Gene editing for controlled expression of additional genes

Abstract

Disclosed herein are methods for modulating expression of a gene located on an epitype vector in a subject in need thereof. In particular, the method comprises administering to the subject one or more gene editors that modify a region of the gene, thereby modulating expression of the gene. Also disclosed are methods of administering to the subject a base editor system that effectuates a base alteration in the region of the gene or the region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority of U.S. Provisional Patent Application 63 / 379,512, filed on October 14, 2022, the disclosure of which is incorporated herein by reference. Reference to a Sequence Listing Submitted Electronically

[0002] This application contains a Sequence Listing submitted electronically. The content of the electronic Sequence Listing (065830 - 15WO1 SequenceListing.xml; Size: 129KB; and Creation Date: October 9, 2023) is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of gene therapy. In particular, the present invention relates to controlling gene expression from episomal vectors by utilizing gene editing agents such as base editing systems. Background Art

[0004] Gene therapy, in its current design, is an irreversible process. It cannot be stopped in the case of unwanted side effects, nor can the expression level of the therapeutic agent be adjusted according to the needs of individual patients. Adeno-associated virus (AAV) vector-mediated gene therapy has great potential in future medical applications. However, to promote safer and broader applicability and to achieve patient-centered care, therapeutic protein expression should be controllable. For example, in certain diseases, gene therapy in which the gene becomes overexpressed has been shown to be toxic (Payne, Mol Ther Methods ClinDev. March 4, 2022:25:1-2; Palmieri et al., Front Neurosci. May 25, 2023:17:1172805). Conversely, in other diseases such as Huntington's disease, too much gene suppression can be toxic (Jung et al., Hum Mol Genet. April 26, 2021;30(3-4):135-148; Wang et al., Proc Natl Acad Sci U S A. March 22, 2016;113(12):3359-64; Murthy et al., PLoS Genet. March 2019;15(3):e1007765). In other cases, AAV gene therapy has been shown to have a low current risk / benefit profile. For example, in diseases with existing treatments, or in diseases in which patient outcomes are better or have less severe consequences (Evan et al., Curr Opin Rheumatol. January 1, 2023;35(1):37-43; Ishikawa et al., Circ Res. August 17, 2018;123(5):601-613). Therefore, a method for regulating gene therapy in such diseases is needed.

[0005] Base editing is a genome editing method that directly generates precise point mutations in genomic DNA or cellular RNA without directly generating double-strand breaks (DSBs), requiring a DNA donor template, or relying on cellular homology-directed repair. Since base editors generally do not generate DSBs, they minimize the formation of DSB-related byproducts (Komor, A.C. et al., (2017) Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity, Sci Adv 3, and Rees, H.A. et al., (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790). Base editors (BEs) are typically fusions of Cas (“CRISPR-associated”) domains and nucleobase modification domains. Two major types of base editors have been developed and widely used. The first type includes cytosine base editors (CBEs), first reported in 2016 (Komor et al., 2016; (2016) Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods, 13, 1029–1035; Nishida et al., (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 353, aaf8729). The second type includes adenine base editors (ABEs), first described in 2017 (Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 551, 464–471).Both CBE and ABE are based on the CRISPR-Cas9 system and utilize cytidine deaminase and adenine deaminase to confer C-to-T and A-to-G base conversion changes in the editing window, respectively. CBE can convert four codons (CGA, CAG, GAG, GAA, and TGG) into stop codons (TGA, TAG, TAA) (Kuscu et al., (2017) CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat. Methods, 14, 710–712; Molla and Yang, (2019) CRISPR / Cas-mediated base editing: technical considerations and practical applications. Trends Biotechnol. 37, 1121–1142). Thus, CBE can be used to knockout protein-coding genes by introducing premature stop codons.

[0006] The present disclosure provides methods for modulating the expression of a gene located on an episomal vector in a subject. In particular, the present disclosure provides methods for modulating the expression of a gene located on an episomal vector using a base editor system. SUMMARY OF THE INVENTION

[0007] The present disclosure provides methods for modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject one or more gene editing agents that modify a region of the gene to modulate the expression of the gene, wherein the region of the gene comprises one or more of a promoter, an enhancer, a silencer, or an insulator, a premature stop codon that can be converted into an amino acid codon by modification of the region, or an amino acid codon that can be converted into a premature stop codon by modification of the region.

[0008] In certain embodiments, the one or more gene editing agents comprise a guide RNA complementary to the region of the gene and a Cas protein or a derivative of the Cas protein.

[0009] In certain embodiments, the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9); CpF1; CasX; CasY; C2c1; C2c2; C2c3; or a variant thereof.

[0010] In certain embodiments, the one or more gene editing agents further comprise a donor nucleic acid that has at least one nucleotide change relative to a region of the gene and is capable of integrating into the region of the gene to modify the region.

[0011] In certain embodiments, the one or more gene editing agents are encoded by one or more nucleic acid molecules administered to a subject, preferably the one or more gene editing agents are encoded by RNA molecules (particularly mRNA molecules) administered to the subject.

[0012] Also provided is a method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject a base editor system that effects a base change in a region of the gene or a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0013] In certain embodiments, the region of the gene comprises one or more of a promoter, enhancer, silencer, or insulator, or the region of the gene or the region of the mRNA transcript comprises a premature stop codon that can be converted to an amino acid codon by a base change or an amino acid codon that can be converted to a premature stop codon by a base change.

[0014] In certain embodiments, the base editor system comprises: (a) a ribonucleic acid complementary to the region of the gene; and (b) a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the polynucleotide programmable DNA binding domain binds to the region of the gene together with the ribonucleic acid to effect the base change.

[0015] In certain embodiments, the polynucleotide programmable DNA binding domain comprises a nuclease-inactivated variant of a Cas protein or a nickase variant of a Cas protein.

[0016] In certain embodiments, the base editor further comprises a uracil-binding protein, such as a uracil glycosylase inhibitor (UGI) domain that inhibits uracil-DNA glycosylase.

[0017] In certain embodiments, (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase 1 (ADA1) and ADA2.

[0018] In certain embodiments, the base editor system comprises: (a) a ribonucleic acid complementary to a region of the mRNA transcript; and (b) a base editor comprising a polynucleotide programmable RNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the polynucleotide programmable RNA-binding domain binds, together with the ribonucleic acid, to a region of the mRNA transcript to effect the base change.

[0019] In certain embodiments, the polynucleotide programmable RNA-binding domain comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13. In certain embodiments, Cas13 is Cas13a and Cas13b.

[0020] In certain embodiments, (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0021] In certain embodiments, the ribonucleic acid is a guide RNA.

[0022] In certain embodiments, the base editor system or its components are encoded by one or more nucleic acid molecules administered to a subject, preferably the ribonucleic acid and the base editor are encoded by one or more RNA molecules (such as one or more mRNA molecules) administered to the subject.

[0023] In certain embodiments, the one or more nucleic acid molecules (such as the one or more mRNA molecules) are administered to the subject using lipid nanoparticles (LNPs), peptide cages, or polymeric nanoparticles.

[0024] In certain embodiments, the base change results in the conversion of an amino acid codon to a premature stop codon, preferably upstream of the splice junction, thereby downregulating the expression of the gene.

[0025] In certain embodiments, wherein the base change results in the conversion of the CGA, CAG, or TGG codon to the premature TGA, TAG, or TAA stop codon, respectively, and the base editor comprises a cytidine deaminase domain, preferably, the CAG codon is located near the 5' end of the gene.

[0026] In certain embodiments, the base change results in the conversion of a premature stop codon to an amino acid codon, thereby upregulating the expression of the gene.

[0027] In certain embodiments, wherein the base change results in the conversion of the premature UAG, UAA, or UGA stop codon to CAG, CAA, or CGA, respectively, and the base editor comprises an adenosine deaminase domain, preferably, the premature UAG stop codon is located near the 5' end of the gene.

[0028] In certain embodiments, it further includes administering to the subject an episomal vector containing the gene. In certain embodiments, the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, or an adenoviral vector. In certain embodiments, wherein the episomal vector is an AAV vector.

[0029] In certain embodiments, the subject is a human, such as a human subject suffering from: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, synucleinopathy, epilepsy, neuropathic pain, wet age-related macular degeneration, Usher syndrome 1F, Usher syndrome 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease.

[0030] Also provided are methods of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the methods comprising administering to the subject an editing agent that effects a change in a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0031] In certain embodiments, the editing agent effects a base change in a region of the mRNA transcript of the gene.

[0032] In certain embodiments, the change in the region of the mRNA transcript of the gene alters the stability of the mRNA transcript, the initiation or level of translation of the mRNA transcript, the stability and / or activity of the translated protein.

[0033] In certain embodiments, the region of the mRNA transcript comprises a premature stop codon that can be converted to an amino acid codon by a base change or an amino acid codon that can be converted to a premature stop codon by a base change.

[0034] In certain embodiments, the base change is (a) within a microRNA target site or a toe-hold switch site, or (b) induces ribosomal frameshifting or alters a codon encoding an amino acid residue critical to the function and / or structure of the encoded protein.

[0035] In certain embodiments, the editing agent comprises a targeting ribonucleic acid complementary to the region of the mRNA transcript.

[0036] In certain embodiments, the targeting ribonucleic acid is linear.

[0037] In certain embodiments, the targeting ribonucleic acid is circular.

[0038] In certain embodiments, the targeting ribonucleic acid effects a base change by binding to an endogenous adenosine deaminase domain.

[0039] In certain embodiments, the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, and ADAR3.

[0040] In certain embodiments, the editing agent further comprises: a base editor comprising a polynucleotide programmable RNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, or a nucleic acid encoding the base editor, wherein the polynucleotide programmable RNA-binding domain effects a base change together with the targeting ribonucleic acid.

[0041] In certain embodiments, the polynucleotide programmable RNA-binding domain comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13.

[0042] In certain embodiments, the Cas13 is Cas13a or Cas13b.

[0043] In certain embodiments, (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0044] In certain embodiments, the target ribonucleic acid is a guide RNA or a trigger RNA.

[0045] In certain embodiments, the base editor or the target ribonucleic acid is encoded by one or more nucleic acid molecules administered to a subject, preferably the base editor is encoded by one or more RNA molecules (such as one or more mRNA molecules) administered to the subject.

[0046] In certain embodiments, the target ribonucleic acid and / or the one or more nucleic acid molecules (such as the one or more mRNA molecules) are administered to the subject using lipid nanoparticles (LNPs), peptide cages, or polymeric nanoparticles.

[0047] In certain embodiments, the base change results in the conversion of an amino acid codon to a premature stop codon, preferably upstream of the splice junction, thereby downregulating the expression of the gene.

[0048] In certain embodiments, the base change results in the conversion of the CGA, CAG, or TGG codons to the premature TGA, TAG, or TAA stop codons, respectively, and the base editor comprises a cytidine deaminase domain, preferably, the CAG codon is near the 5' end of the gene.

[0049] In certain embodiments, wherein the base change results in the conversion of a premature stop codon to an amino acid codon, thereby upregulating the expression of the gene.

[0050] In certain embodiments, the base change results in the conversion of a premature UAG, UAA, or UGA stop codon to CAG, CAA, or CGA, respectively, and the base editor comprises an adenosine deaminase domain. Preferably, the premature UAG stop codon is located near the 5' end of the gene.

[0051] In certain embodiments, different amounts of an editing agent, such as different amounts of a targeting ribonucleic acid, are administered to a subject to obtain different expression levels of a gene.

[0052] In certain embodiments, the method further comprises administering to the subject an episomal vector comprising the gene.

[0053] In certain embodiments, the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector or an adenovirus vector.

[0054] In certain embodiments, the episomal vector is an AAV vector.

[0055] In certain embodiments, the subject is a human, such as a human subject suffering from a disease selected from the group consisting of hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, synucleinopathies, epilepsy, neuropathic pain, wet age-related macular degeneration, Usher syndrome 1F, Usher syndrome 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1A Depicts ADAR-based mRNA editing of a stop codon to a functional amino acid codon in a target mRNA. A single nucleotide change introduces a stop codon at position tyrosine 118 (W118) of FIX40, and a targeting ribonucleic acid ("trigger RNA") targets ADAR to edit TAG (stop) back to TGG (tyrosine).

[0057] Figure 1B Shows FIX40 protein levels measured by WES TM Automated capillary-based immunoassay analysis (ProteinSimple, Bio-Techne). Trigger RNA (CadRNA or CadRNAis) was added at two different concentrations (250 or 100 ng). CadRNA perfectly matches the target mRNA, while CadRNAis has mismatches at the spread loop around the edited position.

[0058] Figure 1C shows FIX40 protein levels in the same samples measured by ELISA Figure 1B from

[0059] Figure 2 Shows the total mRNA levels of all constructs obtained by quantitative polymerase chain reaction (qPCR) using a primer-probe set specific for the FIX sequence. Detailed Description

[0060] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, or the like included in this specification is for the purpose of providing background to the present invention. Such discussion does not admit that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms cited herein have the meanings set forth in this specification.

[0062] It must be noted that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents.

[0063] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising" shall be understood to mean including a recited integer or step or a group of integers or steps but not excluding any other integer or step or a group of integers or steps. When used herein, the term "comprise" may be replaced by the term "contain" or "include" or sometimes when used herein, by the term "have".

[0064] When used herein, "consisting of" does not include any element, step, or ingredient not specified in the claimed element, where such element, step, or ingredient is relevant to the claimed invention. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel features of the claim. Whenever any of the above terms "comprise", "contain", "include", and "have" are used in the context of an aspect or embodiment of the present invention, they may be replaced by the terms "consisting of" or "consisting essentially of" to change the scope of the present disclosure.

[0065] As used herein, the conjunctive term "and / or" between a plurality of recited elements is understood to cover both the alternatives of the individual and the combination. For example, in the case where two elements are joined by "and / or", the first alternative means that the first element applies and the second element does not apply. The second alternative means that the second element applies and the first element does not apply. The third alternative means that the first element and the second element apply together. Any one of these alternatives is understood to fall within the said meaning and thus meets the requirements of the term "and / or" as used herein. The application of more than one of the said alternatives at the same time is also understood to fall within the said meaning and thus meets the requirements of the term "and / or".

[0066] All features disclosed herein can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature for the same, equivalent or similar purpose.

