Gene editing for controlled expression of episomal genes

Abstract

Disclosed herein are methods for regulating the expression of a gene located in an episomal vector in a subject in need thereof. In particular, the method includes administering to the subject one or more gene editing agents that modify a region of the gene, thereby regulating the expression of the gene. Also disclosed are methods for administering to the subject a base editor system that causes a base change in a region of the gene or in a region of the mRNA transcript of the gene, thereby regulating the expression of the gene.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 379,512, filed October 14, 2022, the disclosure of which is incorporated herein by reference.

[0002] [Reference to electronically submitted sequence listing]

[0002] This application contains a sequence listing that has been submitted electronically. The contents of the electronic sequence listing (065830-15WO1 Sequence Listing.xml; size: 129 KB; and creation date: October 9, 2023) are incorporated herein by reference in their entirety.

[0003]

[0003] The present invention relates to the field of gene therapy. In particular, the present invention relates to the control of gene expression from episomal vectors by utilizing gene editing agents (e.g., base editing systems, etc.). [Background technology]

[0004]

[0004] Gene therapy, in its current design, is an irreversible process. It cannot be discontinued if unintended side effects occur, and the expression level of the therapeutic drug cannot be tailored to individual patient needs. Adeno-associated virus (AAV) vector-mediated gene therapy has great potential for future medical applications. However, to be safer and more widely applicable, and to enable patient-centered care, the expression of therapeutic proteins should be controllable. For example, it has been shown that gene overexpression in certain diseases can be toxic (Payne, Mol Ther Methods Clin Dev. 2022 May 4: 25: 1-2; Palmieri et al., Front Neurosci. 2023 May 25: 17: 1172805). Conversely, in other diseases, such as Huntington's Disease, excessive gene silencing can be toxic (Jung et al., Hum Mol Genet. 2021 April 26; 30(3-4):135-148; Wang et al., Proc Natl Acad Sci USA. 2016 May 22; 113(12):3359-64; Murthy et al., PLoS Genet. 2019 March; 15(3):e1007765). In other cases, the current risk-benefit profile for AAV gene therapy has proven to be low. For example, in diseases for which treatments exist or in diseases with relatively better patient outcomes or less severe consequences (Evan et al., Curr Opin Rheumatol. 2023 Jan. 1; Vol. 35(Issue 1): 37-43; Ishikawa et al., Circ Res. 2018 Aug. 17; Vol. 123(Issue 5): 601-613). Thus, there is a need for methods to regulate gene therapy in such diseases.

[0005]

[0005] Base editing is a genome editing method that generates precise point mutations directly in genomic DNA or cellular RNA without directly generating double-strand breaks (DSBs), without requiring a DNA donor template, or without relying on cellular homologous sequence-dependent repair. Because base editors typically do not create DSBs, they minimize the formation of DSB-related by-products (Komor, AC 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, HA 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 generally fusions consisting of a Cas ("CRISPR-associated") domain and a nucleobase-modifying domain. Two main types of base editors have been developed and are widely used. The first type includes cytosine base editors (CBEs), which were first reported in 2016 (Komor et al., 2016; (2016) Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods, Vol. 13, pp. 1029-1035; Nishida et al., (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, Vol. 353, aaf8729).The second type includes adenine base editors (ABEs), which were first described in 2017 (Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, Vol. 551, pp. 464-471). Both CBEs and ABEs are based on the CRISPR-Cas9 system and utilize cytidine deaminase and adenine deaminase to induce C-to-T and A-to-G base transitions, respectively, within the editing window. CBE can convert four codons (CGA, CAG, GAG, GAA, and TGG) into stop codons (TGA, TAG, and TAA) (Kuscu et al., (2017) CRISPR-STOP: Gene silencing through base-editing-induced nonsense mutations. Nat. Methods, Vol. 14, pp. 710-712; Molla and Yang, (2019) CRISPR / Cas-mediated base editing: technical considerations and practical applications. Trends Biotechnol., Vol. 37, pp. 1121-1142). Therefore, CBE can be used to knock out protein-coding genes by introducing premature stop codons.

[0006]

[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]

[0007] The present disclosure provides a method for regulating expression of a gene located on an episomal vector in a subject in need thereof, comprising the step of administering to the subject one or more gene editing agents that modify a region of the gene, thereby regulating 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 via modification of the region, or an amino acid codon that can be converted to a premature stop codon via modification of the region.

