 151).

[0401] Figure 8– Modification of GluR2 adRNA: Scaffold domain modification. Sequence information of adRNA scaffold: ADAR recruitment domain, antisense RNA targeting domain and cytosine mismatches are highlighted. Base pairs mutated to create stable scaffolds are numbered and highlighted in red, while editing-inducing element motifs are shown in green. Quantification of the editing efficiency of the scaffolds thus generated for OTC reporter transcripts, quantified by Sanger sequencing, is shown. Values ​​represent mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0402] Figure 34 - Engineering GluR2 adRNAs: antisense domain engineering. (a) Optimization of adRNA antisense regions using adRNA scaffold 2: length and distance to the ADAR2 recruitment region were systematically varied. Values ​​represent mean + / - SEM (n=3). (b) adRNAs transcribed from the U6 promoter with increasing antisense domain lengths, incorporating 0, 1, or 2 GluR2 domains, were evaluated for their ability to induce targeted RNA editing in the presence or absence of exogenous ADAR2 expression. Values ​​represent mean + / - SEM (n=3). A subset of these data are presented in Fig.32B All experiments above were performed in HEK 293T cells. (c) The expression of endogenous ADAR1 and ADAR2 (relative to GAPDH) in HEK 293T and HeLa cell lines was confirmed. The levels observed were similar to those documented in the Human Protein Atlas (see the global page at proteinatlas.org).

[0403] Figure 35 - Engineering MS2adRNA. (a) Antisense RNA targeting domains of MS2adRNA were systematically evaluated. Values ​​represent mean + / - SEM (n = 3). (b) On-target RNA editing by MCP-ADAR2DD-NLS requires co-expression of MS2adRNA. Values ​​represent mean + / - SEM (n = 3). Experiments were performed in HEK 293T cells.

[0404] Figure 36-Analysis of RNA editing yields for a set of targets. (A) Comparison of the efficiency of RNA editing of OTC reporter transcripts by GluR2adRNA and MS2adRNA guided RNA editing constructs, as well as Cas13b-based REPAIR constructs. Values ​​represent mean + / - SEM (n = 6 for reporter and Cas13b-based constructs, n = 3 for other constructs). (B) Chemically synthesized adRNA forms were tested for a set of mRNAs with or without exogenous ADAR2 expression. The exact chemical modifications and sources of adRNA are described in the figure. Values ​​represent mean + / - SEM (n = 3). (C) Analysis of RNA editing yields for a series of selected endogenous targets to cover a range of expression levels. Long adRNAs transcribed by U6 without or with two GluR2 domains were also evaluated for multiple endogenous mRNA targets with or without exogenous ADAR2 expression. Editing was observed at the tested loci even in the absence of exogenous ADAR2 expression. Values ​​represent mean + / - SEM (n=3). Experiments were performed in HEK 293T cells.

[0405] Figure 37 – ADAR2 variants and their effects on editing and specificity. (A) Comparison of on-target RNA editing of RAB7A transcripts and editing in flanking adenosines by GluR2adRNA and MS2adRNA guide RNA editing constructs, as well as Cas13b-based REPAIR constructs. Mean (n=3) editing yields are depicted. Experiments were performed in HEK293T cells, and editing efficiency was calculated as the ratio of Sanger peak height G / (A+G). (B) In spf ash ADAR2(E488Q) showed higher efficiency than ADAR2 in in vitro editing of OTC reporter transcripts (p=0.037, unpaired t-test, two-tailed; values ​​represent mean + / - SEM (n=3)) and (C) mdx DMD reporter transcripts (p=0.048, p=0.012, unpaired t-test, two-tailed; values ​​represent mean + / - SEM (n=3)). (d) Editing efficiency and specificity profiles of ADAR2, ADAR2(E488Q) and ADAR2(Δ1-138) were compared for OTC reporter transcripts (top) and endogenous RAB7A transcripts (bottom). Heatmap indicates A->G editing near the target (red arrows). Values ​​represent mean + / - SEM (n=3). Experiments were performed in HEK 293T cells and editing efficiency was calculated as the ratio of Sanger peak height G / (A+G).

[0406] Fig.38– Transcriptome-scale specificity profiles of RNA editing approaches (Cas13b-ADAR REPAIR+ / -gRNA). 2D histograms compare the transcriptome-wide A->G editing yields observed with each Cas13b-ADAR2 construct (y-axis) with those observed with control samples (x-axis). Each histogram represents the same set of 8,729,464 reference sites with at least 10 read coverage and at least one putative editing event detected in at least one sample. The statistical bins highlighted in red contain sites with significant changes in A->G editing yield when the treated samples were compared to the control samples. The red crosses in each plot represent the 100 sites with the smallest adjusted p-values. The blue circles represent the expected target A-sites within the RAB7A transcript. The large number of counts in the bins near the lower left corner may correspond not only to lower editing amounts in the test and control samples, but also to sequencing errors and alignment errors. The large number of counts in the box near the upper right corner of each plot likely corresponds to homozygous single nucleotide polymorphisms (SNPs) and other differences between the reference genome and the genome of the HEK293T cell line used in the experiments.

[0407] Fig.39 - Transcriptome-wide specificity profiles of RNA editing approaches (ADAR2+ / -adRNA). The construct used in these studies was GluR2adRNA (1,20,6). 2D histograms compare transcriptome-wide A->G editing yields observed with each ADAR construct (y-axis) to those observed with control samples (x-axis). Fig.38 More details are provided in .

[0408] Fig.40 – Transcriptome-scale specificity profiles of the RNA editing approaches (MCP-ADAR1DD+ / -adRNA). 2D histograms comparing the transcriptome-wide A->G editing yields observed with each ADAR construct (y-axis) to those observed with control samples (x-axis). Fig.38 More details are provided in .

[0409] Fig.41 - Transcriptome-scale specificity profile of the RNA editing approach (MCP-ADAR2DD+ / -adRNA). 2D histograms compare the transcriptome-wide A->G editing yield observed with each ADAR construct (y-axis) to that observed with control samples (x-axis). Fig.38 More details are provided in .