[0067] As used herein, the term "about" means a value within 10% of the base parameter (i.e., plus or minus 10%). For example, "about 1:10" means 1.1:10.1 or 0.9:9.9, and about 5 hours means 4.5 hours or 5.5 hours, etc. The term "about" at the beginning of a string of values will correct each value by 10%.

[0068] Unless the context clearly indicates otherwise, all numerical values or numerical ranges include integers within such ranges and fractions of the integers within the said values or ranges. Thus, by way of illustration, reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., and 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc., and so on. Thus, also by way of illustration, reference to a numerical range (such as "1-4") includes 2, 3, and 1.1, 1.2, 1.3, 1.4, etc., and so on. For example, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days.

[0069] In addition, reference to a numerical range (such as "0.01 to 10") includes 0.011, 0.012, 0.013, etc., and 9.5, 9.6, 9.7, 9.8, 9.9, etc., and so on. For example, a dose of about "0.01 mg / kg to about 10 mg / kg" of the subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., and 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc., and so on.

[0070] Reference to an integer greater than (more than) or less than... includes any number greater than or less than the reference number. Thus, for example, reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., and so on. For example, administering a non-viral vector and / or an immunomodulator "two or more times" includes 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times or more.

[0071] In addition, reference to a numerical range (such as "1 to 90") includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., and 81, 82, 83, 84, 85, etc., and so on. For example, "between about 1 minute and about 90 days" includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., and 1 day, 2 days, 3 days, 4 days, 5 days,..., 81 days, 82 days, 83 days, 84 days, 85 days, etc., and so on.

[0072] In an attempt to assist the reader of this application, the description has been divided into different paragraphs or sections, or for certain embodiments of the invention. These divisions should not be regarded as disconnecting the substance of one paragraph or section or embodiment from the substance of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that the description has broad application and covers all combinations of the different parts, paragraphs, and sentences that can be envisioned. The discussion of any embodiment is meant to be exemplary only and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples.

[0073] The present disclosure provides methods for modulating the expression of a gene located on an episomal vector in a subject in need thereof, the methods comprising administering to the subject one or more gene editing agents that modify a region of the gene, thereby modulating the expression of the gene. The present disclosure also provides methods for modulating the expression of a gene located on an episomal vector in a subject in need thereof, the methods comprising administering to the subject an editing agent that effects a change in a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0074] The methods of the invention modulate the expression of a transgene provided by gene therapy. Modulation of the expression of a gene therapy transgene can be effected in a variety of ways, including but not limited to altering the level, potency, activity, tertiary structure, or folding of the protein product or regulatory RNA product of the gene therapy transgene.

[0075] For example, coding mutations can target catalytic serine, lysine, arginine, or histidine residues in the active site of an enzyme, modification of lysine acetylation or ubiquitination sites (lysine to glutamate or alanine substitutions), serine or threonine phosphorylation sites (threonine or serine to alanine), asparagine-linked (“N-linked”) glycosylation sites (asparagine to aspartic acid) substitutions, or modification of a lipid-binding domain by replacing histidine, lysine, or arginine residues critical for binding to the lipid head group in phosphoinositides (mutation to non-polar or negatively charged residues).

[0076] In certain embodiments, the change in the region of the mRNA transcript of the gene alters the stability of the mRNA transcript, the initiation or level of translation of the mRNA transcript, and / or the stability and / or activity of the translated protein.

[0077] As used herein, the terms “vector” or “expression vector” refer to vectors and particularly episomal vectors. A vector is generally a plasmid used to introduce a particular gene into a target cell and to express the particular gene in the target cell. An expression vector permits the production of large amounts of stable mRNA. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the cell's transcription and translation machinery. The plasmid is engineered such that it contains a highly active promoter that results in the production of large amounts of mRNA. An “episomal vector” is capable of autonomous self-replication within a host cell. In certain embodiments, the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, or an adenoviral vector.

[0078] In certain embodiments, a region of a gene comprises one or more of a promoter, an enhancer, a silencer, or an insulator, or a region of a gene or a region of an mRNA transcript comprises a premature stop codon that can be converted to an amino acid codon by a base change or an amino acid codon that can be converted to a premature stop codon by a base change.

[0079] As used herein, the term "alteration" refers to a change in a polynucleotide or polypeptide sequence or a change in expression level, such as a 10% change, a 25% change, a 40% change, a 50% change, or a greater change.

[0080] As used herein, the term "gene" refers to a set of nucleic acid segments that contain the information necessary to produce a functional RNA product in a controlled manner by the process of transcription. The RNA can then be used directly (such as tRNA, rRNA, snRNA, and other non-coding RNAs (e.g., SRP RNA), antisense RNA, or microRNA) or used to direct the synthesis of a protein. When the phrase "gene encoding a protein" or "protein is encoded by a gene" is used, it means that the gene is transcribed into mRNA, and the mRNA is then translated into a protein, including post-translational and peri-translation that occur in mammalian cells.

[0081] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). When discussing nucleic acids, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0082] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA (polynucleotides and oligonucleotides), chromosomal DNA, spliced or unspliced mRNA, rRNA, tRNA, inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-spliced RNA, or antisense RNA), locked nucleic acid analogs (LNA), oligodeoxynucleotides (ODN) single-stranded and double-stranded, immunostimulatory sequences (ISS), riboswitches, and ribozymes.

[0083] As used herein, the term "mRNA" or sometimes "mRNA transcript" includes, but is not limited to, one or more precursor mRNA transcripts, transcript processing intermediates, one or more mature mRNAs ready for translation, and transcripts of one or more genes, or nucleic acids derived from one or more mRNA transcripts.

[0084] The terms "nucleobase", "nitrogenous base", or "base", which are used interchangeably herein, refer to nitrogen-containing biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on top of each other directly results in long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases (adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)) are referred to as the major or canonical nucleobases. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-major) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, both of which are produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by the deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Y). A "nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.

[0085] The terms "identity", "homology", and their grammatical variants mean that two or more reference entities are the same when they are "aligned" sequences. Thus, for example, when two nucleic acids are identical, they have the same sequence at least within a reference region or reference portion. Identity can be over a defined region (region or domain) of the sequence.

[0086] A "region" or "area" of identity refers to the same portion of two or more reference entities. Thus, in the case where two protein or nucleic acid sequences are identical over one or more sequence regions or sequence domains, they share identity within that region. An "aligned" sequence refers to multiple protein (amino acid) or nucleic acid sequences, typically with corrections (gaps) for deletions or additional bases or amino acids as compared to a reference sequence.

[0087] Identity can extend over the entire length or a portion of a sequence. In certain embodiments, the length of the sequences sharing percent identity is 2, 3, 4, 5, or more contiguous amino acids or nucleic acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous nucleic acids or amino acids. In certain embodiments, the length of the sequences sharing identity is 21 or more contiguous amino acids or nucleic acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc. contiguous amino acids or nucleic acids. In additional embodiments, the length of the sequences sharing identity is 41 or more contiguous amino acids or nucleic acids, such as 42, 43, 44, 45, 45, 47, 48, 49, 50, etc. contiguous amino acids or nucleic acids. In yet additional embodiments, the length of the sequences sharing identity is 50 or more contiguous amino acids or nucleic acids, such as 50 - 55, 55 - 60, 60 - 65, 65 - 70, 70 - 75, 75 - 80, 80 - 85, 85 - 90, 90 - 95, 95 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 500, 500 - 1,000, etc. contiguous amino acids or nucleic acids.

[0088] As used herein, the term "promoter" refers to a sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a nucleic acid molecule is located 3' of the promoter sequence. In certain embodiments, the promoter sequence consists of proximal and more distal upstream elements and may include enhancer elements.

[0089] As used herein, an "enhancer" can refer to a sequence located near a heterologous nucleic acid. Enhancer elements are generally located upstream of promoter elements but also function and can be located downstream of the sequence or within the sequence. Thus, enhancer elements can be located 10 - 50 base pairs, 50 - 100 base pairs, 100 - 200 base pairs, or 200 - 300 base pairs or more base pairs upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements generally increase the expression of an operably linked nucleic acid provided by a promoter element.

[0090] As used herein, the term "silencer" refers to a sequence - specific element that induces a negative effect on gene transcription.

[0091] As used herein, the term "insulator" or "insulating sequence" refers to a class of cis-regulatory elements known as long-range regulatory elements. An insulating sequence is a DNA segment that blocks the interaction or interference of adjacent gene sequences. For example, insulators can reduce transcriptional read-through from the promoter of an adjacent gene or a cryptic promoter in a neighboring nucleotide sequence. Or they block the interaction of an enhancer on one side of the insulating sequence with the promoter of an adjacent gene on the other side of the insulating sequence. Within the meaning of the present invention, the defining feature of an insulating sequence is its ability to insulate or protect a defined transcription unit operably linked to a regulatory element from upstream or downstream interfering genetic elements. For this purpose, the insulating sequence is placed between a (potential) interfering genetic sequence and the regulatory sequence of the transcription unit to be insulated.

[0092] As used herein, the term "stop codon" (also referred to as "termination codon") is a nucleotide triplet within messenger RNA that signals the termination of translation, as opposed to most codons within messenger RNA that correspond to the addition of amino acid residues to a growing polypeptide chain. Thus, the term "premature termination codon" or "premature stop codon" refers to the occurrence of a stop codon instead of a codon corresponding to an amino acid residue. A premature termination codon can be located anywhere upstream of the normal stop codon, which is typically located at the end of the coding nucleic acid sequence of a particular gene. A premature termination codon can be any of the known stop codons, including TAG (transcribed as UAG), TAA (transcribed as UAA), and TGA (transcribed as UGA). Gene editing

[0093] In certain embodiments, the methods of the present invention include administering to a subject one or more gene editing agents that modify a region of the gene, thereby regulating the expression of the gene. Gene editing agents include agents that can target the cell genome to modify gene expression. The term "gene editing agent" as used herein encompasses gene editing agents that cleave targeted DNA to induce mutations (e.g., by homologous directed repair or non-homologous end joining).

[0094] As used herein, the term "mutation" refers to the replacement of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) with another residue, or the deletion or insertion of one or more residues within the sequence. Mutations are typically described herein by identifying the original residue, followed by the position of the residue within the sequence and the identity of the new substituted residue. The various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012)).

[0095] In certain embodiments, the nucleic acid binding protein is a (modified) transcription activator-like effector nuclease (TALEN) system. Transcription activator-like effectors (TALEs) can be engineered to bind virtually any desired DNA sequence. Exemplary methods for genome editing using the TALEN system can be found in, for example, Cermak T., Doyle E.L., Christian M., Wang L., Zhang Y., Schmidt C., et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011;39:e82; Zhang F., Cong L., Lodato S., Kosuri S., Church G.M., Arlotta P. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol. 2011;29:149-153; and U.S. Patent Nos. 8,450,471, 8,440,431, and 8,440,432, which are hereby specifically incorporated by reference in their entirety. By way of further guidance, but not limitation, a naturally occurring TALE or "wild-type TALE" is a nucleic acid binding protein secreted by multiple proteobacterial species. The TALE polypeptide contains a nucleic acid binding domain composed of tandem repeats of highly conserved monomeric polypeptides, the highly conserved monomeric polypeptides being predominantly 33, 34, or 35 amino acids in length and differing from each other predominantly at amino acid positions 12 and 13. In advantageous embodiments, the nucleic acid is DNA. As used herein, the term "polypeptide monomer" or "TALE monomer" will be used to refer to the highly conserved repeat polypeptide sequences within the TALE nucleic acid binding domain, and the term "repeat variable diresidue" or "RVD" will be used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer. As provided throughout this disclosure, the amino acid residues of the RVDs are depicted using the IUPAC single letter code for amino acids. The general representation of a TALE monomer contained within the DNA binding domain is X l-1l -(X 12 X 13 )-X 14-33 or 34 or 35, where the subscript indicates the amino acid position, and X represents any amino acid. X 12 X 13Indicates an RVD. In some polypeptide monomers, the variable amino acid at position 13 is absent or not present, and in such polypeptide monomers, the RVD consists of a single amino acid. In such cases, the RVD can alternatively be represented as X*, where X represents X12 and (*) indicates the absence of X13. The DNA binding domain contains several repeats of the TALE monomer, which can be represented as (X l-ll -(X 12 X 13 )-X 14-33 or 34 or 35 )z, where in a preferred embodiment, z is at least 5 to 40. In a further preferred embodiment, z is at least 10 to 26. The TALE monomer has a nucleotide binding affinity determined by the identity of the amino acid in its RVD. For example, a polypeptide monomer with an RVD of NI preferentially binds to adenine (A), a polypeptide monomer with an RVD of NG preferentially binds to thymine (T), a polypeptide monomer with an RVD of HD preferentially binds to cytosine (C), and a polypeptide monomer with an RVD of NN preferentially binds to both adenine (A) and guanine (G). In yet another embodiment of the present invention, a polypeptide monomer with an RVD of IG preferentially binds to T. Thus, the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of TALE determine its nucleic acid target specificity. In yet another embodiment of the present invention, a polypeptide monomer with an RVD of NS recognizes all four base pairs and can bind to A, T, G, or C. The structure and function of TALE are further described, for example, in Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety. In certain embodiments, targeting is achieved by binding to a polynucleic acid of a TALEN fragment. In certain embodiments, the targeting domain comprises or consists of an inactive TALEN or a nucleic acid binding fragment thereof.

[0096] In certain embodiments, the targeting domain comprises or consists of a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction enzyme generated by fusing a zinc finger DNA-binding domain to a DNA cleavage domain, which can be engineered to target a desired DNA sequence. Exemplary methods for genome editing using ZFNs can be found in, for example, U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, which are specifically incorporated by reference in their entirety. By way of further guidance, but not limitation, artificial zinc finger (ZF) technology involves arrays of ZF modules to target new DNA-binding sites in the genome. Each finger module in the ZF array targets three DNA bases. Custom arrays of individual zinc finger domains are assembled into zinc finger proteins (ZFPs). ZFPs can contain functional domains. The first synthetic zinc finger nuclease (ZFN) was developed by fusing a ZF protein to the catalytic domain of the type IIS restriction enzyme FokI. (Kim, Y.G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A. 91, 883-887; Kim, Y.G. et al., 1996, Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 1156-1160). Increased cleavage specificity and reduced off-target activity can be obtained by using paired ZFN heterodimers, such that each ZFN heterodimer targets different nucleotide sequences separated by a short spacer. (Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods 8, 74-79). ZFPs can also be designed as transcriptional activators and repressors and have been used to target many genes in a variety of organisms. In certain embodiments, the targeting domain comprises or consists of a nucleic acid-binding zinc finger nuclease or a nucleic acid-binding fragment thereof. In certain embodiments, the nucleic acid-binding (fragment) of the zinc finger nuclease is catalytically inactive.