[0008]

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

[0009]

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

[0010]

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

[0011]

[0011] In certain embodiments, 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, particularly mRNA molecules, administered to the subject.

[0012]

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

[0013]

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

[0014]

[0014] In certain embodiments, the base editor system comprises (a) a ribonucleic acid complementary to a region of a 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 in conjunction with the ribonucleic acid to cause a base change.

[0015]

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

[0016]

[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]

[0017] In certain embodiments, (i) the cytidine deaminase domain is selected from the group consisting of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, 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 the group consisting of adenosine deaminase 1 (ADA1) and ADA2.

[0018]

[0018] In certain embodiments, the base editor system includes (a) a ribonucleic acid complementary to a region of an 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 associates with the ribonucleic acid to bind to the region of the mRNA transcript, thereby causing a base change.

[0019] In certain embodiments, the polynucleotide-programmable RNA-binding domain comprises a nuclease-inactive variant of Cas13 or a nickase variant of Cas13. In particular embodiments, the 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) family of deaminases, 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 the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0021]

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

[0022]

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

[0023]

[0023] In certain embodiments, one or more nucleic acid molecules, such as one or more mRNA molecules, are administered to a subject in conjunction with lipid nanoparticles (LNPs), peptide cages, or polymer nanoparticles.

[0024]

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

[0025]

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

[0026]

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

[0027]

[0027] In certain embodiments, the base change results in a 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, and preferably the premature UAG stop codon is located near the 5' end of the gene.

[0028]

[0028] In certain embodiments, the method 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 lentivirus vector, or an adenovirus vector. In certain embodiments, the episomal vector is an AAV vector.

[0029]

[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 macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber's congenital amaurosis, and Stargardt disease.

[0030]

[0030] Also provided is a method for regulating expression of a gene located in an episomal vector in a subject in need thereof, comprising the step of administering to the subject an editing agent that causes a change in a region of the mRNA transcript of the gene, thereby regulating expression of the gene.

[0031]

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

[0032]

[0032] In certain embodiments, the alteration 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]

[0033] In certain embodiments, the region of the mRNA transcript includes a premature stop codon that is convertible to an amino acid codon via a base change or an amino acid codon that is convertible to a premature stop codon via a base change.

[0034]

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

[0035]

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

[0036]

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

[0037]

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

[0038]

[0038] In certain embodiments, the targeted ribonucleic acid induces a base change via binding to an endogenous adenosine deaminase domain.

[0039]

[0039] In certain embodiments, the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, and ADAR3.

[0040]

[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 a basic editor, wherein the polynucleotide-programmable RNA-binding domain associates with a targeted ribonucleic acid to cause a base change.

[0041]

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

[0042]

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

[0043] In certain embodiments, (i) the cytidine deaminase domain is selected from the group consisting of an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; an 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 the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0044]

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

[0045]

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

[0046]

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

[0047]

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

[0048]

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

[0049]

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

[0050]

[0050] In certain embodiments, the base change results in a 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, and preferably the premature UAG stop codon is located near the 5' end of the gene.

[0051]

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

[0052]

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

[0053]

[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]

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

[0055]

[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, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber's congenital amaurosis, and Stargardt disease. [Brief explanation of the drawings]

[0056] [Figure 1A] Diagram showing ADAR-driven 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 tyrosine 118 (W118) of FIX40, and a targeting ribonucleic acid ("trigger RNA") targets the ADAR to back-edit TAG (stop) to TGG (tyrosine). [Figure 1B] Figure 1 shows FIX40 protein levels measured by WES™ automated capillary-based immunoassay (ProteinSimple, Bio-Techne). Trigger RNA (CadRNA or CadRNAis) was added at two different concentrations (250 or 100 ng). CadRNAs perfectly match the target mRNA, while cadRNAis contain mismatches in the interspersed loops surrounding the editing site. [Figure 1C] FIG. 1C shows FIX40 protein levels in the same samples as in FIG. 1B, as measured by ELISA. [Figure 2] FIG. 1 shows total mRNA levels for all constructs obtained by quantitative polymerase chain reaction (qPCR) using primer probe sets specific for the FIX sequence. DETAILED DESCRIPTION OF THE INVENTION

[0057]

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

[0058]

[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 pertains. Unless otherwise defined, certain terms referred to herein have the meanings defined herein.