[0410] FIG42—Changes 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 FIG32. The fraction of edited sites for each construct was calculated by dividing the number of reference sites with a significant change in A- to -G editing yield (see Table 3) by the total number of reference sites considered, 8,729,464. The construct features indicated on the horizontal axis were compared using the Mann-Whitney U test, yielding a p-value of 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). (B) 2D histograms comparing the transcriptome-wide A->G editing yield observed with each construct (y-axis) with the yield observed with the control sample (x-axis). Fig.38 More details are provided in . The illustration shows a violin plot representing the distribution of A->G editing yields observed at the reference site, wherein relative to the control sample, it was found that the editing yield of at least one treated sample had a significant change (Fisher's exact test, FDR=1%). The blue circle represents the editing yield at the target A-site within the RAB7A transcript. In order to better visualize the shape of the distribution, they are equalized on the graph along the maximum range of the y-axis and truncated at 60% of the yield. The sample here corresponds to 293T transfected with a long antisense domain carrying adRNA, which can be recruited for RNA editing via exogenous and / or endogenous ADARs.

[0411] Figure 43 - Optimization and evaluation of dystrophin RNA editing experiments in vitro and in vivo in mdx mice. (A) Schematic diagram of RNA editing using full-length ADAR2 and modified adRNA or reverse adRNA (radRNA); (ii) RNA editing efficiency of amber and ochre stop codons in one-step and two-step approaches. Experiments were performed in HEK293T cells. Values ​​represent mean + / - SEM (n = 3). (B) RNA editing of ochre codons 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 diagram of AAV vectors for in vivo delivery of adRNA and ADAR2, and in vitro optimization of RNA editing of amber and ochre stop codons delivered via AAV vectors in the presence of 1 or 2 copies of adRNA (p = 0.0003, p = 0.0001, p = 0.0015, unpaired t-test, two-tailed, respectively). Experiments were performed in HEK 293T cells. Values ​​represent mean + / - SEM (n = 3 for reporter, n = 6 for other conditions). (D) Representative Sanger sequencing plots showing editing of ochre stop codons (TAA->TGG) in mdx DMD reporter transcripts (quantified by NGS). Experiments were performed in HEK 293T cells (n = 3). (E) Representative examples of in vivo RNA editing analysis of treated mdx mice (quantified using NGS).

[0412] Figure 44 - Analysis of immunofluorescence and immunoblotting 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), AAV8-MCP-ADAR1(E1008Q)NLS). Partial restoration of nNOS localization was also seen in treated samples (scale bar: 250μm). (B) Immunoblot shows partial restoration of dystrophin expression (1-2.5%) in TA muscles of mdx mice (which were injected with both components of the editing tool, enzyme and adRNA), and stable ADAR2 expression in injected TA muscles up to 8 weeks after injection. (C) Immunoblot shows partial restoration of dystrophin expression (10%) using AAV8-CRISPR.

[0413] Figure 45 – In SPF ashOptimization and evaluation of OTC RNA editing experiments in vitro and in vivo in mice. (A) Representative Sanger sequencing profiles show ash Correction of point mutations in OTC reporter transcripts (quantified using NGS). Experiments were performed in HEK 293T cells (n=3). (B) Spf treated ash Representative examples of in vivo RNA editing analysis in mice show correction of point mutations in correctly spliced ​​OTC mRNA (quantified using NGS). (C) In adult Spf treated ash In vivo RNA correction efficiency of OTC pre-mRNA in the liver of mice (retro-orbitally injected with A488-ADAR2 and AAV8-ADAR2(E488Q)). Values ​​represent mean + / - SEM (n=4, 4, 3, 3, 4, 5 independent animals, respectively). (D) PCR products show correctly and incorrectly spliced ​​OTC mRNA. The incorrectly spliced ​​mRNA was extended by 48 base pairs. The fraction of incorrectly spliced ​​mRNA was reduced in mice treated with adRNA+ADAR2(E488Q). (E) Immunoblot for OTC shows that in treated adult spf ash Partial restoration of expression in mice (2.5% to 5%), and stable ADAR2(E488Q) expression three weeks after injection.

[0414] Fig.46 – Toxicity analysis of in vivo RNA editing experiments. A summary of the animal experiments, documenting the route of AAV administration, the construct delivered, and the health of the injected mice 3 weeks after injection.

[0415] Example 5

[0416] The present invention discloses experimental results in the mdx mouse model of Duchenne muscular dystrophy using the E100Q mutant of ADAR1 (contained in MCP-ADAR1(E100Q)). In some cases, this mutant can improve the editing yield in vivo compared to ADAR2 and the E488Q mutant of ADAR2. The present invention also discloses the use of the ADAR system in a manner that changes the splicing pattern by editing splice acceptor sites or branch points in introns, thereby resulting in exon skipping. In addition, methods using APOBEC are contemplated and exemplified. In the embodiments presented herein, local structures of apolipoprotein B mRNA are created on the mRNA of interest, which enables C→T RNA editing, wherein the ACF-APOBEC complex binds to the apolipoprotein B mRNA. The present invention also discloses methods for programmable editing of RNA and DNA using ADAR enzymes.

[0417] In order to utilize the ADAR editing system, adRNA can be generated regardless of whether there is exogenous ADAR1 or ADAR2. The embodiment provides a specific adRNA of interest for a given target. In addition, exemplary chemically synthesized adRNAs are provided herein, which have 2'-O-methyl 3' phosphorothioate modifications in the first and last 3, 6, 9 or 12 nucleotides in the entire antisense region, and / or have 2'-O-methyl modifications except for 3 nucleotides (underlined below) centered on the mismatch site, plus a targeting moiety (such as GalNc or ​​cholesterol or cell-penetrating peptides) that can be used to transform targeted RNA editing in cells or in vivo (especially in the liver, lungs and brain) with or without exogenous ADAR1 / 2 overexpression.

[0418] Overall design:

[0419] adRNA22:

[0420] GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACNNNNN NCN NNNNNNNNNNNNN (SEQ ID NO: 152)

[0421] adRNA32:

[0422] GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCNNNNN NCN NNNNNNNNNNNNN (SEQ ID NO: 153) Specific embodiment:

[0424] adRNA22_RAB7A:

[0425] GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACTGCCG CCA GCTGGATTTCCC (SEQ ID NO: 154)

[0426] adRNA32_RAB7A:

[0427] GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCTGCCG CCA GCTGGATTTCCC (SEQ ID NO: 155)

[0428] adRNA22_CKDN2A:

[0429] GTGGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCCACCTCCT CCA CCCGACCCCGGG (SEQ ID NO: 156)

[0430] adRNA32_CKDN2A:

[0431] GGTGTCGAGAAgAGgAgAACAATATGCTAAATGTTGTTcTcGTcTCCTCGACACCCTCCT CCA CCCGACCCCGGG (SEQ ID NO: 157)

[0432] The examples provided below describe various steps taken to further illustrate the embodiments disclosed herein, such as:

[0433] (1) delivering an MCP-ADAR construct with MS2-adRNA that targets a premature stop codon in the dystrophin transcript of mdx mice;