[0097] In certain embodiments, the targeting domain comprises a (modified) meganuclease, which is an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs). Exemplary methods of using meganucleases can be found in U.S. Patent Nos.: 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134, which are specifically incorporated by reference. In certain embodiments, targeting is achieved by binding a polynucleotide to a meganuclease fragment. In certain embodiments, targeting is achieved by binding a polynucleotide to an inactive catalytic meganuclease (fragment). Thus, in certain embodiments, the targeting domain comprises or consists of a nucleic acid that binds a meganuclease or a nucleic acid binding fragment thereof.

[0098] In certain embodiments, the targeting domain comprises a (modified) CRISPR / Cas complex or system. General information regarding CRISPR / Cas systems, their components, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, and their preparation and use (including amounts and formulations), as well as eukaryotic cells expressing CRISPR / Cas and eukaryotes (such as mice) expressing CRISPR / Cas, is described elsewhere herein. In certain embodiments, targeting is achieved by binding an oligonucleotide and / or gRNA to a CRISPR protein fragment. In certain embodiments, targeting is achieved by binding a nucleic acid to an inactive catalytic CRISPR protein (fragment). Thus, in certain embodiments, the targeting domain comprises an oligonucleotide that binds a CRISPR protein or an oligonucleotide that binds a fragment of a CRISPR protein and / or gRNA.

[0099] In certain embodiments, the one or more gene editing agents comprise a guide RNA complementary to a region of the gene and a Cas protein or a derivative of the Cas protein.

[0100] The term "guide RNA" or "gRNA" refers to a polynucleotide that can be specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cas13). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. Although "gRNA" is used interchangeably to refer to guide RNAs that exist as a single molecule or as a complex of two or more molecules, a gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA). Generally, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and directs binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire content of which is incorporated herein by reference.

[0101] As used herein, the term “Cas” generally refers to the (modified) effector protein of the CRISPR / Cas system or complex. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas13a / C2c2, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, e.g., Makarova et al.

[0102] The term "Cas" may be used interchangeably in this text with the terms "CRISPR" protein, "CRISPR / Cas protein", "CRISPR effector", "CRISPR / Cas effector", "CRISPR enzyme", "CRISPR / Cas enzyme", etc., unless otherwise apparent, such as by specific and exclusive reference to Cas9. It should be understood that the term "CRISPR protein" may be used interchangeably with "CRISPR enzyme", regardless of whether the CRISPR protein has been altered compared to the wild-type CRISPR protein, such as increased or decreased (or no) enzyme activity. Similarly, as used herein, in certain embodiments, and where appropriate and will be apparent to those skilled in the art, the term "nuclease" may refer to a modified nuclease in which the catalytic activity has been altered (such as having increased or decreased nuclease activity, or no nuclease activity at all, as well as nickase activity), and other modified nucleases as defined elsewhere herein, unless otherwise apparent, such as by specific and exclusive reference to an unmodified nuclease.

[0103] In some embodiments, the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9); CpF1; CasX; CasY; C2c1; C2c2; C2c3; or variants thereof, or Cas13a, Cas13b, Cas13c or Cas13d. In some embodiments, the Cas protein is a DNA-targeting CRISPR effector protein. In some embodiments, the Cas protein is a type II CRISPR effector protein, such as Cas9. In some embodiments, the CRISPR effector protein is a type V CRISPR effector protein, such as Cpf1 or C2c1. In some embodiments, the Cas protein is an RNA-targeting CRISPR effector protein. In some embodiments, the CRISPR effector protein is a type VI CRISPR effector protein, such as Cas13a, Cas13b, Cas13c or Cas13d.

[0104] In certain embodiments, the one or more gene editing agents further comprise a donor nucleic acid that has at least one nucleotide change relative to a region of the gene and is capable of integrating into the region of the gene to modify the region.

[0105] In certain embodiments, the one or more gene editing agents are encoded by one or more nucleic acid molecules administered to a subject, preferably the one or more gene editing agents are encoded by RNA molecules (particularly mRNA molecules) administered to a subject.

[0106] As used herein, the term "donor DNA" or "donor nucleic acid" refers to a nucleic acid that is designed to be introduced into a locus by homologous recombination. The donor nucleic acid will have at least one region of sequence homology with the locus. In many cases, the donor nucleic acid will have two regions of sequence homology with the locus. These regions of homology can be at one of the two ends or can be internal to the donor nucleic acid. In many cases, and the "insertion" region of the nucleic acid that one wishes to introduce into the nucleic acid molecule present in the cell will be located between the two regions of homology. Base editing

[0107] Also provided is a method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject a base editor system that effects a base change in a region of the gene or a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0108] Also provided is a method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject an editing agent that effects a change in a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0109] In certain embodiments, the editing agent effects a base change in a region of the mRNA transcript of the gene.

[0110] The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In certain embodiments, the base editor system comprises: a ribonucleic acid complementary to a region of the gene; and a base editor comprising a polynucleotide programmable DNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the polynucleotide programmable DNA-binding domain binds, together with the ribonucleic acid, to the region of the gene to effect the base change.

[0111] The term "base editor (BE)" or "nucleobase editor (NBE)" refers to an agent comprising a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. "Base editing activity" means the ability to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C*G to T*A. In another embodiment, the base editing activity is adenosine deaminase activity, e.g., converting an A*T to G*C.

[0112] The term "nucleic acid programmable DNA-binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide-binding domain" and refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that directs the napDNAbp to a specific nucleic acid sequence. Non-limiting examples of polynucleotide programmable nucleotide-binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the polynucleotide programmable nucleotide-binding domain is a polynucleotide programmable DNA-binding domain. In some embodiments, the polynucleotide programmable nucleotide-binding domain is a polynucleotide programmable RNA-binding domain. In some embodiments, the polynucleotide programmable nucleotide-binding domain is a Cas9 protein. The Cas9 protein can associate with a guide RNA that directs the Cas9 protein to a specific DNA sequence complementary to the guide RNA.

[0113] In some embodiments, the polynucleotide programmable DNA-binding domain comprises a nuclease-inactivated variant of a Cas protein or a nickase variant of a Cas protein. In certain embodiments, the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9); CpF1; CasX; CasY; C2c1; C2c2; C2c3; or variants thereof.

[0114] In certain embodiments, the base editor further comprises a base repair inhibitor. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to UGI or UGI fragments. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease". Without wishing to be bound by any particular theory, a catalytically inactive inosine glycosylase (e.g., alkyladenine glycosylase (AAG)) can bind inosine but cannot generate an abasic site or remove inosine, thereby spatially blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, the catalytically inactive inosine-specific nuclease is capable of binding inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include, for example, catalytically inactive alkyladenosine glycosylase from humans (AAG nuclease) and, for example, catalytically inactive endonuclease V from Escherichia coli (Endo V nuclease). In some embodiments, the catalytically inactive AAG nuclease comprises the E125Q mutation or a corresponding mutation in another AAG nuclease. In some embodiments, the base editor further comprises a uracil-binding protein, such as the uracil glycosylase inhibitor (UGI) domain that inhibits uracil-DNA glycosylase.

[0115] As used herein, the term "deaminase" or "deaminase domain" or "deaminase moiety" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine or adenosine (e.g., an engineered adenosine deaminase that deaminates adenosine in DNA). In some embodiments, the deaminase or deaminase domain is a cytidine deaminase that catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain that catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism that does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a naturally occurring deaminase from an organism. The term deaminase also includes any genetically engineered deaminase that may contain genetic modifications (e.g., one or more mutations) that result in a variant deaminase having an amino acid sequence that contains one or more changes relative to the wild-type corresponding deaminase. Examples of deaminases are given herein, and the term is not meant to be limiting.

[0116] The term "adenosine deaminase" refers to a polypeptide or a fragment thereof that is capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or the hydrolytic deamination of deoxyadenosine to deoxyinosine. Base editors comprising adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids (Zheng et al. Nucleic Acids Res. 2017, 45(6):3369-3377). Base editors comprising an adenosine deaminase domain are capable of deaminating the A nucleobase of a DNA polynucleotide. In one embodiment, the adenosine deaminase domain of the base editor comprises all or a portion of an adenosine deaminase that acts on DNA (e.g., adenosine deaminase 1 (ADA1) or ADA2). In certain embodiments, a base editor comprising adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA. In one embodiment, the adenosine deaminase incorporated into the base editor comprises all or a portion of an adenosine deaminase that acts on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, the adenosine deaminase incorporated into the base editor comprises all or a portion of an adenosine deaminase that acts on tRNA (ADAT, e.g., ADAT1, ADAT2, or ADAT3, or a naturally occurring or engineered tRNA-specific adenosine deaminase (TadA)). In certain embodiments, TadA is any of the TadAs described in PCT / US 2017 / 045381, which is incorporated herein by reference in its entirety. The following table provides exemplary sequences; other sequences may also be used. Table 1.

[0117] The term "cytidine deaminase" refers to a polypeptide or a fragment thereof that is capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. In certain embodiments, the cytidine deaminase is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytosine deaminase 1 (CDA1) or CDA2; or a cytosine deaminase that acts on tRNA (CDAT). The following table provides exemplary sequences; other sequences may also be used. Table 2 *from Saccharomyces cerevisiae S288C

[0118] In some embodiments, the base editor is Cas9 fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is nuclease-inactivated Cas9 (dCas9) fused to a deaminase (e.g., adenosine deaminase or cytidine deaminase).

[0119] As used herein, the term "linker" can refer to a covalent linker (e.g., covalent bond), non-covalent linker, chemical group, or molecule that connects two molecules or moieties, such as two components of a protein complex or ribonucleic acid complex, or two domains of a fusion protein (e.g., a polynucleotide-programmable DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase; see PCT / US2019 / 044935, PCT / US2020 / 016288, each of which is incorporated herein by reference in its entirety)). The linker can connect different components of the base editor system or different portions of the components of the base editor system. For example, in some embodiments, the linker can connect the guide polynucleotide-binding domain of the polynucleotide-programmable nucleotide-binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can connect the CRISPR polypeptide and the deaminase. In some embodiments, the linker can connect Cas9 and the deaminase. In some embodiments, the linker can connect dCas9 and the deaminase. In some embodiments, the linker can connect nCas9 and the deaminase. In some embodiments, the linker can connect the guide polynucleotide and the deaminase. In some embodiments, the linker can connect the deaminating component of the base editor system and the polynucleotide-programmable nucleotide-binding component of the base editor system. In some embodiments, the linker can connect the RNA-binding portion of the deaminating component of the base editor system and the polynucleotide-programmable nucleotide-binding component of the base editor system. In some embodiments, the linker can connect the RNA-binding portion of the deaminating component of the base editor system and the RNA-binding portion of the polynucleotide-programmable nucleotide-binding component of the base editor system. The linker can be located between or flanked by two groups, molecules, or other moieties and is connected to each group, molecule, or other moiety by a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker.

[0120] "Targeting ribonucleic acid" or "targeting RNA" is a ribonucleic acid that is complementary to a region targeting mRNA.

[0121] In certain embodiments, the editing agent comprises a targeting ribonucleic acid that is complementary to a region of the target mRNA transcript.

[0122] In certain embodiments, the targeting ribonucleic acid is a guide RNA. In certain embodiments, the targeting ribonucleic acid is a trigger RNA.

[0123] In certain embodiments, the trigger RNA is an adRNA or a cadRNA.

[0124] As used herein, the term "adRNA" refers to an ADAR recruitment guide. AdRNAs contain a programmable antisense region complementary to the target RNA sequence, which has a mismatched cytidine opposite the target adenosine. Additionally, they contain zero, one, or two ADAR recruitment domains engineered from the naturally occurring ADAR substrate GluR2 pre-mRNA, see, e.g., Katrekar et al., Nat Methods. March 2019;16(3):239–242, the contents of which are incorporated by reference in their entirety.

[0125] As used herein, the term "cadRNA" refers to a circular ADAR recruitment RNA. Like adRNAs, cadRNAs contain a recruitment domain derived from a natural RNA site known to be highly edited by ADAR, which recruits endogenous ADAR to the target site, see, e.g., Katrekar et al., Nat Biotechnol. June 2022;40(6):938-945, the contents of which are incorporated by reference in their entirety.

[0126] In certain embodiments, the trigger RNA comprises two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and directs the binding of the deaminase to the target); and (2) a domain that binds the deaminase. In certain embodiments, the targeting ribonucleic acid is circular. In certain embodiments, the targeting ribonucleic acid is linear.

[0127] In certain embodiments, the targeting ribonucleic acid effects base changes by binding to an endogenous deaminase domain (e.g., adenosine deaminase or cytidine deaminase).

[0128] In certain embodiments, the base editor system comprises: a targeting ribonucleic acid complementary to a region of an mRNA transcript; and a base editor comprising a programmable RNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the programmable RNA-binding domain of the polynucleotide binds, together with the ribonucleic acid, to a region of the mRNA transcript to effect a base change.

[0129] In certain embodiments, the programmable RNA-binding domain of the polynucleotide comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13. Nickase variants of Cas13 are known in the art. For example, those described in WO 2019 / 005884, the content of which is incorporated herein by reference in its entirety.

[0130] In certain embodiments, the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT).