[0059]

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

[0060]

[0063] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to refer to the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be substituted for the terms "containing" or "including," and, occasionally, when used herein, with the term "having."

[0061]

[0064] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element, if such element, step, or ingredient is relevant to the claimed invention. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the above terms "comprising," "containing," "including," and "having," when used herein in the context of aspects or embodiments of the invention, may be substituted with the terms "consisting of" or "consisting essentially of" to modify the scope of the disclosure.

[0062]

[0065] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and joint alternatives. For example, when two elements are connected by "and / or," the first alternative indicates that the first element applies when the second element is not present. The second alternative indicates that the second element applies when the first element is not present. The third alternative indicates that both the first and second elements apply. Any one of these alternatives is understood to fall within the meaning and thus satisfy the requirements of the term "and / or" as used herein. Two or more alternatives that apply simultaneously are also understood to fall within the meaning and thus satisfy the requirements of the term "and / or."

[0063]

[0066] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose.

[0064]

[0067] The term "about," as used herein, refers to a numerical value within 10% (i.e., ±10%) of the underlying parameter. 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 series of numerical values ​​modifies each of the numerical values ​​by 10%.

[0065]

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

[0066]

[0069] Furthermore, when a numerical range is stated, such as "0.01 to 10," it includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. (the same applies below). For example, a dosage of about "0.01 mg / kg to about 10 mg / kg" per subject body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc. (the same applies below).

[0067]

[0070] When a greater or lesser number is followed by an integer, it includes any number greater or less than the referenced number, respectively. Thus, for example, more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., and so forth. For example, "two or more" administrations of a non-viral vector and / or immune cell modulator include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more administrations.

[0068]

[0071] Furthermore, when a numerical range is stated, such as "1 to 90," it includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc. (the same applies below). For example, "about 1 minute to about 90 days" includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days, ..., 81 days, 82 days, 83 days, 84 days, 85 days, etc. (the same applies below).

[0069]

[0072] To assist the reader in understanding this application, the description has been divided into various paragraphs or sections, or has been described in relation to certain specific embodiments of the present invention. Such division should not be considered to separate the substance of one paragraph, section, or embodiment from the substance of another paragraph, section, or embodiment. To the contrary, those skilled in the art will understand that the description has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that may be subject to discussion. Any discussion of any embodiment is intended to be illustrative only and is not intended to suggest that the scope of the present disclosure, including the claims, is limited to these examples.

[0070]

[0073] Provided herein is a method for regulating the expression of a gene located in an episomal vector in a subject in need thereof, comprising administering to the subject one or more gene editing agents that modify a region of the gene, thereby regulating the expression of the gene. Also provided herein is a method for regulating the expression of a gene located in an episomal vector in a subject in need thereof, comprising administering to the subject an editing agent that causes a change in a region of the mRNA transcript of the gene, thereby regulating the expression of the gene.

[0071]

[0074] The methods of the present invention modulate the expression of a transgene provided by gene therapy. Modulation of gene therapy transgene expression 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.

[0072]

[0075] For example, coding mutations may target catalytic serine, lysine, arginine, or histidine residues within the active site of the enzyme; altering lysine acetylation or ubiquitination sites (lysine to glutamic acid or alanine substitutions); substituting serine or threonine phosphorylation sites (threonine or serine to alanine), substituting asparagine-linked ("N-linked") glycosylation sites (asparagine to aspartic acid); or altering the lipid-binding domain by substituting histidine, lysine, or arginine residues important for binding to the lipid head group in phosphoinositides (mutation to a non-polar or negatively charged residue).

[0073]

[0076] In certain embodiments, an alteration in a region of the mRNA transcript of a 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.

[0074]

[0077] The term "vector" or "expression vector," as used herein, refers to a vector, particularly an episomal vector. A vector is generally a plasmid used to introduce and express a specific gene into a target cell. An expression vector allows for the production of large amounts of stable mRNA. Once the expression vector is transferred inside the cell, the cell's transcription and translation machinery produces the protein encoded by the gene. The plasmid is engineered to contain a highly active promoter that causes large amounts of mRNA to be produced. An "episomal vector" has the ability to autonomously replicate 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 lentivirus vector, or an adenovirus vector.