[0434] (2) testing exon skipping by creating point mutations in the mdx mouse model of Duchenne muscular dystrophy and delivering adRNA with ADAR2 / ADAR2(E488Q), or ms-adRNA with MCP-ADAR1 / ADAR(E1008Q), or ms-adRNA with MCP-ADAR2 / ADAR2(E488Q) – to generate A→G substitutions at splice sites;

[0435] (3) testing editing of splice site mutations in the OTC transcripts of spc-ash mice;

[0436] (4) Testing editing of mutant splice sites to alter splicing patterns in the spf-ash mouse model of ornithine transcarbamylase deficiency;

[0437] (5) knocking down SCN9A transcripts by generating A→G substitutions at the splice sites and targeting SCN9A transcripts in mice to engineer pain insensitivity;

[0438] (6) engineering the APOBEC gRNA to create a localized structure of adipoprotein B mRNA on the target mRNA of interest; and

[0439] (7) Testing editing of ADARs in RNA and DNA by recruiting ADARs using RNA, DNA, and RNA-DNA hybrids.

[0440] In the examples, it was observed that when editing the premature stop codon in the dystrophin transcript of mdx mice, enhanced RNA yield and protein expression were obtained using MS2-adRNA and MCP-ADAR1 (E1008Q). After using adRNA to deliver ADAR2 (E488Q), a reduction in mis-splicing OTC mRNA in the spf-ash mouse model was also confirmed.

[0441] Thus, the present disclosure describes a system that enables site-specific A->G editing of RNA. This 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->G editing of DNA. Additionally, the present disclosure provides the potential for C->T editing in RNA by using APOBEC1 expressed together with ACF1.

[0442] This disclosure describes the first in vivo site-specific RNA editing. Indeed, in the mdx mouse model of muscular dystrophy, use of MCP-ADAR1(E1008Q) showed higher editing efficiency than previous constructs. Further in vitro and in vivo testing examined the efficacy of (1) RNA editing for altered splicing, as well as (2) C->T editing by APOBEC in conjunction with overexpressed ACF, and (3) ADARs for editing DNA.

[0443] The current ADAR system is unique compared to other ADAR2 systems (such as those from the Stafforst, Zhang, and Rosenthal labs) and Cas13d-based splicing inhibition.

[0444] As described herein, the above-mentioned ADAR system includes the following items, or is essentially composed of the following items, or is further composed of the following items: an RNA targeting domain complementary to the target RNA, and one or more ADAR recruitment domains capable of recruiting ADARs. After 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 its hyperactive mutants to create A->G editing at the splicing acceptor site and / or branch point of the intron to transform exon skipping. The skipped symmetric exons lead to the formation of truncated proteins. This strategy can be used to skip exon 23 in the dystrophin transcript of the mdx mouse model of DMD, which carries an early stop codon in the exon. Skipping exon 23 leads to the translation of functionally truncated dystrophin. In addition, skipping asymmetric exons can lead to frameshift mutations. Therefore, by skipping essential exons or asymmetric exons, gene knockdown can be transformed. In addition, editing the start codon ATG and Kozak / Shine-Dalgarno sequences can also help change translation efficiency and lead to gene knockdown. Exon skipping strategies can be used to target SCN9A transcripts to engineer insensitivity to pain. Local structures of apolipoprotein B mRNA can be created at the mRNA target of interest to carefully position the C to be edited in a position similar to the naturally occurring site in apolipoprotein B mRNA, where the ACF-APOBEC complex binds to the apolipoprotein B mRNA. This can be achieved by overexpressing paired adRNAs to create an apolipoprotein B mRNA-like structure that can be edited by overexpressing MCP-ACF1 and APOBEC1.

[0445] Example 6 - Exon skipping via creation of splice acceptor and / or branch point mutations

[0446] The present disclosure demonstrates the use of ADAR2 or MCP-ADAR1 / 2-NLS or their hyperactive mutants to create A->G editing at the splice acceptor site and / or branch point of the intron to transform exon skipping. Skipping symmetric exons results in the formation of truncated proteins. In the present embodiment, the strategy can be used to skip exon 23 in the dystrophin transcript of the mdx mouse model of DMD, which carries an early stop codon in the exon. Skipping exon 23 results in the translation of functionally truncated dystrophin. In addition, skipping asymmetric exons can lead to frameshift mutations. Therefore, by skipping essential exons or asymmetric exons, gene knockdown can be transformed. In addition, editing the start codon ATG and Kozak / Shine-Dalgarno sequence helps to change the translation efficiency and lead to gene knockdown. This strategy can be used to target SCN9A transcripts to transform insensitivity to pain.

[0447] An exemplary sequence for accomplishing this is provided below.

[0448] Sequence Set 1: Exemplary adRNA sequences

[0449] adRNA sequence for exon 23 skipping in the dystrophin transcript of mdx mice (SEQ ID NOs: 158-159):

[0450]

[0451] MS2-adRNA sequence for exon 23 skipping in the dystrophin transcript of mdx mice (SEQ ID NOs: 160-161):

[0452]

[0453]

[0454] MS2-adRNA sequence for editing premature stop codon in mdx mice (SEQ ID 162):

[0455]

[0456] adRNA sequences for exon skipping in mouse SCN9A transcripts (SEQ ID NOs: 163-167)

[0457]

[0458] MS2-adRNA sequences for exon skipping in mouse SCN9A transcripts (SEQ ID NOs: 168-172)

[0459]

[0460] Example 7 - C→T Editing via APOBEC

[0461] Local structures of the apolipoprotein B mRNA can be created to carefully position the C to be edited at a location similar to the site naturally present in the apolipoprotein B mRNA, where the ACF-APOBEC complex binds the apolipoprotein B mRNA at the mRNA target of interest. This is achieved by overexpressing paired gRNAs to create an apolipoprotein B mRNA-like structure that can be edited by overexpressing one or more combinations of the following:

[0462] 1. MCP-ACF1 and APOBEC1

[0463] 2. MCP-ACF1 and MCP-APOBEC1

[0464] 3.MCP-connector-ACF-connector-APOBEC1

[0465] 4. MCP-APOBEC1 and ACF1

[0466] Sequence Set 2: Exemplary C→T Editing Sequences

[0467] MS2-adRNA sequence for CT editing (SEQ ID NO: 173-174)

[0468]

[0469]

[0470] Example 8 - Creation of cancer-associated point mutations

[0471] Several genes involved in cancer pathways carry single amino acid substitutions. By creating A->G substitutions in the mRNA sequences of these genes, dominant negative mutants, constitutively active mutants, and catalytically inactive mutants can be created. Some of these genes include KRAS, HRAS, JAK2, GSK3β, β-catenin, SmoM2, cysteine-containing aspartate proteinase (Caspase) 3, Caspase 8, TGF-β, and p53.