[0131] In certain embodiments, the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0132] Exemplary base editors that can be used to implement the methods of the present invention can include, for example, those described in the following references and / or patent publications, each of which is incorporated by reference in its entirety: (a) WO 2015 / 089406 and its equivalents in the United States or around the world; (b) WO 2017 / 070632 and its equivalents in the United States or around the world; (c) WO 2017 / 070633 and its equivalents in the United States or around the world; (d) WO 2018 / 027078 and its equivalents in the United States or around the world; (e) WO 2018 / 071868 and its equivalents in the United States or around the world; (f) W0 2017 / 048390 and its equivalents in the United States or around the world; (f) WO 2018 / 119359 and its equivalents in the United States or around the world; (g) WO 2018 / 119354 and its equivalents in the United States or around the world; (h) WO 2018 / 031683 and its equivalents in the United States or around the world; (i) WO 2018 / 176009 and its equivalents in the United States or around the world; (j) WO 2018 / 021878 and its equivalents in the United States or around the world; (k) WO 2019 / 060746 and its equivalents in the United States or around the world; (l) WO 2020 / 160517 and its equivalents in the United States or around the world; (m) WO 2020 / 168132 and its equivalents in the United States or around the world; (n) WO2020 / 028823 and its equivalents in the United States or around the world; (o) WO 2019 / 226953 and its equivalents in the United States or around the world; (p) WO 2019 / 005884 and its equivalents in the United States or around the world; (q) Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. and Liu, D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420 - (2016); (r) Gaudelli, N.M. et al. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 551, 464 - (2017); (s) Any reference listed in this specification or describing base editors known in the art.

[0133] The term "target site" refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase.

[0134] Some aspects of the present disclosure are based on the recognition that any base editor provided herein is capable of efficiently generating a desired mutation in a nucleic acid (e.g., a nucleic acid located on an episomal vector) without generating a significant number of undesired mutations. In some embodiments, the desired mutation is a mutation generated by a specific base editor that binds to a gRNA, and the specific base editor is specifically designed to alter the desired mutation. In some embodiments, the desired mutation is a mutation that generates a stop codon (e.g., a premature stop codon) within the coding region of a gene. In some embodiments, the desired mutation is a mutation that eliminates a stop codon. In some embodiments, the desired mutation is a mutation that alters gene splicing. In some embodiments, the desired mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promoter or a gene repressor).

[0135] In certain embodiments, the base change results in the conversion of an amino acid codon to a premature stop codon, preferably upstream of the splice junction, thereby downregulating the expression of the gene. As used herein, "splice junction" includes the region where the 3' end of the first exon is joined to the 5' end of the second exon in a mature mRNA transcript or the encoded polypeptide. The size of the region can vary and can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more (including all integers therebetween) nucleotide or amino acid residues on either side of the exact residue at which the 3' end of one exon is joined to the 5' end of another exon. An "exon" refers to a nucleic acid sequence represented in the mature form of an RNA molecule after any portion (intron) of the precursor RNA has been removed by cis-splicing or after two or more precursor RNA molecules have been joined by trans-splicing.

[0136] In certain embodiments, the base change results in the conversion of the CGA, CAG or TGG codon to a premature TGA, TAG or TAA stop codon, respectively, and the base editor comprises a cytidine deaminase domain, preferably, the CAG codon is located near the 5' end of the gene.

[0137] In certain embodiments, the base change results in the conversion of a premature stop codon to an amino acid codon, thereby upregulating the expression of the gene.

[0138] In certain embodiments, the base change results in the conversion of premature UAG, UAA, or UGA stop codons to CAG, CAA, or CGA, respectively, and the base editor comprises an adenosine deaminase domain. Preferably, the premature UAG stop codon is located near the 5' end of the gene. Vector

[0139] In certain embodiments, the methods of the invention further comprise administering to a subject an episomal vector comprising the gene.

[0140] In certain embodiments, the episomal vector is a non-viral vector, including but not limited to plasmids.

[0141] In certain embodiments, the episomal vector is a viral vector. Examples of viral vectors include but are not limited to adeno-associated virus (AAV) vectors, lentiviral vectors, and adenoviral vectors.

[0142] The vector nucleic acid sequence generally contains at least an origin of replication for proliferation in a cell and optionally additional elements such as a heterologous polynucleotide sequence, expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selectable markers (e.g., antibiotic resistance), polyadenylation signals.

[0143] As used herein, the term "expression cassette" refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a polynucleotide molecule. Generally, the expression cassette comprises a polynucleotide molecule operably linked to a promoter sequence.

[0144] "Expression control element" refers to one or more nucleic acid sequences that affect the expression of an operably linked nucleic acid. Expression control elements as set forth herein include promoters and enhancers. Vector sequences (including AAV vectors and non-viral vectors) may include one or more "expression control elements". Generally, such elements are included to facilitate proper transcription of the heterologous polynucleotide and, where appropriate, translation (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of the mRNA and stop codons, etc.). Such elements generally act in cis and are referred to as "cis-acting" elements, but may also act in trans.

[0145] Expression control can be affected at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). Expression control elements can be located near or at a distance from the transcribed sequence (e.g., 1-10, 10-25, 25-50, 50-100, 100-500 or more nucleotides from the polynucleotide), even at a considerable distance. However, due to the length limitation of AAV vectors, the expression control elements in AAV vectors are typically within 1 to 1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0146] Functionally, the expression of an operably linked nucleic acid can be controlled at least in part by an element (e.g., a promoter) such that the element regulates the transcription of the nucleic acid and, where appropriate, the translation of the transcript. Specific examples of expression control elements are promoters, which are typically located at the 5' of the transcribed nucleic acid sequence. A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed when no promoter is present.

[0147] The term "operably linked" means that regulatory sequences necessary for the expression of a nucleic acid sequence are positioned relative to the sequence in an appropriate position to mediate the expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector (e.g., an rAAV vector or a non-viral vector). The coding sequence can be operably linked to the regulatory sequence in a sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter.

[0148] As used herein, the term "heterologous promoter" refers to a promoter not found in nature to be operably linked to a given coding sequence. In certain embodiments, the expression cassette can contain additional elements, such as introns, enhancers, polyadenylation sites, woodchuck response elements (WREs), and / or other elements known to affect the level of expression of a coding sequence.

[0149] As used herein, the term "promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the nucleic acid molecule of the invention is located 3' of the promoter sequence. In certain embodiments, the promoter sequence consists of proximal and more distal upstream elements and can contain enhancer elements.

[0150] As used herein, an "enhancer" can refer to a sequence located near a heterologous nucleic acid. Enhancer elements are typically located upstream of the promoter element, but also function and can be located downstream of the sequence or within the sequence. Thus, an enhancer element can be located 10 - 50 base pairs, 50 - 100 base pairs, 100 - 200 base pairs, or 200 - 300 base pairs or more base pairs upstream or downstream of the heterologous nucleic acid sequence. Enhancer elements typically increase the expression of an operably linked nucleic acid provided by the promoter element.

[0151] An expression construct can contain regulatory elements for driving expression in a particular cell or tissue type. Expression control elements (e.g., promoters) include those that are active in a particular tissue or cell type, referred to herein as "tissue - specific expression control elements / promoters". Tissue - specific expression control elements are typically active in a particular cell or tissue (e.g., the liver). Expression control elements are typically active in a particular cell, tissue, or organ because they are recognized by transcriptional activator proteins or other transcriptional regulatory factors that are unique to the particular cell, tissue, or organ type. Such regulatory elements are known to those of skill in the art (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed., Volume II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al. (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0152] Incorporating tissue-specific regulatory elements into an expression construct provides at least partial tissue tropism for the expression of heterologous nucleic acids encoding proteins or inhibitory RNAs. Examples of promoters active in the liver are the thyroxine-binding globulin (TTR) gene promoter; the human α1-antitrypsin (hAAT) promoter; the apolipoprotein A-I promoter; albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); the hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996); the human factor IX promoter; the thyroxine-binding globulin (TBG) promoter; the TTR minimal enhancer / promoter; the alpha-antitrypsin promoter; LSP (845 nt) (requires intronless scAAV); and the LSP1 promoter, etc. Examples of enhancers active in the liver are apolipoprotein E (apoE) HCR-l and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0153] Expression control elements also include ubiquitous or broad-host-range promoters / enhancers capable of driving the expression of polynucleotides in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers active in a variety of mammalian cell types, or synthetic elements not found in nature (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic b-actin promoter, and the phosphoglycerate kinase (PGK) promoter.

[0154] Expression control elements can also be rendered to express in an adjustable manner, i.e., a signal or stimulus increases or decreases the expression of an operably linked heterologous polynucleotide. A regulatory element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also referred to as an “inducible element” (i.e., induced by a signal). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. Generally, the amount of increase or decrease conferred by such an element is proportional to the amount of signal or stimulus present; the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Specific non-limiting examples include the zinc-inducible sheep metallothionein (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98 / 10088); the tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science. 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512-518 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997); Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other regulatory control elements that can be used in this context are those that are regulated by specific physiological states (e.g., temperature, acute phase, development).

[0155] Other examples of promoters include, but are not limited to, the phosphoglycerate kinase (PGK) promoter, CAG (a complex of the CMV enhancer, the chicken β-actin promoter (CBA), and the rabbit β-globin intron), and other constitutive promoters, the NSE (neuron-specific enolase), synapsin, or NeuN promoter, the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the SFFV promoter, the Rous sarcoma virus (RSV) promoter, the rat insulin promoter, the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1α promoter, synthetic promoters, hybrid promoters, promoters with multi-tissue specificity, etc., all of which are well-known and readily available to those skilled in the art. Other promoters can be of human origin or from other species, including from mice.

[0156] The expression control element also includes the natural element of the heterologous polynucleotide. When it is desired that the expression of the heterologous polynucleotide should mimic the natural expression, natural control elements (e.g., promoters) can be used. When the expression of the heterologous polynucleotide is to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus, natural elements can be used. Other natural expression control elements, such as introns, polyadenylation sites, or Kozak consensus sequences, can also be used.

[0157] In an example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates the expression of the nucleic acid. More specifically, for example, two operably linked DNA sequences mean that the two DNAs are arranged in such a relationship (cis or trans) that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[0158] Thus, additional elements of the vector include, but are not limited to, expression control (e.g., promoter / enhancer) elements, transcription termination signals or stop codons, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences) flanking the sequence (such as one or more copies of the AAV ITR sequence), or introns.

[0159] Additional elements include, for example, filler or stuffer polynucleotide sequences, for example to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept inserts of DNA with a size range generally from about 4 kb to about 5.2 kb or slightly larger. Thus, for shorter sequences, fillers are included to adjust the length to be close to or within the normal size of the viral genome sequence acceptable for packaging the AAV vector into viral particles. In certain embodiments, the filler nucleic acid sequence is a non-translated (non-protein-coding) segment of the nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler polynucleotide sequence has a length such that when combined with the sequence (e.g., inserted into a vector), the total length is between about 3.0 - 5.5 kb, or between about 4.0 - 5.0 kb, or between about 4.3 - 4.8 kb.

[0160] As used herein, the term "gene transfer system" refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue. For example, the gene transfer system can be a viral gene transfer system, such as intact viruses, modified viruses, and VLPs, to facilitate delivery of the viral vector to the desired cell or tissue. The gene transfer system can also be a non-viral delivery system that does not contain viral coat proteins or does not form viral particles or VLPs, such as liposome-based systems, polymer-based systems, protein-based systems, metal particle-based systems, peptide cage systems, etc.

[0161] Viral vectors are derived from or based on one or more nucleic acid elements containing a viral genome. Specific viral vectors include retroviral, lentiviral, and adeno-associated virus (AAV) vectors.

[0162] Retroviruses are enveloped single-stranded RNA viruses that contain 5' and 3' LTRs and a signal packaging sequence located just outside the LTRs. Different types of retroviral vectors can contain different amounts of the viral genome. In certain embodiments, the retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences necessary for viral RNA packaging, reverse transcription, and proviral DNA integration while removing all HIV protein-coding genes. Lentiviral vectors have a packaging capacity of up to approximately 9 kb. If desired, filler sequences can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be trans-produced using appropriate plasmids and cell lines by providing the viral proteins necessary for vector production. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0163] The term "recombinant," as a modifier of vectors (such as recombinant AAV (rAAV) vectors) and sequences (such as recombinant polynucleotides and polypeptides), means to manipulate (i.e., engineer) a composition in a manner that does not normally occur in nature. Although the term "recombinant" is not always used herein with respect to AAV vectors and sequences (such as polynucleotides), any such omission notwithstanding, recombinant forms of polynucleotides are expressly included.

[0164] Recombinant adeno-associated virus vectors (also referred to herein as "rAAV") are based on adeno-associated virus. Adeno-associated virus is a single-stranded DNA virus containing a 4.7-kb genome that is flanked at both ends of the genome by 145-nt ITRs. ITR activity is important for self-priming and packaging and can also provide additional activities, such as promoter activity.

[0165] rAAV contains an AAV recombinant nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. Recombinant adeno-associated virus vectors typically accept DNA inserts in a size range generally from about 4 kb to about 5.2 kb. If desired, filler sequences can be used to increase rAAV nucleic acid size and packaging efficiency. In different embodiments, the rAAV nucleic acid including the filler is 4 - 5.2 kb, 3.0 - 5.5 kb, 4.0 - 5.0 kb, 4.3 - 4.8 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0166] In certain embodiments, the rAAV nucleic acid comprises a 5' ITR and / or a 3' ITR, independently selected from the 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3BITR. In additional embodiments, there are both a 5' and a 3' ITR, and the two ITRs are from the same serotype genome.

[0167] Naturally occurring AAV capsids contain the viral proteins VP1, VP2, and VP3 in a ratio of approximately 1:1:10. AAV vectors can be produced in the context of all three viral proteins being based on a particular serotype or in the context of one, two, or all three viral proteins being based on different serotypes or variants thereof.

[0168] There are different serotypes among different types of viruses. Different serotypes can confer different activities, such as cell or tissue tropism and the likelihood of generating a host immune response. The term "serotype" broadly refers to both serologically distinct viruses and non-serologically distinct viruses that can be within a subgroup or variant of a given serotype. Serological distinctiveness can be determined based on the lack of cross-reactivity between antibodies against one capsid compared to another. Such differences in cross-reactivity are typically due to differences in capsid protein sequences / epitopes (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0169] As more naturally occurring virus isolates are discovered or capsid mutants are generated, there may or may not be serological differences from any currently existing serotype. Thus, in the case where the new virus has no serological differences, the new virus will be a subgroup or variant of the corresponding serotype.

[0170] In certain embodiments, the AAV capsid is based on a VP1, VP2, or VP3 having at least 80% sequence identity to the VP1, VP2, or VP3 of any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10; SEQ ID NO:1 and SEQ ID NO:2; and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions). (See, e.g., U.S. Patent Nos. 9,909,142 and 9,840,719, which disclose RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6; U.S. Patent Nos. 2013 / 0059732 and 9,169,299, which disclose LK01, LK02, and LK03; and U.S. Patent No. 11,110,153; the disclosures of which patents are incorporated herein by reference in their entirety).