[0075]

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

[0076]

[0079] The term "alteration," as used herein, refers to a change in 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 more.

[0077]

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

[0078]

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

[0079]

[0082] In certain embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA, (poly- and oligonucleotide), 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-splicing RNA, or antisense RNA), locked nucleic acid analogs (LNA), single and double stranded oligonucleotide DNA (ODN), immunostimulatory sequences (ISS), riboswitches, and ribozymes.

[0080]

[0083] The term "mRNA" or sometimes "mRNA transcript," as used herein, includes, but is not limited to, pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) that are readily translated, and transcripts of a gene or genes, or nucleic acids derived from mRNA transcript(s).

[0081]

[0084] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein to refer to nitrogenous biological compounds that form nucleosides and, thus, are components of nucleotides. The ability of nucleobases to base pair and stack one base on another directly leads to 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 primary or canonical bases. Adenine and guanine are derived from purines, and cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain modified or other (non-primary) bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be created in the presence of mutagens, both via deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from the deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (either ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides having 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 (either ribose or deoxyribose), and at least one phosphate group.

[0082]

[0085] The terms "identity," "homology," and grammatical variations thereof mean that two or more referenced entities are identical when their sequences are "aligned." Thus, for example, when two nucleic acids are identical, they have the same sequence at least in the referenced region or portion. Identity is considered to cover a defined area (region or domain) of the sequence.

[0083]

[0086] An "area" or "region" of identity refers to a portion of two or more referenced entities that is identical. Thus, if two protein or nucleic acid sequences are identical over one or more sequence areas or regions, they share identity within that region. An "aligned" sequence refers to two or more protein (amino acid) or nucleic acid sequences (often including corrections for missing or added bases or amino acids (gaps) compared to a reference sequence).

[0084]

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

[0085]

[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 may also include enhancer elements, consisting of proximal elements and more distal upstream elements.

[0086]

[0089] As used herein, the term "enhancer" refers to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements are generally located upstream of, but function at, a promoter element and may also be located downstream 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, upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements generally increase expression of an operably linked nucleic acid conferred by a promoter element.

[0087]

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

[0088]

[0091] As used herein, the term "insulator" or "insulating sequence" refers to a type of cis-regulatory element known as a long-range regulatory element. An insulating sequence is a segment of DNA that blocks the interaction or interference of adjacent gene sequences. For example, an insulator can reduce transcription readthrough through the promoter of an adjacent gene or a spurious promoter within an adjacent nucleotide sequence. Alternatively, an insulator can block an enhancer on one side of the insulating sequence from interacting with the promoter of an adjacent gene on the other side of the insulating sequence. A given characteristic of an insulating sequence as intended by the present invention is its ability to insulate or protect a defined transcription unit operably linked to a regulatory element from the influence of interfering genetic elements located upstream or downstream. To this end, the insulating sequence is placed between a (potentially) interfering gene sequence and the regulatory sequence of the transcription unit to be insulated.

[0089]

[0092] As used herein, the term "stop codon" (also called a termination codon) is a nucleotide triplet in a messenger RNA that signals the end of translation and is different from most codons in a messenger RNA that correspond to the addition of an amino acid residue to a polypeptide chain during elongation. Thus, the term "premature stop codon" or "premature termination codon" refers to the occurrence of a stop codon in place of a codon that corresponds to an amino acid residue. A premature termination codon can be located anywhere upstream relative to the normal termination codon (usually located at the end of the coding nucleic acid sequence of a particular gene). A premature termination codon can be any one of the known termination codons, including TAG (transcribed as UAG), TAA (transcribed as UAA), and TGA (transcribed as UGA).

[0090] 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 a gene, thereby regulating the expression of the gene. Gene editing agents include agents that can modify the expression of a gene by targeting the genome of a cell. The term "gene editing agent" as used herein encompasses gene editing agents that induce mutations by cleaving targeted DNA (e.g., via homologous sequence-dependent repair or non-homologous end joining).