[0472] Example 9 - Editing both strands of DNA and DNA / RNA hybrids

[0473] Since ADARs have been shown to edit both strands of double-stranded RNA as well as DNA-RNA hybrids, ADARs can be recruited via single-stranded DNA or DNA-RNA hybrids to edit DNA and RNA. This can be used to modify current adenine base editing approaches for Cas9 (or Cpf1)-ADAR deaminase domain fusions (ADAR1, ADAR2, and their catalytically active mutants E1008Q and E488Q) so that, instead of targeting the ssDNA displacement strand through current base editors, the gRNA binding strand with the AC bulge can be targeted, ideally in the first 10 bp close to the 5' end of the gRNA.

[0474] Example 10 - Ornithine Transcarbamylase Deficiency

[0475] In spf ash ADAR2(E488Q) and adRNA were delivered to mice. In addition to the correctly spliced ​​mRNA, spf ash The mice also carried an extended mRNA variant that was mis-spliced, which was formed due to the use of a cryptic splice site (48 base pairs) in exon 4. After delivery of ADAR2 (E488Q) with adRNA, a reduction in mis-splicing products was confirmed. Efficient RNA editing yields of up to 33.9% of spliced ​​mRNA were also observed. In pre-mRNA, yields of up to 8% were observed. It was also observed that within 3 weeks after injection, the protein recovered by 2.5-5%. This demonstrates the utility of RNA editing in the correction of splice site mutations and in changing splicing patterns. (Figures 50A to 50D)

[0476] Example 11 - Duchenne Muscular Dystrophy

[0477] MCP-ADAR1(E1008Q) was tested together with MS2-adRNA in mdx mice. Enhanced RNA editing yield and restoration of dystrophin expression were observed compared to ADAR2 and ADAR2(E488Q) enzymes. (Figure 51A-Figure 51B)

[0478] Equivalent

[0479] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0480] The techniques illustratively described herein can be appropriately practiced in the absence of any element or elements, one limitation or multiple limitations not specifically disclosed herein. Thus, for example, the terms "include," "comprise," "contain," etc. should be understood broadly and without limitation. In addition, the terms and expressions employed herein have been used as descriptive terms, rather than restrictive terms, and are not intended to exclude any equivalents of the features shown and described or parts thereof when using these terms and expressions, but it should be recognized that various modifications can be made within the scope of the claimed technology.

[0481] Therefore, it should be understood that the materials, methods, and examples provided herein are representative of preferred aspects, are exemplary, and are not intended to limit the scope of the technology.

[0482] The technology has been described broadly and generically herein. Each of the narrower generic and subgeneric groupings falling within the generic disclosure also form part of the technology. This includes the generic description of the technology, but with a qualification or negative limitation to remove any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0483] In addition, where features or aspects of the technology are described in terms of Markush groups, those skilled in the art will recognize that the technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0484] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the specification, including definitions, will control. DETAILED DESCRIPTION

[0486] Many compositions, methods and systems are disclosed herein. Specific exemplary embodiments of these compositions, methods and systems are disclosed below.

[0487] Part 1

[0488] Embodiment 1. A modified ADAR1 or ADAR2 guide RNA ("adRNA"), comprising: a sequence complementary to a target RNA.

[0489] Embodiment 2. The modified adRNA described in Embodiment 1 further comprises an ADAR2 recruitment domain derived from GluR2 mRNA.

[0490] Embodiment 3. The modified adRNA described in embodiment 1 further comprises two MS2 hairpins on both sides of the sequence complementary to the target RNA.

[0491] Embodiment 4. The engineered adRNA of any one of embodiments 1 to 3, wherein the sequence complementary to the target RNA comprises about 20 to 100 base pairs.

[0492] Embodiment 5. The engineered adRNA of embodiment 2 or 4, wherein the ADAR2 recruitment domain derived from GluR2 mRNA is located at the 5' end or the 3' end of the engineered adRNA.

[0493] Embodiment 6. The modified adRNA described in Embodiment 5 comprises GluR2 mRNA at both the 5' end and the 3' end of the modified adRNA.

[0494] Embodiment 7. The modified adRNA described in embodiment 5 or 6 further includes an editing inducing element.

[0495] Embodiment 8. The engineered adRNA of any one of the above embodiments, wherein the target RNA is ornithine transcarbamylase.

[0496] Embodiment 9. A modified ADAR2 guide RNA ("adRNA") selected from Table 1 or Figure 2 The polynucleotide sequences in the provided sequence groups or their respective equivalents are encoded.

[0497] Embodiment 10. An isolated polynucleotide encoding the modified adRNA according to any one of embodiments 1 to 9, or their respective equivalents.

[0498] Embodiment 11. A vector comprising one or more of the isolated polynucleotides described in embodiment 10, or a polynucleotide sequence encoding the modified adRNA described in embodiment 9, and optionally a regulatory sequence operably linked to the isolated polynucleotide.

[0499] Embodiment 12. The vector of embodiment 11, wherein the regulatory sequence comprises a promoter, an enhancer element and / or a reporter factor.

[0500] Embodiment 13. The vector of embodiment 12, wherein the promoter is human U6, mouse U6 promoter or CMV promoter.

[0501] Embodiment 14. The vector of any one of Embodiments 11 to 13, further comprising a detectable marker or a purification marker.

[0502] Embodiment 15. The vector of embodiment 14, wherein the vector is a plasmid or a viral vector.

[0503] 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.

[0504] Embodiment 17. A recombinant cell further comprising the vector of any one of embodiments 11 to 16, wherein the modified adRNA is recombinantly expressed.

[0505] Embodiment 18. A composition comprising a carrier, and one or more modified 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.

[0506] Embodiment 19. The composition of embodiment 18, further comprising a chemotherapeutic agent or drug.

[0507] Embodiment 20. The composition of embodiment 18 or 19, wherein the carrier is a pharmaceutically acceptable carrier or solid support.

[0508] Embodiment 21. A method for modifying protein expression, comprising contacting a polynucleotide encoding the protein with the modified adRNA of any one of embodiments 1 to 9.

[0509] Embodiment 22. The method of embodiment 21, wherein the contacting is in vitro or in vivo.