[0171] The recombinant AAV capsid and nucleic acid can be based on the same serotype (or subgroup or variant), or can be different from each other. In certain embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the encapsidated capsid protein.

[0172] In various embodiments, the rAAV capsid comprises a protein having a sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9%, or 100% identity to the VP1, VP2, or VP3 of any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10; and the VP1 of SEQ ID NO:1 or SEQ ID NO:2. Table 3. Exemplary AAV Capsid Protein Sequences

[0173] Recombinant AAV can be produced by different types of cell lines. In certain embodiments, human HEK293 cells (American Type Culture Collection accession number ATCC CRL1573) are used. Other host cell lines suitable for rAAV production are described, for example, in Robert et al., (2017) Biotechnol. J., 12:1600193; and International Application PCT / US2017 / 024951, the disclosures of which are incorporated herein by reference in their entirety.

[0174] In certain embodiments, AAV helper functions are introduced into host cells by transfecting the host cells with an AAV helper construct before or in parallel with transfection of the AAV expression vector. Host cells with AAV helper functions can be referred to as "helper cells" or "packaging helper cells". Thus, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to complement the missing AAV functions necessary for productive AAV transduction. AAV helper constructs typically lack AAV ITRs and are neither capable of self-replication nor self-packaging. These constructs can be in the form of, for example, plasmids, phages, transposons, cosmids, viruses, or virus particles. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 that encode both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known. For example, recombinant AAV can be produced as described in U.S. Patent 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein by reference in their entirety.

[0175] When used as a modifier of a composition, the term "isolated" means that the composition is prepared artificially or is separated completely or at least in part from its natural in vivo environment. Generally, an isolated composition is substantially free of one or more substances with which it is normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes.

[0176] The term "isolated" does not exclude combinations produced artificially, such as rAAV sequences or rAAV particles that package or encapsidate an AAV vector genome (vg) and pharmaceutical formulations. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, polymeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced artificially.

[0177] The term "substantially pure" refers to a preparation comprising at least 50%-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation may comprise at least 75% by weight, or at least 85% by weight, or about 90%-99% by weight of the compound of interest. Purity is measured by a method suitable for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.). Therapeutic protein

[0178] Episomal vectors can deliver multiple different genes, which can be expressed to provide proteins with desired activities. Examples of genes include those that provide a healthy copy of a gene in a subject in which the gene is defective, or new modified genes that can help treat a disease or disorder, or new genes that encode proteins that provide a beneficial effect.

[0179] In different embodiments, the gene encodes GAA (acid alpha-glucosidase) for the treatment of Pompe disease; TPP1 (tripeptidyl peptidase 1) for the treatment of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2); ATP7B (copper-transporting ATPase 2) for the treatment of Wilson's disease; alpha-galactosidase for the treatment of Fabry disease; ASS1 (argininosuccinate synthase) for the treatment of citrullinemia type 1; beta-glucocerebrosidase for the treatment of Gaucher disease type 1; beta-hexosaminidase A for the treatment of Tay Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE) (also known as type I and type II C1 inhibitor deficiency); or glucose-6-phosphatase for the treatment of glycogen storage disease type I (GSDI).

[0180] In various embodiments, the gene encodes insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin-like growth factor I or II (IGF-I or IGF-II), TGFβ, activin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 or NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 or netrin-2, hepatocyte growth factor (HGF), ephrin, dickkopf, sonic hedgehog or tyrosine hydroxylase.

[0181] In various embodiments, the gene encodes thrombopoietin (TPO), interleukins (IL-1 through IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor alpha or beta, interferon alpha, beta or gamma, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD or IgE, chimeric immunoglobulins, antibodies, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I or class II MHC molecules. For example, antibodies and immunoglobulins that target cancer cells or other cells that cause disease or disorder can be provided.

[0182] In various embodiments, the gene encodes CFTR (cystic fibrosis transmembrane regulator), blood coagulation (clotting) factors (Factor XIII, Factor IX (FIX), Factor VIII (FVIII), Factor X, Factor VII, Factor VIIa, or Protein C), gain-of-function coagulation factors, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globin, alpha-globin, spectrin, alpha-1 antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine-guanine phosphoribosyltransferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophins 3 and 4, brain-derived neurotrophic factor, glial cell-derived growth factor, transforming growth factor alpha and beta, cytokines, alpha interferon, beta interferon, interferon gamma, interleukin 2, interleukin 4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel–Lindau (VHL), adenomatous polyposis coli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (choroideremia), LCA 5 (LCA-lebercilin), ornithine ketoacid transaminase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), X-linked retinitis pigmentosa GTPase (XLRP), MER proto-oncogene tyrosine kinase (MERTK) (autosomal recessive (AR) form of retinitis pigmentosa (RP)), ABCA4 (Stargardt disease), ACHM 2, 3, and 4 (achromatopsia), anti-vascular endothelial growth factor (VEGF) agent polypeptides (e.g., bevacizumab, brolucizumab, ranibizumab, aflibercept), DFNB1 (connexin 26 deafness), USH1C (Usher syndrome 1C), PKD-1 or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase 1), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator proteins,One or more donor sequences for use as repair templates for genome editing.,

[0183] In various embodiments, the gene encodes erythropoietin (EPO) for treating anemia; interferons α, β, and γ for treating various immune disorders, viral infections, and cancers; interleukins (ILs) for treating various inflammatory diseases or immune deficiencies, including any one of IL-1 through IL-36 and corresponding receptors; chemokines for treating immune disorders, including chemokine (C-X-C motif) ligand 5 (CXCL5); granulocyte colony-stimulating factor (G-CSF) for treating immune disorders such as Crohn's disease; granulocyte-macrophage colony-stimulating factor (GM-CSF) for treating various human inflammatory diseases; macrophage colony-stimulating factor (M-CSF) for treating various human inflammatory diseases; keratinocyte growth factor (KGF) for treating epithelial tissue damage; chemokines such as monocyte chemoattractant protein 1 (MCP-1) for treating recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for treating various immune disorders; α1-antitrypsin for treating emphysema or chronic obstructive pulmonary disease (COPD); α-L-iduronidase for treating mucopolysaccharidosis I (MPS I); ornithine transcarbamylase (OTC) for treating OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for treating phenylketonuria (PKU); lipoprotein lipase for treating lipoprotein lipase deficiency; apolipoprotein for treating apolipoprotein (Apo) A-I deficiency; low-density lipoprotein receptor (LDL-R) for treating familial hypercholesterolemia (FH); albumin for treating hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl phosphate synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine β-synthase for treating homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase; propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; liver phosphorylase; phosphorylase kinase; glycine decarboxylase; H protein; T protein; cystic fibrosis transmembrane conductance regulator (CFTR); member 4 of ATP-binding cassette subfamily A (ABC1) (ABCA4) for treating Stargardt disease; or dystrophin.

[0184] In additional embodiments, the gene encodes a protein for treating a disease or disorder selected from the group consisting of: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington disease, Parkinson disease, Alzheimer disease, synucleinopathy, epilepsy, neuropathic pain, wet age-related macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease. Inhibitory nucleic acid

[0185] An episomal vector can provide multiple different genes encoding multiple different inhibitory nucleic acids, such as short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), RNA i, ribozymes, and antisense RNAs. In various embodiments, the inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease, wherein the gene associated with the polynucleotide repeat disease is selected from: the huntingtin (HTT) gene, the gene associated with dentatorubral-pallidoluysian atrophy (ataxin-1, ATN1), the androgen receptor on the X chromosome in spinal bulbar muscular atrophy, human ataxias 1, 2, 3, and 7, the Cav2.1 P / Q voltage-dependent calcium channel (CACNA1A), the TATA-binding protein, the ataxin 8 opposite strand (ATXN8OS), the serine / threonine protein phosphatase 2A 55 kDa regulatory subunit Bβ isoform in spinocerebellar ataxias (type 1, 2, 3, 6, 7, 8, 12, 17), FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; the myotonic dystrophy protein kinase (MT-PK) in myotonic dystrophy; the frataxin in Friedreich ataxia; the superoxide dismutase 1 (SOD1) gene mutation in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, the human immunodeficiency virus transactivator of transcription gene in HIV infection; HIV TAR, the human immunodeficiency virus transactivator response element gene in HIV TAR, HIV infection; the C-C chemokine receptor (CCR5) in HIV infection; the Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; the liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function in kidney transplantation or acute renal failure in kidney injury; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 type (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors; the apoptosis inhibitor B cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors;Polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, β-catenin in familial adenomatous polyposis; β2-adrenergic receptor, glaucoma; RTP801 / Redd1, also known as DNA damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6AN17K mutant protein in congenital pachyonychia; influenza A virus genome / gene sequence in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence in SARS infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola filovirus genome / gene sequence in Ebola infection; hepatitis B and C virus genomes / gene sequences in hepatitis B and C infections; HSV genome / gene sequence in herpes simplex virus (HSV) infection; coxsackievirus B3 genome / gene sequence in coxsackievirus B3 infection; pathogenic allele silencing (allele-specific silencing)-like torsin A (TOR1A) of genes in primary dystonia, pan-class I and HLA allele-specific in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP).; Gene editing

[0186] An episomal vector can provide multiple different genes encoding multiple different gene editing nucleic acids, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9. In different embodiments, the gene editing nucleic acids edit the DNA of a subject to provide a therapeutic protein as provided hereinbefore or disrupt a gene as provided hereinbefore. Delivery system

[0187] In certain embodiments, a base editor system or its components are encoded by one or more nucleic acid molecules administered to a subject, preferably the ribonucleic acid and the base editor are encoded by one or more RNA molecules (such as one or more messenger RNA (mRNA) molecules) administered to the subject.

[0188] Any RNA of the system, such as guide RNA or mRNA encoding a base editor, can be delivered in the form of RNA. mRNA encoding a base editor can be generated by in vitro transcription. For example, nuclease mRNA can be synthesized using a PCR cassette containing the following elements: a T7 promoter, an optional kozak sequence (GCCACC), a nuclease sequence, and a 3' UTR (such as the 3' UTR from β-globin-polyA tail). The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g., gRNA) can also be transcribed by in vitro transcription from a cassette containing a T7 promoter, followed by the sequence "GG" and a guide polynucleotide sequence. To enhance expression and reduce possible toxicity, for example, pseudouridine or 5-methyl-C modified base editor coding sequences and / or guide nucleic acids can be used to include one or more modified nucleosides.

[0189] The nucleic acid encoding a nuclear base editor according to the present disclosure can be administered to a subject or delivered into cells in vitro or in vivo by methods known in the art or as described herein. In one embodiment, the nuclear base editor can be delivered by, for example, a vector (e.g., a viral vector or a non-viral vector), a non-vector-based method (e.g., using naked DNA, DNA complexes, lipid nanoparticles), or a combination thereof.

[0190] The nucleic acid encoding a nuclear base editor can be directly delivered to cells as naked DNA or RNA, for example, by transfection or electroporation, or can be conjugated to a molecule that promotes uptake by target cells (e.g., N-acetylgalactosamine). Nucleic acid carriers can also be used, such as the carriers described herein.

[0191] The nucleic acid carrier can contain one or more sequences encoding the domains of the base editing system described herein. The carrier can also contain a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), which is associated with the sequence encoding the protein (e.g., inserted into the sequence encoding the protein or fused to the sequence encoding the protein). As an example, the nucleic acid carrier can include a Cas9 coding sequence containing one or more nuclear localization sequences (e.g., the nuclear localization sequence from SV40) and an adenosine deaminase. Non-viral methods

[0192] In certain embodiments, one or more nucleic acid molecules encoding the base editor system of the present invention or its components are administered by a non-viral delivery system, including, for example, encapsulation in lipid nanoparticles (LNPs).

[0193] In certain embodiments, one or more nucleic acid molecules encoding the base editor system of the present invention or its components are delivered or administered using a non-viral delivery system. Non-viral delivery systems include, for example, chemical methods such as non-viral vectors or extracellular vesicles; and physical methods such as gene guns, electroporation, particle bombardment, sonoporation, and magnetofection.

[0194] In certain embodiments, one or more nucleic acid molecules encoding the base editor system of the present invention or its components are delivered as naked DNA, minicircles, transposons, or closed-ended linear double-stranded DNA.

[0195] In certain embodiments, one or more nucleic acid molecules encoding the base editor system of the present invention or its components are delivered or administered in AAV vector particles or other viral particles that are further encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles, or extracellular vesicles.

[0196] In certain embodiments, one or more nucleic acid molecules encoding the base editor system of the present invention or its components are delivered or administered using a non-viral vector.

[0197] As used herein, a "non-viral vector" refers to a vector that is not delivered by a viral particle or by a virus-like particle (VLP). According to certain embodiments, a non-viral vector is a vector that is not delivered via a capsid. The vector can be encapsulated with, mixed with, or otherwise associated with non-viral delivery nanoparticles.

[0198] In view of the present disclosure, any suitable non-viral delivery system known to those of skill in the art can be used in the present invention. Non-viral delivery nanoparticles can be, for example, lipid-based nanoparticles, polymer-based nanoparticles, protein-based nanoparticles, microparticles, microcapsules, metal particle-based nanoparticles, peptide cage nanoparticles, and the like.

[0199] The non-viral delivery nanoparticles of the present invention can be constructed by any method known in the art, and the non-viral vectors of the present invention can be constructed by any method known in the art. Lipid-based delivery systems

[0200] Lipid-based delivery systems are well known in the art, and in view of the present disclosure, any suitable lipid-based delivery system known to those of skill in the art can be used in the present invention. Examples of lipid-based delivery systems include, for example, liposomes, lipid nanoparticles, micelles, or extracellular vesicles.

[0201] "Lipid nanoparticle" or "LNP" refers to lipid-based vesicles that can be used to deliver AAV and non-viral vectors, which have nanoscale dimensions, i.e., about 10 nm to about 1000 nm, or about 50 nm to about 500 nm, or about 75 nm to about 127 nm. Without being bound by theory, it is believed that LNPs provide partial or complete shielding of polynucleotides, expression cassettes, AAV vectors or non-viral vectors from the immune system. The shielding allows the delivery of polynucleotides, expression cassettes, AAV vectors or non-viral vectors to tissues or cells while avoiding inducing a substantial immune response against the polynucleotides, expression cassettes, AAV vectors or non-viral vectors in vivo. The shielding can also allow repeated administration without inducing a substantial immune response against the polynucleotides, expression vectors, AAV vectors or non-viral vectors in vivo (e.g., in a subject such as a human). The shielding can also improve or increase the delivery efficiency of polynucleotides, expression cassettes, AAV vectors or non-viral vectors in vivo.