[0091]

[0094] The term "mutation" as used herein refers to the substitution of a residue for another residue in a sequence, such as a nucleic acid sequence or an amino acid sequence, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue in the sequence, followed by the position of that residue, and by identifying the newly substituted residue. Various methods for making the amino acid substitutions (mutations) presented herein are well known in the art and are presented, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0092]

[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 functionally bind to any desired DNA sequence. Exemplary methods of genome editing using the TALEN system can be found, for example, in Cermak T. Doyle EL. 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 GM. 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, all of which are specifically incorporated by reference. By way of further guidance, and without limitation, naturally occurring TALEs or "wild-type TALEs" are nucleic acid-binding proteins secreted by numerous Proteobacterial species. TALE polypeptides are primarily 33, 34, or 35 amino acids in length and contain nucleic acid-binding domains composed of tandem repeats of highly conserved monomeric polypeptides that differ from each other primarily 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" is used to refer to the highly conserved, repetitive polypeptide sequence within a TALE nucleic acid-binding domain, and the term "repeated variable diresidue" or "RVD" is used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer.When presented throughout this disclosure, the amino acid residues of the RVD are represented using the IUPAC single letter code for amino acids. A common representation of a TALE monomer contained within a DNA binding domain is X. l ~ 1l -(X 12 X 13 )-X 14 ~ 33又は34又は35 where the subscripts represent amino acid positions and X represents any amino acid. 12 X 13 represents the RVD. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such polypeptide monomers, the RVD consists of a single amino acid. In such cases, the RVD is represented by X * where X represents X12 and ( * ) indicates the absence of X13. The DNA binding domain contains several repeats of TALE monomers, which are (X 1~11 -(X 12 X 13 )-X 14~33又は34又は35 ) zwhere z is at least 5-40 in advantageous embodiments. In even more advantageous embodiments, z is at least 10-26. TALE monomers have nucleotide binding affinity determined by the identity of the amino acids within their RVDs. For example, a polypeptide monomer with an RVD of NI preferentially binds adenine (A), a polypeptide monomer with an RVD of NG preferentially binds thymine (T), a polypeptide monomer with an RVD of HD preferentially binds cytosine (C), and a polypeptide monomer with an RVD of NN preferentially binds both adenine (A) and guanine (G). In yet another embodiment of the invention, a polypeptide monomer with an RVD of IG preferentially binds T. Thus, the number and order of polypeptide monomer repeats present within the nucleic acid binding domain of a TALE determine its nucleic acid target specificity. In yet a further embodiment of the invention, a polypeptide monomer with an RVD of NS can recognize all four base pairs and bind A, T, G, or C. The structure and function of TALEs are further described, for example, in Moscour 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 effected by a polynucleic acid-binding TALEN fragment. In certain embodiments, the targeting domain comprises or consists of a catalytically inactive TALEN or a nucleic acid-binding fragment thereof.

[0093]

[0096] In certain embodiments, the targeting domain comprises or consists of (modified) zinc finger nuclease (ZFN) system.ZFN system uses artificial restriction enzymes, which are created by fusing zinc finger DNA binding domains with DNA cleavage domains (which can be engineered to target desired DNA sequences).Exemplary methods of genome editing using ZFN are described 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, 7,013,21 9, 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, all of which are specifically incorporated by reference. Further guidance, but not limited to, artificial zinc finger (ZF) technology involves an array of ZF modules that target novel DNA binding sites in the genome. Each finger module in the ZF array targets three DNA bases. The customized array of individual zinc finger domains is assembled into a ZF protein (ZFP). The ZFP can include 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, YG et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. USA, vol. 91, pp. 883-887; Kim, YG et al., 1996, Hybrid restriction enzymes: zinc finger fusions to the FokI cleavage domain, Proc. Natl. Acad. Sci. USA, vol. 93, pp. 1156-1160).Increased cleavage specificity can be achieved by using paired ZFN heterodimers (each targeting a different nucleotide sequence separated by a short spacer) to reduce off-target activity. (Doyon, Y. et al., 2011, Enhancing zinc-finger nuclease activity with improved obligate heterodimeric architectures. Nat. Methods, Vol. 8, pp. 74-79.) ZFPs can also be engineered as transcriptional activators and repressors and have been used to target many genes in a wide 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 zinc finger nuclease (or fragment thereof) is catalytically inactive.