[0510] Embodiment 23. A method for treating a disease or disorder associated with abnormal protein expression, comprising administering to a subject in need of such treatment an effective amount of one or more modified adRNAs of any one of embodiments 1 to 9.

[0511] Embodiment 24. The method of embodiment 23, wherein the disease or disorder is Duchenne muscular dystrophy.

[0512] Embodiment 25. The method of embodiment 23 or 24, wherein the subject is an animal.

[0513] Embodiment 26. The method of embodiment 23 or 24, wherein the animal is a mammal.

[0514] Embodiment 27. Use of an effective amount of one or more modified adRNAs according to any one of embodiments 1 to 9 for treating a disease or disorder associated with abnormal protein expression.

[0515] Embodiment 28. The use of embodiment 27, wherein the disease or disorder is Duchenne muscular dystrophy.

[0516] Embodiment 29. A kit comprising: the modified adRNA described in any one of embodiments 1 to 9, the isolated polynucleotide described in embodiment 10, the vector described in any one of embodiments 11 to 16, the recombinant cell described in embodiment 17 or the composition described in any one of embodiments 18 to 20, and instructions for use.

[0517] Embodiment 30. The kit of embodiment 19, wherein the instructions describe the method of any one of embodiments 21 to 26.

[0518] Embodiment 31. A complex comprising the adRNA according to any one of embodiments 1 to 9, wherein the adRNA hybridizes to a complementary polynucleotide under high stringency conditions.

[0519] Part 2

[0520] Embodiment 1. A modified ADAR2 guide RNA ("adRNA") comprises: a sequence complementary to a target RNA, and an ADAR2 recruitment domain derived from GluR2 mRNA.

[0521] Embodiment 2. The engineered adRNA of embodiment 1, wherein the sequence complementary to the target RNA comprises about 20 to 100 base pairs.

[0522] Embodiment 3. The engineered adRNA according to embodiment 1 or embodiment 2, wherein the ADAR2 recruitment domain derived from GluR2 mRNA is located at the 5' end or the 3' end of the engineered adRNA.

[0523] Embodiment 4. The modified adRNA described in Embodiment 3, wherein both the 5' end and the 3' end of the modified adRNA include GluR2 mRNA.

[0524] Embodiment 5. The engineered adRNA of any of the preceding embodiments, wherein the target RNA is ornithine transcarbamylase.

[0525] Embodiment 6. A modified ADAR2 guide RNA ("adRNA") selected from Table 1 or Figure 2 The sequence codes in the sequence group provided in .

[0526] Embodiment 7. A method for modifying protein expression, comprising contacting a polynucleotide encoding the protein with the modified adRNA described in any one of embodiments 1 to 6.

[0527] Embodiment 8. The method described in embodiment 7, wherein the contacting is in vitro or in vivo.

[0528] Embodiment 9. A method for treating a disease or disorder associated with abnormal protein expression, comprising administering to a subject in need of such treatment an effective amount of one or more of the engineered adRNAs of any one of embodiments 1 to 6.

[0529] Embodiment 10. The method of embodiment 9, wherein the subject is an animal.

[0530] Embodiment 11. The method of embodiment 9, wherein the animal is a mammal.

[0531] Embodiment 12. Use of an effective amount of one or more engineered adRNAs according to any one of embodiments 1 to 6 for treating a disease or disorder associated with abnormal protein expression.

[0532] Embodiment 13. A kit comprising the modified adRNA according to any one of embodiments 1 to 6 and instructions for use.

[0533] Embodiment 14. The kit described in embodiment 13, wherein the instructions record the method described in embodiment 7 or embodiment 9.

[0534] Embodiment 15. A composition comprising the AdRNA according to any one of embodiments 1 to 6 and a carrier.

[0535] Embodiment 16. The composition of embodiment 15, wherein the carrier is a pharmaceutically acceptable carrier.

[0536] Embodiment 17. A complex comprising the AdRNA according to any one of embodiments 1 to 6, wherein the AdRNA hybridizes to a complementary polynucleotide under high stringency conditions.

[0537] Part 3

[0538] Embodiment 1. An ADAR system for exon skipping, comprising an adRNA targeting a splice acceptor and / or a branch point in an intron, and optionally an ADAR enzyme.

[0539] Embodiment 2. The ADAR system of claim 1, wherein the ADAR enzyme is ADAR1, ADAR2, or a mutant or variant thereof.

[0540] Embodiment 3. The ADAR system of claim 2, wherein the mutant or variant is selected from ADAR1 (E1008Q) and ADAR2 (E488Q).

[0541] Embodiment 4. The ADAR system of any one of claims 1 to 3, wherein the intron is contained in a gene selected from dystrophin, SCN9A, or ornithine transcarbamylase.

[0542] Embodiment 5. The ADAR system of any one of claims 1 to 4, wherein the adRNA is selected from Sequence Set 1.

[0543] 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 claims 1 to 5.

[0544] Embodiment 7. The method of claim 6, wherein the disease, disorder or condition is selected from Duchenne muscular dystrophy or ornithine transcarbamylase deficiency.

[0545] Embodiment 8. The method of claim 6 or claim 7, wherein the disease, disorder or condition is associated with pain.

[0546] Embodiment 9. An APOBEC system for cytosine to thymine editing, comprising a gRNA that creates a lipoprotein B mRNA-like structure, and optionally an APOBEC enzyme.

[0547] Embodiment 10. The APOBEC system of claim 9, wherein the paired gRNAs are paired sequences provided in sequence set 2.

[0548] In general, the present invention provides the following exemplary embodiments.

[0549] 1. A vector comprising a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity recruitment domain, wherein:

[0550] (a) the polynucleotide sequence encoding at least one RNA editing entity recruitment domain lacks a stem-loop containing secondary structure, or

[0551] (b) the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity to at least one sequence selected from the group consisting of an Alu domain encoding sequence, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain encoding sequence, and combinations thereof.

[0552] 2. A vector according to embodiment 1, wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity with the Alu domain sequence.

[0553] 3. A vector according to embodiment 1, wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity with the APOBEC recruitment domain encoding sequence.

[0554] 4. The vector according to embodiment 1, wherein the vector is a viral vector.

[0555] 5. The vector according to embodiment 1, wherein the vector is a liposome.

[0556] 6. The carrier according to embodiment 1, wherein the carrier is a nanoparticle.

[0557] 7. A vector according to embodiment 1, wherein at least one RNA editing entity recruitment domain is configured to recruit ADAR protein.

[0558] 8. The vector according to embodiment 7, wherein the ADAR protein is ADAR1, ADAR2 or ADAR3 protein.