[0202] The pI (isoelectric point) of AAV is in the pH range of about 6 to about 6.5. Thus, AAV carries a slight negative charge on its surface. Therefore, it may be beneficial for the LNP to contain cationic lipids (e.g., amino lipids). Exemplary amino lipids have been described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122 and 7,745,651 and U.S. Patent Publication Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, 2014 / 0288146, 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411 and 2010 / 0117125, the disclosures of which are incorporated herein in their entirety.

[0203] The terms "cationic lipid" and "amino lipid" are used interchangeably herein and include those lipids having one, two, three or more fatty acid or fatty alkyl chains and pH-titratable amino groups (e.g., alkylamino or dialkylamino groups) and their salts. Cationic lipids are generally protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and are substantially neutral at a pH above the pKa. Cationic lipids can also be titratable cationic lipids. In certain embodiments, the cationic lipid comprises: a protonatable tertiary amine (e.g., pH-titratable) group; C18 alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2 or 3) double bonds; and an ether, ester or ketal linkage between the head group and the alkyl chain.

[0204] Cationic lipids can include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K-DMA, XTC2, and C2K), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleoylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-M-C3-DMA, also known as MC3), its salts, and mixtures thereof. Other cationic lipids also include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleoyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0205] Additional cationic lipids can include, but are not limited to, 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleoylaminocarbonyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-trifluoroacetate propylammonium (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilino leylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0206] Many commercial formulations of cationic lipids can be used, such as (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECT (containing DOSPA and DOPE, available from GIBCO / BRL).

[0207] In certain embodiments, the cationic lipid can be present in an amount of about 10% to about 85% by weight of the LNP, or about 50% to about 75% by weight of the LNP.

[0208] Sterols can confer fluidity to LNPs. As used herein, "sterol" refers to any naturally occurring sterol of plant (phytosterol) or animal (zoosterol) origin as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A-ring. The sterol can be any sterol conventionally used in the field of liposome, lipid vesicle or lipid particle formulations, most commonly cholesterol. Phytosterols can include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication 2011 / 0177156, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, the sterol can be present in an amount of about 5% to about 50% by weight of the LNP, or about 10% to about 25% by weight of the LNP, based on the weight of the lipid nanoparticles.

[0209] The LNP can comprise neutral lipids. Neutral lipids can comprise any lipidic substance that exists in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalins, and cerebrosides. The choice of neutral lipid is generally guided by considerations such as, in particular, particle size and the desired stability. In certain embodiments, the neutral lipid component can be a lipid having two acyl groups (e.g., diacyl phosphatidylcholine and diacyl phosphatidylethanolamine).

[0210] Lipids having various acyl chain groups of different chain lengths and degrees of saturation are available or can be isolated or synthesized by well-known techniques. In certain embodiments, lipids containing saturated fatty acids with a carbon chain length in the range of C14 to C22 can be used. In another group of embodiments, lipids having mono-unsaturated or di-unsaturated fatty acids with a carbon chain length in the range of C14 to C22 are used. Additionally, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or any related phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids having other head groups such as serine and inositol.

[0211] In certain embodiments, the neutral lipid can be present in an amount of about 0.1% to about 75% by weight of the lipid nanoparticles, or about 5% to about 15% by weight of the LNP.

[0212] LNP-encapsulated nucleic acids, expression cassettes, AAV vectors, and non-viral vectors can be incorporated into pharmaceutical compositions, such as pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions can be used, inter alia, for in vivo or ex vivo administration and delivery of LNP-encapsulated nucleic acids, expression cassettes, AAV vectors, and non-viral vectors to a subject.

[0213] Formulations of LNPs can be combined with additional components. Non-limiting examples include polyethylene glycol (PEG) and sterols.

[0214] The term "PEG" refers to polyethylene glycol, which is a linear water-soluble polymer of ethylene glycol repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies and includes, for example, the following functional PEGs: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-tosylate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0215] In certain embodiments, the PEG can be polyethylene glycol having an average molecular weight of from about 550 to about 10,000 daltons and optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the PEG can be substituted with a methyl group at the terminal hydroxyl position. In certain embodiments, the PEG can have an average molecular weight of from about 750 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons, or from about 1,500 to about 3,000 daltons, or about 2,000 daltons or about 750 daltons. The PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the terminal hydroxyl group can be substituted with a methoxy or methyl group.

[0216] PEGylated lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) as described in U.S. Patent Nos. 8,936,942 and 7,803,397, the disclosures of which are incorporated herein in their entirety. Useful PEGylated lipids (or lipid-polyoxyethylene conjugates) can have a variety of "anchoring" lipid moieties to attach the PEG moiety to the surface of the lipid vesicle. Examples of suitable PEGylated lipids include PEGylated phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20) as described in U.S. Patent No. 5,820,873, the disclosure of which is incorporated herein in its entirety, PEGylated dialkylamines, and PEGylated 1,2-diacyl-oxypropyl-3-amines. In certain embodiments, the PEGylated lipid can be PEGylated diacylglycerol and dialkylglycerol. In certain embodiments, the amount of PEG can be from about 0.5% to about 20% by weight of the LNP, or from about 5% to about 15% by weight of the LNP.

[0217] In addition, the LNP can be a PEGylated and sterol-modified LNP. The LNP combined with additional components can be the same LNP or a separate LNP. In other words, the same LNP can be PEGylated and sterol-modified, or alternatively, the first LNP can be PEGylated and the second LNP can be sterol-modified. Optionally, the first modified LNP and the second modified LNP can be combined.

[0218] In certain embodiments, prior to encapsulation, the LNP can have a size in the range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or 75 nm to about 125 nm. In certain embodiments, the nucleic acid, expression vector, AAV vector, or non-viral vector encapsulated by the LNP can have a size in the range of about 10 nm to 500 nm. Polymer-based systems

[0219] Polymer-based delivery systems are well known in the art, and in view of the present disclosure, any suitable polymer-based delivery system or polymer nanoparticle known to those skilled in the art can be used in the present invention. DNA can be entrapped in the polymer matrix of the polymer nanoparticle, or can be adsorbed or conjugated on the surface of the nanoparticle. Examples of commonly used polymers for gene delivery include, for example, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI), chitosan, dendrimers, polyanhydrides, polycaprolactone, and polymethacrylates.

[0220] Polymer-based non-viral vectors can have different sizes, ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally from about 100 nm to about 150 nm, optionally about 150 nm or smaller. Protein-based systems

[0221] Protein-based delivery systems are well known in the art, and any suitable protein-based delivery system or cell-penetrating peptide (CPP) known to those skilled in the art in view of the present disclosure can be used in the present invention.

[0222] CPPs are short peptides (6 - 30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules. Most CPPs are mainly composed of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., Tat, HIV-1 protein), or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018; 25(1):1996 - 2006). Examples of CPPs include, for example, cationic CPPs (high positive charge) (e.g., Tat peptide, penetratin, protamine, poly-L-lysine, polyarginine, etc.); amphiphilic CPPs (chimeric or fusion peptides constructed from different sources, containing both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin 7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPP) 3 , TP10, pep-1, MPG, etc.); amphipathic CPPs (exhibiting both hydrophobic and amphiphilic properties and containing both large aromatic residues and small residues) (e.g., gH625, SPIONs-PEG-CPP NP, etc.); and hydrophobic CPPs (containing only non-polar motifs or residues) (e.g., SG3, PFVYLI, pep-7, fibroblast growth factor (FGF), etc.).

[0223] Protein-based non-viral vectors can have different sizes, ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally from about 100 nm to about 150 nm, optionally about 150 nm or smaller. Peptide cage systems

[0224] Peptide cage-based delivery systems are well known in the art, and in view of the present disclosure, any suitable peptide cage-based delivery system known to those skilled in the art can be used in the present invention. Generally, any protein material capable of assembling into a cage-like structure to form a confined internal environment can be used. Several different types of protein "shells" can be assembled and loaded with different types of materials. For example, protein cages containing viral coat proteins encapsulating non-viral materials (e.g., the protein coat from Cowpea chlorotic mottle virus (CCMV)) have been described, as well as protein cages formed from non-viral proteins (see, e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Application 20040028694, and U.S. Patent Application 20090035389, the disclosures of which are incorporated herein by reference in their entirety). Peptide cages can comprise a protein shell that self-assembles to form a protein cage (e.g., a structure having an internal cavity that is naturally accessible to solvent or can be made accessible by changing solvent concentration, pH, equilibrium ratios).

[0225] Examples of protein cages derived from non-viral proteins include, for example, ferritin and apoferritin derived from eukaryotic and prokaryotic species, such as 12- and 24-subunit ferritins; and protein cages formed from heat shock proteins (HSPs), such as the class of 24-subunit heat shock proteins that form an internal core space, small HSPs from Methanococcus jannaschii, dodecameric Dsp HSP from Escherichia coli, MrgA proteins, etc. As will be understood by those skilled in the art, the monomers of the protein cages can be in their native or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments) that can be made.

[0226] Protein cages can have different core sizes, ranging from about 1 nm to about 1000 nm, optionally about 10 nm to about 500 nm, optionally about 50 nm to about 200 nm, optionally about 100 nm to about 150 nm, optionally about 150 nm or less. Administration and treatment

[0227] The present invention can be used in human and veterinary medical applications. Thus, suitable subjects include mammals (such as humans) as well as non-human mammals. The term "subject" refers to an animal, typically a mammal, such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent, and adult subjects.

[0228] As used herein, the terms “administering” and “administration” refer to providing to a patient or subject one or more of the compositions described herein. By way of example, and not limitation, administration of the composition (e.g., injection) can be by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by infusion over time. Alternatively or concomitantly, administration can be by the oral route.

[0229] The terms “treatment,” “treat,” and “treating” refer to a clinical intervention that is intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease or disorder or one or more of its symptoms, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention that is intended to reverse, alleviate, delay the onset of, or inhibit the progression of a disease or disorder or one or more of its symptoms, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed and / or after the disease has been diagnosed. In other embodiments, treatment can be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., based on a symptom history and / or based on genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0230] Although reducing, decreasing, inhibiting, suppressing, limiting, or controlling the progression or worsening of a disease is a desirable outcome, an “effective amount” or “sufficient amount” of a dose for treatment (e.g., to improve or provide a therapeutic benefit or improvement) generally effectively provides a measurable response to one, more than one, or all of the adverse symptoms, consequences, or complications of the disease, one or more adverse symptoms, disorders, afflictions, pathologies, or complications (e.g., caused by or associated with the disease).

[0231] An effective amount or sufficient amount may or may not be provided in a single administration, may require multiple administrations, and may or may not be administered alone or in combination with another composition (e.g., an agent), treatment, regimen, or treatment protocol. For example, the amount may be increased proportionally as indicated by the needs of the subject, the type, status, and severity of the disease being treated, or the side effects (if any) of the treatment. Additionally, if administered in a single dose or multiple doses without a second composition (e.g., another drug or agent), treatment, regimen, or treatment protocol, the effective amount or sufficient amount need not be effective or sufficient, as additional doses, amounts, or durations, or additional compositions (e.g., drugs or agents), treatments, regimens, or treatment protocols above and beyond such doses may be included in order to be considered effective or sufficient in a given subject.

[0232] An effective amount or sufficient amount need not be effective for each and every subject treated, nor need it be effective for most of the subjects treated in a given group or population. An effective amount or sufficient amount refers to effectiveness or sufficiency in a particular subject, rather than effectiveness or sufficiency in a group or general population. It is typical for such methods that some subjects will exhibit a greater response, or a lesser response, or no response to a given method of treatment or use.

[0233] As used herein, in the context of administering two or more therapies or components to a subject, the terms and phrases "co - delivery" and "administered with" refer to the simultaneous administration of two or more therapies or components. "Simultaneous administration" can be the administration of at least two components within the same day. When two components are "administered with" or "administered in combination with" each other, they can be administered sequentially in separate compositions within a short time period (such as 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours), or within 1 hour, or within 30 minutes, or within 10 minutes, or within 5 minutes, or within 2 minutes, or they can be administered simultaneously in a single composition.

[0234] The order and timing of administration of one or more gene editing agents and episomal vectors can vary depending on the type and severity of the disease being treated. For example, the episomal vector can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of one or more gene editing agents.

[0235] The therapeutic dose of the episomal vector can vary and depends on the type, onset, progression, severity, frequency, duration, or probability of the disease or disorder being treated, the desired clinical endpoint, prior or concurrent treatments, general health status, age, sex, race, or immune capacity of the subject, and other factors that will be understood by one of ordinary skill in the art. The dose amount, number, frequency, or duration can be increased or decreased proportionally as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy and the status of the subject.

[0236] The dose to achieve a therapeutic effect (e.g., the episomal vector dose in milligrams per kilogram body weight (mg / kg)) will also vary based on several factors, including the route of administration, the level of gene expression necessary to achieve a therapeutic effect, the specific disease or disorder being treated, the host immune response to the DNA, the host immune response to the transgenic expression product, and the stability of the protein, peptide, or nucleic acid being expressed. Based on the guidance provided herein, one of ordinary skill in the art can determine an appropriate range of episomal vector doses to treat a patient with a specific disease or disorder.

[0237] The overall level of gene expression can vary depending on the use of the episomal vector. In different embodiments of gene therapy providing a therapeutic protein, the expression or activity provided is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% of the normal expression of the subject protein.

[0238] The dosage of the editing agent for achieving expression modulation (e.g., dosage in milligrams per kilogram body weight (mg / kg)) will also vary based on several factors, including the route of administration, the specific disease or disorder being treated, whether the editing agent targets DNA or RNA, whether gene or mRNA expression is increased or decreased, or the stability of the guide RNA. Based on the guidance provided herein, one of ordinary skill in the art can determine the appropriate dosage range of the editing agent to treat a patient suffering from a specific disease or disorder.