[0094]

[0097] In certain embodiments, the targeting domain comprises a (modified) meganuclease, an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence consisting of 12 to 40 base pairs). Exemplary methods for 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 provided by a polynucleic acid-binding meganuclease fragment. In certain embodiments, targeting is provided by a polynucleic acid-binding, catalytically inactive meganuclease (fragment). Thus, in particular embodiments, the targeting domain comprises or consists of a nucleic acid-binding meganuclease or a nucleic acid-binding fragment thereof.

[0095]

[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), as well as their production and use (including amounts and formulations, as well as information regarding CRISPR / Cas-expressing eukaryotic cells and CRISPR / Cas-expressing eukaryotic organisms (e.g., mice)) is described elsewhere herein. In certain embodiments, targeting is achieved by an oligonucleic acid-binding CRISPR protein fragment and / or gRNA. In certain embodiments, targeting is achieved by a nucleic acid-binding, catalytically inactive CRISPR protein (fragment). Thus, in particular embodiments, the targeting domain comprises an oligonucleic acid-binding CRISPR protein or an oligonucleic acid-binding fragment of a CRISPR protein, and / or gRNA.

[0096]

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

[0097]

[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). A gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule is sometimes referred to as a single guide RNA (sgRNA), while "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. 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., directs binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to tracrRNA as set forth in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

[0098]

[0101] As used herein, the term "Cas" generally refers to the (engineered) effector proteins of a 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 / Cpf, and the like. l, 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, C sc2, 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, Cs f4, 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 within the scope of this disclosure, but are not specifically listed in this disclosure. See, e.g., Makarova et al.

[0099]

[0102] The term "Cas," as used herein, can be used interchangeably with the terms "CRISPR" protein, "CRISPR / Cas protein," "CRISPR effector," "CRISPR / Cas effector," "CRISPR enzyme," "CRISPR / Cas enzyme," etc., except where otherwise clear (e.g., by specific and exclusive reference to Cas9). The term "CRISPR protein" can be used interchangeably with "CRISPR enzyme," and it is understood that the enzymatic activity of the CRISPR protein is altered, e.g., increased or decreased (or absent), compared to a wild-type CRISPR protein. Similarly, as used herein, in certain embodiments, where applicable and clear to one of skill in the art, the term "nuclease" can refer to a modified nuclease having altered catalytic activity (e.g., increased or decreased nuclease activity, or complete loss of nuclease and nickase activity), or otherwise as otherwise defined herein, unless otherwise clear, such as by specific and exclusive reference to an unmodified nuclease.

[0100]

[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.

[0101]

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

[0102]

[0105] In certain embodiments, 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 an RNA molecule, particularly an mRNA molecule, administered to the subject.

[0103]

[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 has at least one region of sequence homology to the locus. In many cases, the donor nucleic acid has two regions of sequence homology to the locus. Such regions of homology can be at either end or can be internal to the donor nucleic acid. In many cases, an "insertion" region carrying the nucleic acid that would be desired to be introduced into a nucleic acid molecule present in a cell is located between the two regions of homology.

[0104] Base Editing

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

[0105]

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

[0106]

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

[0107]

[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 a 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 associates with the ribonucleic acid to bind to the region of the gene, thereby causing a base change.

[0108]

[0111] The term "base editor (BE)" or "nuclear base editor (NBE)" refers to an agent comprising a polypeptide capable of causing a modification to a base (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, a base editor has the ability to deaminate a base in a nucleic acid. In some embodiments, a base editor has the ability to deaminate a base in a DNA molecule. "Base editing activity" means acting to chemically change a base in a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, base editing activity is achieved by, for example, modifying a target C * G to T * In another embodiment, the base editing activity is, for example, cytidine deaminase activity converting A * T to G * adenosine deaminase activity that converts C

[0109]

[0112] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" may be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that guides the napDNAbp to a specific nucleic acid sequence. Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include domains from CRISPR proteins, 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 guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA.

[0110]

[0113] In some embodiments, the polynucleotide-programmable DNA-binding domain comprises a nuclease-inactive 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.

[0111]

[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 "inactive inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylases (AAG)) can bind to inosine but cannot create abasic sites or remove inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, a catalytically inactive inosine-specific nuclease may have the ability to bind to 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 alkyl adenosine glycosylase (AAG nuclease) from humans and catalytically inactive endonuclease V (EndoV nuclease) from, for example, Escherichia coli (E. coli). In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation, or a corresponding mutation in another AAG nuclease. In some embodiments, the base editor further comprises a uracil-binding protein, such as a uracil glycosylase inhibitor (UGI) domain that inhibits uracil-DNA glycosylase.