[0559] 9. The vector according to embodiment 7 or 8, wherein the ADAR protein is a human ADAR protein.

[0560] 10. The vector according to embodiment 7 or 8, wherein the ADAR protein is a recombinant ADAR protein.

[0561] 11. The vector according to embodiment 7 or 8, wherein the ADAR protein is a modified ADAR protein.

[0562] 12. A vector according to embodiment 1, wherein at least one RNA editing entity recruitment domain is configured to recruit APOBEC protein.

[0563] 13. The vector of embodiment 12, wherein the APOBEC protein is an APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4 protein.

[0564] 14. The vector according to embodiment 12 or 13, wherein the ADAR protein is a human ADAR protein.

[0565] 15. The vector according to embodiment 12 or 13, wherein the ADAR protein is a recombinant ADAR protein.

[0566] 16. The vector of embodiment 12 or 13, wherein the ADAR protein is a modified ADAR protein.

[0567] 17. A vector according to embodiment 1, wherein at least one RNA editing entity recruitment domain lacks a stem-loop containing secondary structure.

[0568] 18. A vector according to embodiment 1, wherein the polynucleotide sequence encodes at least two RNA editing recruitment domains.

[0569] 19. A vector according to embodiment 18, wherein at least one of the at least two RNA editing recruitment domains is an Alu domain.

[0570] 20. A vector according to embodiment 19, wherein the sequence of the Alu domain forms a secondary structure containing at least one stem loop.

[0571] 21. A vector according to embodiment 19, wherein the Alu domain encoding sequence includes multiple Alu repeat sequences.

[0572] 22. A vector according to embodiment 19, wherein the Alu domain encoding sequence is at least partially single-stranded.

[0573] 23. A vector according to embodiment 18, wherein at least one of the at least two RNA editing recruitment domains is an APOBEC recruitment domain.

[0574] 24. A vector according to embodiment 18, wherein at least one of the at least two RNA editing recruitment domain encoding sequences comprises at least about 80% sequence identity with a GluR2 domain encoding sequence.

[0575] 25. A vector according to embodiment 24, wherein at least one of the at least two RNA editing recruitment domains is a GluR2 domain.

[0576] 26. A vector according to embodiment 18, wherein at least one of the at least two RNA editing recruitment domains is a Cas13 domain.

[0577] 27. The vector of embodiment 19 or 23, wherein the at least two RNA editing recruitment domains are an Alu domain and an APOBEC recruitment domain.

[0578] 28. The vector of any one of embodiments 1 to 27, further comprising a nucleic acid encoding an RNA complementary to at least a portion of the target RNA.

[0579] 29. The vector of embodiment 28, wherein the nucleic acid encoding RNA complementary to at least a portion of the target RNA is about 10 base pairs (bp) to about 1000 bp in length.

[0580] 30. A vector according to embodiment 28, wherein the nucleic acid encoding at least one RNA editing entity recruitment domain, and the nucleic acid encoding RNA complementary to at least a portion of the target RNA, comprise a continuous nucleic acid of at least about 200 bp in length.

[0581] 31. The vector of any one of embodiments 1 to 30, wherein the nucleic acid is chemically synthesized.

[0582] 32. The vector of any one of embodiments 1 to 30, wherein the nucleic acid is genetically encoded.

[0583] 33. The vector of any one of embodiments 1 to 30, wherein the vector comprises DNA.

[0584] 34. A vector according to embodiment 33, wherein the DNA is double-stranded.

[0585] 35. The vector of embodiment 33, wherein the DNA is single-stranded.

[0586] 36. The vector of any one of embodiments 1 to 30, wherein the vector comprises RNA.

[0587] 37. The vector of any one of embodiments 1 to 30, wherein the RNA comprises a base modification.

[0588] 38. The vector of any one of embodiments 1 to 37, wherein the vector is an adeno-associated virus (AAV) vector.

[0589] 39. The vector of embodiment 38, wherein the AAV is a recombinant AAV (rAAV).

[0590] 40. A vector according to embodiment 38, wherein the AAV is selected from the group consisting of: AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, derivatives of any of these, and combinations of any of these.

[0591] 41. The vector of embodiment 38, wherein the AAV is an AAV5 serotype or a derivative thereof.

[0592] 42. The vector of embodiment 40, comprising a derivative of the AAV, wherein the derivative of the AAV comprises a modified VP1 protein.

[0593] 43. A vector according to embodiment 3, wherein the APOBEC recruitment domain is selected from the group consisting of: APOBEC1 recruitment domain, APOBEC2 recruitment domain, APOBEC3A recruitment domain, APOBEC3B recruitment domain, APOBEC3C recruitment domain, APOBEC3E recruitment domain, APOBEC3F recruitment domain, APOBEC3G recruitment domain, APOBEC3H recruitment domain, APOBEC4 recruitment domain and any combination thereof.

[0594] 44. A vector according to any one of embodiments 1 to 43, wherein the at least one RNA editing entity recruitment domain recruits at least two RNA editing entities, and wherein at least one of the at least two polynucleotide sequences encoding the RNA editing entities comprises at least about 80% identity with an APOBEC protein coding sequence.

[0595] 45. A vector according to any one of embodiments 1 to 43, wherein the at least one RNA editing entity recruitment domain recruits at least two RNA editing entities, and wherein at least one of the at least two polynucleotide sequences encoding the RNA editing entities comprises at least about 80% identity with an ADAR protein coding sequence.

[0596] 46. ​​The vector of any one of embodiments 1 to 45, wherein the RNA recruitment domain encoded by the nucleic acid comprises at least one stem-loop.

[0597] 47. A vector according to any one of embodiments 1 to 46, wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises a secondary structure that is substantially cross-shaped.

[0598] 48. A vector according to any one of embodiments 1 to 46, wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises at least two substantially cruciform secondary structures.

[0599] 49. A vector according to embodiment 48, wherein the polynucleotide sequence encoding at least one RNA editing entity recruitment domain is located between the polynucleotide sequences forming the at least two substantially cross-shaped secondary structures.

[0600] 50. A vector according to any one of embodiments 47 to 49, wherein the cruciform secondary structure comprises a stem-loop adjacent to at least one pair of at least partially complementary strands of the cruciform secondary structure.

[0601] 51. The vector of any one of embodiments 1 to 46, wherein the polynucleotide sequence encoding at least one RNA editing recruitment domain comprises a secondary structure that is substantially toehold-shaped.

[0602] 52. A vector comprising a nucleic acid encoding an RNA having a substantially cross-shaped two-dimensional shape, wherein the RNA comprises at least one sequence encoding a recruitment domain for an RNA editing entity.