[0239] In certain embodiments, different amounts of the targeted ribonucleic acid are administered to a subject to obtain different expression levels of a gene. In certain embodiments, different amounts of the base editor are administered to obtain different expression levels of a gene. In certain embodiments, different amounts of the targeted ribonucleic acid and the base editor are administered to obtain different expression levels of a gene.

[0240] The modulation of expression by one or more editing agents can occur within 1 month of administering one or more nucleic acid molecules encoding one or more gene editing agents, e.g., within 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 72 hours, 48 hours, 24 hours, 12 hours, 8 hours, 4 hours, 2 hours of administering one or more nucleic acid molecules encoding one or more editing agents. Exemplary diseases and disorders

[0241] Diseases and disorders that can be treated include lung diseases (e.g., cystic fibrosis), blood disorders (e.g., anemia), CNS diseases and disorders, epilepsy, lysosomal storage diseases (e.g., aspartylglucosaminuria), Rett syndrome, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), type II (I-cell disease), type III (pseudo-Hurler disease), and type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter disease, Sanfilippo syndrome types A, B, C, D, Morquio syndrome types A and B, Maroteaux-Lamy disease, and Sly disease), Niemann-Pick disease types A / B, C1, and C2, and Schindler disease types I and II, hereditary angioedema (HAE), copper or iron storage diseases (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neurodegenerative or neurological disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), and solid organ (e.g., brain, liver, kidney, heart) diseases.

[0242] Synucleinopathies can be treated by the methods according to the present invention. Synucleinopathies are neurodegenerative diseases or disorders characterized by neuronal and / or glial inclusions. Pathologically, synucleinopathies can be divided into two main disease groups: Lewy body diseases or disorders and multiple system atrophy (MSA). Lewy body diseases and disorders are characterized by aggregated α-synuclein and include Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, adult-onset dystonia-Parkinsonism, autosomal recessive early-onset Parkinsonism, POLG-related neurodegeneration, Niemann-Pick disease type C1, and Krabbe disease. (Koga et al. Molecular Neurodegeneration (2021) 16:83).

[0243] Parkinson's disease can be treated by the method according to the present invention. Parkinson's disease is an age-related progressive neurodegenerative disorder. Parkinson's disease is characterized by the abnormal accumulation of misfolded α-synuclein aggregates in various regions of the brain. The loss of dopaminergic neurons in the substantia nigra is a pathological hallmark of Parkinson's disease. (Lee et al. Neuroimmunol. Neuroinflammation (2021) 8:222-44; and Koga et al. Molecular Neurodegeneration (2021) 16:83.)

[0244] Glycogen storage disease type II, also known as Pompe disease, can be treated by the method according to the present invention. Pompe disease is an autosomal recessive disorder caused by mutations in the gene encoding the lysosomal enzyme acid α-glucosidase (GAA) that catalyzes glycogen degradation. The resulting enzyme deficiency leads to the pathological accumulation of glycogen and lysosomal alterations in body tissues, resulting in cardiac, respiratory, and skeletal muscle dysfunction.

[0245] Coagulation disorders that can be treated include hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any of the coagulation factors VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or γ-carboxylase deficiency.

[0246] Other diseases and disorders that can be treated include bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors); or platelet disorders such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0247] Other diseases and disorders that can be treated include proliferative diseases (e.g., cancer, tumors, and dysplasia), Crigler-Najjar and metabolic diseases, such as metabolic diseases of the liver; Friedreich's ataxia; infectious diseases; viral diseases induced by, for example, hepatitis B or C virus, HIV, herpes, and retroviruses; genetic diseases such as cystic fibrosis, dystroglycanopathy, myopathies such as Duchenne myopathy or dystrophy, myotubular myopathy, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubularin myopathy, motor neuron diseases such as spinal muscular atrophy (SMA); spinal bulbar muscular atrophy, or Charcot-Marie-Tooth disease; arthritis; severe combined immunodeficiency, such as RS-SCID, ADA-SCID, or X-SCID; Wiskott-Aldrich syndrome; X-linked thrombocytopenia; X-linked congenital neutropenia; chronic granulomatous disease; coagulation factor deficiencies; cardiovascular diseases, such as restenosis, ischemia, dyslipidemia, and homozygous familial hypercholesterolemia; eye or ocular diseases, such as retinitis pigmentosa, X-linked retinitis pigmentosa, autosomal dominant retinitis pigmentosa, recessive retinitis pigmentosa, choroideremia, choroidal neovascularization, gyrate atrophy, retinoschisis, X-linked retinoschisis, macular degeneration, diabetic macular edema (DME), diabetic retinopathy associated with DME, wet age-related macular degeneration (wet AMD or wAMD), macular edema after retinal vein occlusion, non-arteritic ischemic optic neuropathy, Leber congenital amaurosis, Leber hereditary optic neuropathy, achromatopsia, and Stargardt disease; lysosomal storage diseases, such as Sanfilippo syndrome; hyperbilirubinemia, such as CN type I or II or Gilbert syndrome; glycogen storage diseases, such as GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or lethal congenital cardiac glycogen storage disease.

[0248] In certain embodiments, the subject has a disease or disorder that affects or originates from the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease of the nervous system. Non-limiting examples of CNS or neurodegenerative diseases of the nervous system include Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the disease is a mental illness, addiction (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan's disease, or adrenoleukodystrophy. In certain embodiments, the CNS or neurodegenerative disease of the nervous system is a polyglutamine repeat disease, such as spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0249] In certain embodiments, the subject has a disease or disorder associated with pain, such as chronic pain or neuropathic pain. Gene therapy targets are described in Ovespian and Waxman, Nat Rev Neurosci. April 2023;24(4):252-265, the content of which is incorporated herein by reference in its entirety.

[0250] Throughout this application, many different aspects and embodiments of the invention have been described. However, various changes and modifications can be made to the invention by those skilled in the art without departing from the spirit and scope of the invention to adapt it to various uses and conditions. Embodiments Example A

[0251] 1. A method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject one or more gene editing agents that modify a region of the gene to modulate the expression of the gene, wherein the region of the gene comprises one or more of a promoter, an enhancer, a silencer, or an insulator, a premature stop codon that can be converted to an amino acid codon by modification of the region, or an amino acid codon that can be converted to a premature stop codon by modification of the region.

[0252] 2. The method according to embodiment 1, wherein the one or more gene editing agents comprise a guide RNA complementary to the region of the gene and a Cas protein or a derivative of the Cas protein.

[0253] 3. The method according to embodiment 2, wherein the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9); CpF1; CasX; CasY; C2c1; C2c2; C2c3; or a variant thereof.

[0254] 4. The method according to any one of the foregoing embodiments, wherein the one or more gene editing agents further comprise a donor nucleic acid, the donor nucleic acid having at least one nucleotide change relative to the region of the gene and being capable of integrating into the region of the gene to modify the region.

[0255] 5. The method according to any one of the foregoing embodiments, wherein the one or more gene editing agents are encoded by one or more nucleic acid molecules administered to the subject, preferably the one or more gene editing agents are encoded by RNA molecules (in particular mRNA molecules) administered to the subject.

[0256] 6. The method according to embodiment 5, wherein the one or more nucleic acid molecules (such as one or more mRNA molecules) are administered to the subject using liposomes, lipid nanoparticles (LNP), peptide cages or polymer nanoparticles.

[0257] 7. A method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject a base editor system that effects a base change in a region of the gene or a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0258] 8. The method according to embodiment 7, wherein the region of the gene comprises one or more of a promoter, enhancer, silencer or insulator, or the region of the gene or the region of the mRNA transcript comprises a premature stop codon that can be converted to an amino acid codon by the base change or an amino acid codon that can be converted to a premature stop codon by the base change.

[0259] 9. The method according to embodiment 7 or 8, wherein the base editor system comprises: a ribonucleic acid complementary to the region of the gene; and a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the polynucleotide programmable DNA binding domain binds to the region of the gene together with the ribonucleic acid to effect the base change.

[0260] 10. The method according to embodiment 9, wherein the polynucleotide programmable DNA binding domain comprises a nuclease-inactivated variant of a Cas protein or a nickase variant of a Cas protein.

[0261] 11. The method according to embodiment 10, wherein the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9); CpF1; CasX; CasY; C2c1; C2c2; C2c3; or a variant thereof.

[0262] 12. The method according to any one of embodiments 9-11, wherein the base editor further comprises a uracil-binding protein, such as a uracil glycosylase inhibitor (UGI) domain that inhibits uracil-DNA glycosylase.

[0263] 13. The method according to any one of embodiments 9-12, wherein (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase 1 (ADA1) and ADA2.

[0264] 14. The method according to embodiment 7 or 8, wherein the base editor system comprises: a ribonucleic acid complementary to a region of the mRNA transcript; and a base editor comprising a polynucleotide programmable RNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain, wherein the polynucleotide programmable RNA binding domain binds to the region of the mRNA transcript together with the ribonucleic acid to effect the base change.

[0265] 15. The method according to embodiment 14, wherein the polynucleotide programmable RNA binding domain comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13.

[0266] 16. The method according to embodiment 15, wherein the Cas13 is Cas13a and Cas13b.

[0267] 17. The method according to any one of embodiments 14-16, wherein (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4; activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytosine deaminase 1 (CDA1) or CDA2; or cytosine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0268] 18. The method according to any one of embodiments 9-17, wherein the ribonucleic acid is a guide RNA.

[0269] 19. The method according to any one of embodiments 7-18, wherein the base editor system or its components are encoded by one or more nucleic acid molecules administered to the subject, preferably the ribonucleic acid and the base editor are encoded by one or more RNA molecules (such as one or more mRNA molecules) administered to the subject.

[0270] 20. The method according to embodiment 19, wherein the one or more nucleic acid molecules, such as the one or more mRNA molecules, are administered to the subject using lipid nanoparticles (LNP), peptide cages or polymer nanoparticles.

[0271] 21. The method according to any one of embodiments 7-20, wherein the base change causes the amino acid codon to be converted to the premature termination codon, preferably upstream of the splice junction, thereby downregulating the expression of the gene.

[0272] 22. The method according to embodiment 21, wherein the base change causes the CGA, CAG or TGG codon to be converted to the premature TGA, TAG or TAA termination codon, respectively, and the base editor comprises the cytidine deaminase domain, preferably, the CAG codon is located near the 5' end of the gene.

[0273] 23. The method according to any one of embodiments 7-20, wherein the base change causes the premature termination codon to be converted to an amino acid codon, thereby upregulating the expression of the gene.

[0274] 24. The method according to embodiment 23, wherein the base change causes an early UAG, UAA or UGA stop codon to be respectively converted into CAG, CAA or CGA, and the base editor comprises the adenosine deaminase domain. Preferably, the early UAG stop codon is located near the 5' end of the gene.

[0275] 25. The method according to any one of the preceding claims, the method further comprising administering to the subject an episomal vector comprising the gene.

[0276] 26. The method according to any one of the preceding claims, wherein the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector or an adenoviral vector.

[0277] 27. The method according to embodiment 26, wherein the episomal vector is an AAV vector.

[0278] 28. The method according to any one of the preceding, wherein the subject is a human, such as a human subject suffering from a disease selected from the following: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome 1F, Usher syndrome 1B, glaucoma, Leber congenital amaurosis and Stargardt disease. Example B

[0279] 1. A method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject an editing agent that effects a change in a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

[0280] 2. The method according to embodiment 1, wherein the editing agent effects a base change in a region of the mRNA transcript of the gene.

[0281] 3. The method according to embodiment 1, wherein the change in the region of the mRNA transcript of the gene alters the stability of the mRNA transcript, the translation initiation or level of the mRNA transcript, the stability and / or activity of the translated protein.

[0282] 4. The method according to embodiment 2, wherein the region of the mRNA transcript comprises a premature stop codon that can be converted into an amino acid codon by the base change or an amino acid codon that can be converted into a premature stop codon by the base change.

[0283] 5. The method according to embodiment 2, wherein the base change (a) is within a microRNA target site or a toehold switch site, or (b) induces ribosomal frameshifting or alters a codon encoding an amino acid residue that is crucial for the function and / or structure of the encoded protein.

[0284] 6. The method according to any one of embodiments 2-5, wherein the editing agent comprises a targeting ribonucleic acid that is complementary to a region of the mRNA transcript.

[0285] 7. The method according to embodiment 6, wherein the targeting ribonucleic acid is linear.

[0286] 8. The method according to embodiment 6, wherein the targeting ribonucleic acid is circular.

[0287] 9. The method according to any one of embodiments 6-8, wherein the targeting ribonucleic acid effects the base change by binding to an endogenous adenosine deaminase domain.

[0288] 10. The method according to embodiment 9, wherein the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, and ADAR3.

[0289] 11. The method according to any one of embodiments 6-10, wherein the editing agent further comprises: a base editor comprising a polynucleotide programmable RNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, or a nucleic acid encoding the base editor, wherein the polynucleotide programmable RNA-binding domain effects the base change together with the targeting ribonucleic acid.

[0290] 12. The method according to embodiment 11, wherein the polynucleotide programmable RNA-binding domain comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13.

[0291] 13. The method according to embodiment 12, wherein the Cas13 is Cas13a or Cas13b.

[0292] 14. The method according to any one of embodiments 11 - 13, wherein (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide - like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4; activation - induced cytidine deaminase (AID), such as activation - induced cytidine deaminase (AICDA); cytosine deaminase 1 (CDA1) or CDA2; or cytosine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA - specific adenosine deaminase (TadA).

[0293] 15. The method according to any one of embodiments 6 - 14, wherein the targeted ribonucleic acid is a guide RNA or a trigger RNA.

[0294] 16. The method according to any one of embodiments 11 - 14, wherein the base editor or the targeted ribonucleic acid is encoded by one or more nucleic acid molecules administered to the subject, preferably the base editor is encoded by one or more RNA molecules (such as one or more mRNA molecules) administered to the subject.

[0295] 17. The method according to embodiment 16, wherein the targeted ribonucleic acid and / or the one or more nucleic acid molecules such as the one or more mRNA molecules are administered to the subject using lipid nanoparticles (LNP), peptide cages or polymer nanoparticles.

[0296] 18. The method according to any one of claims 2 - 17, wherein the base change results in the conversion of an amino acid codon to a premature termination codon, preferably upstream of the splice junction, thereby down - regulating the expression of the gene.