[0603] 53. The vector of embodiment 52, further comprising a nucleic acid encoding an RNA having at least one targeting domain encoding sequence, wherein the at least one targeting domain encoding sequence is complementary to at least a portion of the target RNA sequence.

[0604] 54. The vector of embodiment 53, wherein the nucleic acid encoding an RNA having at least one targeting domain further comprises a substantially linear two-dimensional structure, the at least one targeting domain being complementary to at least a portion of the target RNA sequence.

[0605] 55. A non-naturally occurring RNA encoded by the vector of any one of embodiments 1 to 54.

[0606] 56. A non-naturally occurring RNA comprising: a first domain sequence comprising a substantially cross-shaped two-dimensional shape; and a second domain sequence having a substantially linear two-dimensional structure connected to the first domain sequence,

[0607] Wherein the first domain sequence encodes an RNA editing entity recruitment domain, and the second domain sequence encodes a targeting domain, wherein the second domain sequence is complementary to at least a portion of a target RNA.

[0608] 57. The non-naturally occurring RNA of embodiment 56, further comprising a third domain sequence attached to the second domain sequence.

[0609] 58. A non-naturally occurring RNA according to embodiment 57, wherein the third domain sequence includes an RNA editing entity recruitment domain encoding sequence, and the RNA editing entity recruitment domain encoding sequence forms a secondary structure having a two-dimensional shape that is essentially a cross.

[0610] 59. The non-naturally occurring RNA of any one of embodiments 56 to 59, wherein at least one base in the non-naturally occurring RNA comprises a chemical modification.

[0611] 60. The non-naturally occurring RNA of any one of embodiments 56 to 59, wherein at least one sugar in the non-naturally occurring RNA comprises a chemical modification.

[0612] 61. A nucleic acid comprising an RNA editing entity recruitment domain and an antisense domain sequence, wherein, 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, at least one base pair of the target nucleic acid is modified with an efficiency at least about 4 times higher 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.

[0613] 62. A nucleic acid comprising an RNA editing entity recruitment domain and an antisense domain, wherein, 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 modifies at least one base pair of the target nucleic acid with an efficiency at least about 4 times greater than a comparable nucleic acid complexed with a GluR2 domain and the antisense domain, as determined by Sanger sequencing of the target nucleic acid.

[0614] 63. A nucleic acid according to embodiment 61 or 62, wherein the nucleic acid comprises RNA.

[0615] 64. A nucleic acid according to any one of embodiments 51 to 63, wherein the target nucleic acid comprises RNA.

[0616] 65. The nucleic acid of embodiment 64, wherein the RNA is mRNA.

[0617] 66. The nucleic acid of embodiment 65, wherein the mRNA encodes a protein or a portion thereof.

[0618] 67. A nucleic acid according to embodiment 66, wherein dysfunction of the protein or a portion thereof is associated with a disease or condition.

[0619] 68. The nucleic acid of embodiment 67, wherein the disease or condition is selected from the group consisting of a neurodegenerative disorder, a muscle disorder, a metabolic disorder, an eye disorder, a cell proliferative disorder, and any combination thereof.

[0620] 69. The nucleic acid of embodiment 64, wherein the RNA is a small interfering RNA (siRNA).

[0621] 70. A nucleic acid according to any one of embodiments 61 to 69, wherein the RNA editing entity recruitment domain comprises at least about 80% identity with the GluR2 domain.

[0622] 71. The nucleic acid of any one of embodiments 61 to 69, wherein the RNA editing entity recruitment domain comprises at least about 80% identity to an Alu domain.

[0623] 72. A nucleic acid according to any one of embodiments 61 to 69, wherein the RNA editing entity recruitment domain comprises at least about 80% identity with the APOBEC recruitment domain.

[0624] 73. A nucleic acid according to any one of embodiments 61 to 72, wherein the RNA editing entity recruitment domain is configured to recruit ADAR protein.

[0625] 74. The nucleic acid of embodiment 73, wherein the ADAR protein is ADAR1, ADAR2 or ADAR3 protein.

[0626] 75. The nucleic acid of embodiment 73 or 74, wherein the ADAR protein is a human ADAR protein.

[0627] 76. The nucleic acid of embodiment 73 or 74, wherein the ADAR protein is a recombinant ADAR protein.

[0628] 77. The nucleic acid of embodiment 73 or 74, wherein the ADAR protein is a modified ADAR protein.

[0629] 78. A nucleic acid according to any one of embodiments 61 to 72, wherein the RNA editing entity recruitment domain is configured to recruit APOBEC protein.

[0630] 79. The nucleic acid of embodiment 78, wherein the APOBEC protein is an APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4 protein.

[0631] 80. The nucleic acid of embodiment 78 or 79, wherein the ADAR protein is a human ADAR protein.

[0632] 81. The nucleic acid of embodiment 78 or 79, wherein the ADAR protein is a recombinant ADAR protein.

[0633] 82. The nucleic acid of embodiment 78 or 79, wherein the ADAR protein is a modified ADAR protein.

[0634] 83. The nucleic acid of any one of embodiments 61 to 82, wherein the nucleic acid is chemically synthesized.

[0635] 84. The nucleic acid of any one of embodiments 61 to 82, wherein the nucleic acid is genetically encoded.

[0636] 85. A nucleic acid comprising a sequence comprising an antisense domain, a first stem-loop forming sequence, and a second stem-loop forming sequence, wherein the nucleic acid modifies at least one base pair of a target nucleic acid when contacted with: (a) a first polypeptide comprising a first portion of an RNA editing entity, and a first polynucleotide binding domain configured to bind to the first stem-loop forming sequence; and, (b) a second polypeptide comprising a second portion of an RNA editing entity, and a second polynucleotide binding domain configured to bind to the second stem-loop forming sequence; and, (c) a target nucleic acid that is complementary to at least a portion of the antisense domain.

[0637] 86. A nucleic acid according to embodiment 85, wherein the first stem-loop or the second stem-loop is an MS2 stem-loop.

[0638] 87. The nucleic acid of embodiment 85, wherein the first stem-loop or the second stem-loop is a BoxB stem-loop.

[0639] 88. A nucleic acid according to embodiment 85, wherein the first stem-loop or the second stem-loop is a U1A stem-loop.

[0640] 89. The nucleic acid of any one of embodiments 85 to 88, wherein the first portion of the RNA editing entity or the second portion of the RNA editing entity comprises an N-terminal fragment of an ADAR deaminase domain encoding sequence.

[0641] 90. The nucleic acid of any one of embodiments 85 to 88, wherein the first portion of the RNA editing entity or the second portion of the RNA editing entity comprises a C-terminal fragment of an ADAR deaminase domain encoding sequence.

[0642] 91. The nucleic acid of any one of embodiments 85 to 90, wherein the first polynucleotide binding domain or the second polynucleotide binding domain comprises an MS2 coat protein.

[0643] 92. The nucleic acid of any one of embodiments 85 to 90, wherein the first polynucleotide binding domain or the second polynucleotide binding domain comprises a λN peptide.

[0644] 93. A nucleic acid according to any one of embodiments 85 to 90, wherein the first polynucleotide binding domain or the second polynucleotide binding domain comprises a human nucleic acid binding protein.

[0645] 94. A nucleic acid according to embodiment 93, wherein the human nucleic acid binding protein is U1A protein, TBP6.7 protein, human histone stem-loop binding protein or the DNA binding domain of the glucocorticoid receptor.

[0646] 95. A nucleic acid according to any one of embodiments 85 to 94, wherein the RNA editing entity is capable of performing an adenosine to inosine mutation on the target nucleic acid.

[0647] 96. A nucleic acid according to any one of embodiments 85 to 94, wherein the RNA editing entity is capable of performing a cytosine to thymine mutation on the target nucleic acid.

[0648] 97. A kit comprising the vector of any one of embodiments 1 to 54 in a container, the non-naturally occurring RNA of any one of embodiments 55 to 60 in a container, or the nucleic acid of any one of embodiments 61 to 96 in a container.

[0649] 98. The kit according to embodiment 97 further comprises a syringe.

[0650] 99. The kit of embodiment 98, wherein the container is the syringe.

[0651] 100. An isolated cell comprising the vector of any one of embodiments 1 to 54, the non-naturally occurring RNA of any one of embodiments 55 to 60, or the nucleic acid of any one of embodiments 61 to 96.

[0652] 101. A pharmaceutical composition comprising the vector of any one of embodiments 1 to 54 in a unit dosage form, the non-naturally occurring RNA of any one of embodiments 55 to 60 in a unit dosage form, or the nucleic acid of any one of embodiments 61 to 96 in a unit dosage form.

[0653] 102. The pharmaceutical composition according to embodim...

Claims

1. A vector comprising a nucleic acid having a polynucleotide sequence encoding at least one RNA editing entity recruitment domain, wherein: (a) the polynucleotide sequence encoding at least one RNA editing entity recruitment domain lacks a stem-loop containing secondary structure, or (b) the polynucleotide sequence encoding at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity to at least one sequence selected from the group consisting of an Alu domain encoding sequence, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain encoding sequence, and combinations thereof.

2. A vector comprising a nucleic acid encoding an RNA having a substantially cross-shaped two-dimensional shape, wherein the RNA comprises at least one sequence encoding a recruitment domain for an RNA editing entity.

3. A non-naturally occurring RNA encoded by the vector of any one of claims 1 to 2.

4. A non-naturally occurring RNA comprising: a first domain sequence comprising a substantially cross-shaped two-dimensional shape; and a second domain sequence having a substantially linear two-dimensional structure connected to the first domain sequence, Wherein the first domain sequence encodes an RNA editing entity recruitment domain, and the second domain sequence encodes a targeting domain, wherein the second domain sequence is complementary to at least a portion of a target RNA.

5. A nucleic acid comprising an RNA editing entity recruitment domain and an antisense domain sequence, wherein: When the nucleic acid is contacted with an RNA editing entity and a target nucleic acid that is complementary to at least a portion of the antisense domain, at least one base pair of the target nucleic acid is modified with an efficiency at least about 4 times higher than that of a comparable nucleic acid complexed with a Cas13b protein or an active fragment thereof, as determined by Sanger sequencing of the target nucleic acid.

6. A nucleic acid comprising an RNA editing entity recruitment domain and an antisense domain, wherein: The nucleic acid, when contacted with an RNA editing entity and 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 an efficiency at least about 4 times greater than that of a comparable nucleic acid complexed with a GluR2 domain and the antisense domain, as determined by Sanger sequencing of the target nucleic acid.

7. A nucleic acid comprising a sequence comprising an antisense domain, a first stem-loop forming sequence, and a second stem-loop forming sequence, wherein the nucleic acid modifies at least one base pair of a target nucleic acid when in contact with: (a) a first polypeptide comprising a first portion of an RNA editing entity, and a first polynucleotide binding domain configured to bind to the first stem-loop forming sequence; and, (b) a second polypeptide comprising a second portion of an RNA editing entity, and a second polynucleotide binding domain configured to bind to the second stem-loop forming sequence; and, (c) a target nucleic acid that is complementary to at least a portion of the antisense domain.

8. A kit comprising the vector of any one of claims 1 to 2 in a container, the non-naturally occurring RNA of any one of claims 3 to 4 in a container, or the nucleic acid of any one of claims 5 to 7 in a container.

9. An isolated cell comprising the vector of any one of claims 1 to 2, the non-naturally occurring RNA of any one of claims 3 to 4, or the nucleic acid of any one of claims 5 to 7.

10. A pharmaceutical composition comprising the vector of any one of claims 1 to 2 in a unit dosage form, the non-naturally occurring RNA of any one of claims 3 to 4 in a unit dosage form, or the nucleic acid of any one of claims 5 to 7 in a unit dosage form.

11. A method of treating a disease or condition in a subject, comprising administering to the subject the vector of any one of claims 1 to 2, the non-naturally occurring RNA of any one of claims 3 to 4, or the nucleic acid of any one of claims 5 to 7.

12. A method for treating muscular dystrophy in a subject, comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a first nucleic acid encoding a second nucleic acid, wherein the second nucleic acid comprises (a) an antisense region that is at least partially complementary to an RNA sequence associated with muscular dystrophy, and (b) at least one RNA editing entity recruitment domain, wherein the at least one RNA editing entity recruitment domain does not include a stem-loop, or wherein the at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity with at least one of the following: an Alu domain, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain, and any combination thereof.

13. A method for manufacturing a carrier, comprising: At least one copy of a nucleic acid is cloned into the vector, wherein the nucleic acid encodes at least one RNA editing entity recruitment domain, and wherein the sequence encoding the at least one RNA editing entity recruitment domain does not form a secondary structure comprising a stem-loop, or wherein the nucleic acid encoding the at least one RNA editing entity recruitment domain comprises at least about 80% sequence identity to a sequence selected from the following items: an Alu domain encoding sequence, an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) recruitment domain encoding sequence, and any combination thereof.

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