[0297] 19. The method according to embodiment 18, wherein the base change results in the conversion of the CGA, CAG or TGG codons to premature TGA, TAG or TAA termination codons respectively, and the base editor comprises the cytidine deaminase domain, preferably, the CAG codon is near the 5' end of the gene.

[0298] 20. The method according to any one of embodiments 2 - 17, wherein the base change results in the conversion of a premature termination codon to an amino acid codon, thereby up - regulating the expression of the gene.

[0299] 21. The method according to embodiment 20, wherein the base change causes the premature UAG, UAA or UGA stop codon to be respectively converted to CAG, CAA or CGA, and the base editor comprises the adenosine deaminase domain. Preferably, the premature UAG stop codon is located near the 5' end of the gene.

[0300] 22. The method according to any one of the foregoing embodiments, wherein different amounts of the editing agent, such as different amounts of the targeted ribonucleic acid, are administered to the subject to obtain different expression levels of the gene.

[0301] 23. The method according to any one of the foregoing embodiments, the method further comprising administering to the subject an episomal vector comprising the gene.

[0302] 24. The method according to any one of the foregoing embodiments, wherein the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector or an adenovirus vector.

[0303] 25. The method according to embodiment 24, wherein the episomal vector is an AAV vector.

[0304] 26. The method according to any one of the foregoing embodiments, wherein the subject is a human, such as a human subject suffering from a disease selected from the following: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington's disease, Parkinson's disease, Alzheimer's disease, synucleinopathy, epilepsy, neuropathic pain, wet age-related macular degeneration, Usher syndrome 1F, Usher syndrome 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease. Examples

[0305] Examples are provided below that further illustrate the different features of the present invention and the methods of practicing the present invention. The provided examples do not limit the claimed invention. Example 1. In vivo administration of base editor to reduce expression levels

[0306] Recombinant AAV (rAAV) particles expressing human factor IX (FIX) were administered to mice (Balb / c; Jackson Laboratory) by intravenous administration at 5e10 vg / mouse and 5e9 vg / mouse to achieve steady-state serum levels of FIX expression of approximately 50,000 ng / ml and 5,000 ng / ml, respectively, 4 weeks after administration. The expression cassette was packaged in AAV viral particles by encapsidation into the AAV capsid. Viral particles were typically produced using a triple transfection protocol.

[0307] At week 5 after administration, cytosine base editor (CBE) and gRNA targeting the glutamine codon in FIX exon 1 were administered, which converted the CAG codon into a premature UAG stop codon. The CBE mRNA and gRNA were formulated in a single LNP for administration. Mice were divided into 5 groups and administered 0.25 mpk, 0.5 mpk, 1.0 mpk, 2.0 mpk of mRNA / gRNA LNP, or 2.0 mpk of control mRNA / gRNA LNP. One day before administration by intravenous injection, the first plasma samples were collected from the mice by serial sampling. In all groups, samples were collected at 4 hours, 24 hours, 72 hours, and 1 week after administration. A total of 80 μL of whole blood was drawn by orbital bleeding. The blood was transferred to lithium heparin tubes and centrifuged at 9,800 x g for 10 minutes at 2°C - 5°C. After collection, the samples were frozen and stored at ≤ -70°C for further analysis. The transgenic hFIX protein in blood plasma was measured by ELISA. Example 2. In vivo administration of base editor to increase expression levels

[0308] A non-expressing variant form of recombinant AAV (rAAV) particles expressing human factor IX (FIX) was administered to mice (Balb / c; Jackson Laboratory) by intravenous injection. The variant form included a UAG premature termination codon (PTC) in FIX exon 1. The expression cassette was packaged in AAV viral particles by encapsidation into the AAV capsid. Viral particles were typically produced using a triple transfection protocol.

[0309] At week 5 after administration, adenine base editor (ABE) and gRNA targeting the FIX exon 1 PTC codon were administered, which converted the UAG PTC codon into a glutamine CAG codon. The ABE mRNA and gRNA were formulated in a single LNP for administration. Mice were divided into 5 groups and administered 0.25 mpk, 0.5 mpk, 1.0 mpk, 2.0 mpk of mRNA / gRNA LNP, or 2.0 mpk of control mRNA / gRNA LNP. One day before administration by intravenous injection, the first plasma samples were collected from the mice by serial sampling. In all groups, samples were collected at 4 hours, 24 hours, 72 hours, and 1 week after administration. A total of 80 μL of whole blood was drawn by orbital bleeding. The blood was transferred to lithium heparin tubes and centrifuged at 9,800 x g for 10 minutes at 2°C - 5°C. After collection, the samples were frozen and stored at ≤ -70°C for further analysis. The transgenic hFIX protein in blood plasma was measured by ELISA. Example 3. ADAR-based RNA editing of AAV-delivered transgene

[0310] Editing of early termination codons in mRNA transcripts permits mRNA translation and protein expression of exogenously delivered transgenes. A human factor IX (FIX) (also referred to herein as FIX40) transgene construct (FIX40_W118STOP) was generated that has a G to A point mutation in the TGG codon of tryptophan at amino acid position 118 of the FIX sequence (SEQ ID NO: 53), converting it to an early or premature TAG stop codon ( Figure 1A ). In a Huh7 cell transfection experiment, the point mutation completely abolished the expression of the FIX40 protein, as demonstrated by WES TM automated capillary-based immunoassay analysis (ProteinSimple, Bio-Techne)( Figure 1B ) and enzyme-linked immunosorbent assay (ELISA) (Figure 1C).

[0311] The ability of endogenous ADAR to perform A-to-I editing on FIX40_W118STOP mRNA was tested in Huh7 cells. Briefly, co-transfection experiments of plasmids carrying the FIX40_W118STOP construct and trigger RNAs resulted in FIX expression, as detected by WES( Figure 1B ) and ELISA (Figure 1C) at 48 hours, reaching between 30% - 45% of the FIX levels from the wild-type FIX expression construct, depending on the FIX construct and trigger RNA concentrations. The trigger RNAs were driven by pol3 from a human U6 promoter plasmid. The trigger RNAs were 200 nucleotides long and self-circularized, and were either fully complementary to the mRNA target (cadRNA; SEQ ID NO: 54) or carried scattered mismatches (cadRNAis; SEQ ID NO: 55). Without wishing to be bound by any theory, the scattered mismatches may contribute to a reduction in bystander (off-target) editing. Collectively, these results demonstrate that transgene expression can be switched on in vitro by ADAR-based RNA editing.

[0312] To determine whether the introduction of the stop codon had any effect on mRNA abundance and to determine the efficiency of the RNA editing event at the molecular level, RNA was extracted from transfected Huh7 cells. Total FIX40_W118STOP mRNA (FIX40 mRNA) was quantified by qPCR. Quantification of FIX40 mRNA expressed as copy number showed that the introduction of the stop codon resulted in a ~35% decrease in the level of FIX40 mRNA compared to the mRNA from the wild-type FIX expression construct ( Figure 2, lane 1 relative to lane 6). Without wishing to be bound by any theory, the decrease in FIX40 mRNA levels may be driven by nonsense-mediated decay. In addition, comparison of protein levels and mRNA abundance in base-edited samples shows a strong correlation between the measured values ( Figure 1B and Figure 1C relative to Figure 2 , lanes 2-5). Finally, Sanger sequencing of the mRNA shows that 100% of the mRNA molecules have been edited from TAG to TGG at position W118 (not shown), demonstrating that no off-target editing has occurred. These results are consistent with the high correlation observed between protein and mRNA levels. Conclusion

[0313] The results indicate that endogenous ADAR can be used as a switch to control the expression of exogenously delivered transgenes, which can be applied to gene therapy. These in vitro experiments demonstrate that the trigger RNA specifically targets ADAR to edit the stop codon in each mRNA molecule produced from the delivered transgene to allow protein expression. Example 4. In vivo study in mice

[0314] To demonstrate the functionality of the ADAR-mediated base editing system in animals, an in vivo mouse study was designed using recombinant AAV (rAAV) particles to deliver the FIX expression cassette payload. AAV-encapsulated FIX40 or AAV-encapsulated FIX40_W118STOP (Table 4) was administered intravenously (tail vein) to mice. To generate non-limiting levels of FIX40_W118STOP mRNA, low and high doses of rAAV were tested. Table 4.

[0315] In animals administered at 5e9 or 5e10 vg / animal, the expression of FIX40 in plasma was measured by ELISA on days 0, 7, and 14. No FIX40 expression was detected in animals administered FIX40_W118STOP (Table 5; BQL: below the limit of detection). Expression of the control FIX40 construct showed an expected dose response and time-dependent increase from day 7 to day 14. For 3 animals in each group, the values are expressed as mean ng / ml. Table 5.

[0316] After establishing the basal level of FIX40 expression in each group, RNA was triggered by LNP delivery. Based on in vitro results, the addition of circular trigger RNA was expected to recruit endogenous ADAR to edit the W118STOP codon to the TGG tryptophan codon. Plasma FIX40 expression levels were measured daily for 4 weeks to determine editing efficiency and editing persistence. At the end of the study, animals were sacrificed and liver RNA was extracted to determine FIX40 RNA levels and editing efficiency. Example 5. In vivo study to determine the editing efficiency and persistence induced by FIX40W118STOP via the ADAR-cadRNAis complex

[0317] In the follow-up study, animals were dosed with the rAAV vector on day 0 and with LNP on day 14. FIX40 levels were measured daily to determine the ability of the triggered cadRNAis and endogenous or exogenous ADAR to edit FIX40W118STOP mRNA and allow protein expression. Table 6.

[0318] Although the invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adjustments, changes, modifications, substitutions, deletions or additions can be made to the procedures and protocols without departing from the spirit and scope of the invention.

Claims

1. A method of modulating the expression of a gene located on an episomal vector in a subject in need thereof, the method comprising administering to the subject an editing agent that effects a change in a region of the mRNA transcript of the gene, thereby modulating the expression of the gene.

2. The method according to claim 1, wherein the editing agent effects a base change in a region of the mRNA transcript of the gene.

3. The method according to claim 1, wherein the change in the region of the mRNA transcript of the gene alters the stability of the mRNA transcript, the translation initiation or level of the mRNA transcript, the stability and / or activity of the translated protein.

4. The method according to claim 2, wherein the region of the mRNA transcript comprises a premature termination codon that can be converted to an amino acid codon by the base change or an amino acid codon that can be converted to a premature termination codon by the base change.

5. The method according to claim 2, wherein the base change (a) is within a microRNA target site or a toehold switch site, or (b) induces ribosomal frameshifting or alters a codon encoding an amino acid residue critical for the function and / or structure of the encoded protein.

6. The method according to any one of claims 2-5, wherein the editing agent comprises a targeting ribonucleic acid complementary to the region of the mRNA transcript.

7. The method according to claim 6, wherein the targeting ribonucleic acid is linear.

8. The method according to claim 6, wherein the targeting ribonucleic acid is circular.

9. The method according to any one of claims 6-8, wherein the targeting ribonucleic acid effects the base change by binding to an endogenous adenosine deaminase domain.

10. The method according to claim 9, wherein the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, and ADAR3.

11. The method according to any one of claims 6-10, wherein the editing agent further comprises: a base editor comprising a polynucleotide programmable RNA-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, or a nucleic acid encoding the base editor, wherein the polynucleotide programmable RNA-binding domain effects the base change together with the targeting ribonucleic acid.

12. The method according to claim 11, wherein the polynucleotide programmable RNA-binding domain comprises a nuclease-inactivated variant of Cas13 or a nickase variant of Cas13.

13. The method according to claim 12, wherein the Cas13 is Cas13a or Cas13b.

14. The method according to any one of claims 11 - 13, wherein (i) the cytidine deaminase domain is selected from the apolipoprotein B mRNA editing enzyme catalytic polypeptide - like (APOBEC) deaminase family, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H or APOBEC4; activation - induced cytidine deaminase (AID), such as activation - induced cytidine deaminase (AICDA); cytidine deaminase 1 (CDA1) or CDA2; or cytidine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA - specific adenosine deaminase (TadA).

15. The method according to any one of claims 6 - 14, wherein the target ribonucleic acid is a guide RNA or a trigger RNA.

16. The method according to any one of claims 11 - 14, wherein the base editor or the target ribonucleic acid is encoded by one or more nucleic acid molecules administered to the subject, preferably the base editor is encoded by one or more RNA molecules, such as one or more mRNA molecules, administered to the subject.

17. The method according to claim 16, wherein the target ribonucleic acid and / or the one or more nucleic acid molecules, such as the one or more mRNA molecules, are administered to the subject using lipid nanoparticles (LNP), peptide cages or polymer nanoparticles.

18. The method according to any one of claims 2 - 17, wherein the base change results in the conversion of an amino acid codon into a premature termination codon, preferably upstream of the splice junction, thereby down - regulating the expression of the gene.

19. The method according to claim 18, wherein the base change results in the conversion of the CGA, CAG or TGG codon into the premature TGA, TAG or TAA termination codon respectively, and the base editor comprises the cytidine deaminase domain, preferably, the CAG codon is located near the 5' end of the gene.

20. The method according to any one of claims 2 - 17, wherein the base change results in the conversion of a premature termination codon into an amino acid codon, thereby up - regulating the expression of the gene.

21. The method according to claim 20, wherein the base change results in the conversion of the premature UAG, UAA or UGA termination codon into CAG, CAA or CGA respectively, and the base editor comprises the adenosine deaminase domain, preferably, the premature UAG termination codon is located near the 5' end of the gene.

22. The method according to any one of the preceding claims, wherein different amounts of the editing agent, such as different amounts of the targeted ribonucleic acid, are administered to the subject to obtain different expression levels of the gene.

23. The method according to any one of the preceding claims, the method further comprising administering to the subject an episomal vector comprising the gene.

24. The method according to any one of the preceding claims, wherein the episomal vector is a non-viral vector, such as a plasmid; or a viral vector, such as an adeno-associated virus (AAV) vector or an adenovirus vector.

25. The method according to claim 24, wherein the episomal vector is an AAV vector.

26. The method according to any one of the preceding claims, wherein the subject is a human, such as a human subject suffering from a disease selected from the following: hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, Huntington disease, Parkinson's disease, Alzheimer's disease, synucleinopathy, epilepsy, neuropathic pain, wet age-related macular degeneration, Usher syndrome 1F, Usher syndrome 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease.

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