[0112]

[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 (not naturally occurring) of a naturally occurring deaminase from an organism. For example, in some embodiments, the deaminase or deaminase domain is 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% identical to a naturally occurring deaminase from an organism. The term deaminase also encompasses any genetically engineered deaminase, which may contain genetic modifications (e.g., one or more mutations) that result in a variant deaminase having an amino acid sequence containing one or more changes compared to a wild-type counterpart deaminase. Examples of deaminases are described herein, but the term is not meant to be limiting.

[0113]

[0116] The term "adenosine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or 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 can be capable of deaminating the A nucleobase of a DNA polynucleotide. In one embodiment, the adenosine deaminase domain of a 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 an adenosine deaminase is capable of deaminating 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 particular embodiments, the TadA is any one of the TadAs described in International Application PCT / US2017 / 045381 (herein incorporated by reference in its entirety). The table below provides exemplary sequences; other sequences may also be used.

[0114] [Table 1]

[0115]

[0117] The term "cytidine deaminase" refers to a polypeptide or fragment thereof having the ability to catalyze 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 group consisting of an apolipoprotein B mRNA editing enzyme, the catalytic polypeptide-like (APOBEC) family of deaminases, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; an activation-triggered cytidine deaminase (AID), such as activation-triggered cytidine deaminase (AICDA); cytosine deaminase 1 (CDA1) or CDA2; or a cytosine deaminase acting on tRNA (CDAT). The table below provides exemplary sequences; other sequences may also be used.

[0116] [Table 2]

[0117]

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

[0118]

[0119] The term "linker," as used herein, can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that links two molecules or moieties, such as two components of a protein complex or ribonucleoconjugate, or two domains of a fusion protein, such as 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 International Application No. PCT / US2019 / 044935 and International Application No. PCT / US2020 / 016288, each of which is incorporated by reference herein in its entirety). A linker can connect different components of a base editor system, or different portions of its components. For example, in some embodiments, a linker can connect the guide polynucleotide-binding domain of a polynucleotide-programmable nucleotide-binding domain and the catalytic domain of a deaminase. In some embodiments, a linker can link a CRISPR polypeptide and a deaminase. In some embodiments, a linker can link a Cas9 and a deaminase. In some embodiments, a linker can link a dCas9 and a deaminase. In some embodiments, a linker can link an nCas9 and a deaminase. In some embodiments, a linker can link a guide polynucleotide and a deaminase. In some embodiments, a linker can link a deamination component of a base editor system and a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can link an RNA-binding portion of a deamination component of a base editor system and a polynucleotide-programmable nucleotide-binding component. In some embodiments, a linker can link an RNA-binding portion of a deamination component of a base editor system and an RNA-binding portion of a polynucleotide-programmable nucleotide-binding component.A linker can be positioned between or adjacent to two groups, molecules, or other moieties and can be connected to one another through a covalent or non-covalent interaction, thus linking 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.

[0119]

[0120] A "targeting ribonucleic acid" or "targeting RNA" is a ribonucleic acid that is complementary to a region of an mRNA that is targeted.

[0120]

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

[0121]

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

[0122]

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

[0123]

[0124] The term "adRNA" as used herein refers to an ADAR recruitment guide. adRNAs contain a programmable antisense region that is complementary to a target RNA sequence with a mismatched cytidine opposite the target adenosine. In addition, adRNAs contain zero, one, or two ADAR recruitment domains engineered from the naturally occurring ADAR substrate GluR2 pre-mRNA. See, for example, Katrekar et al., Nat Methods. 2019 March;16(3):239-242, the contents of which are incorporated by reference in their entirety.

[0124]

[0125] The term "cadRNA" as used herein refers to circular ADAR recruiting RNA.Like adRNA, cadRNA is derived from the natural RNA site known to be highly edited by ADAR, and contains a recruiting domain that introduces endogenous ADAR to target site.See, for example, Katrekar et al., Nat Biotechnol.2022 June;40(6):938-945, the contents of which are incorporated by reference in their entirety.

[0125]

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

[0126]

[0127] In certain embodiments, the targeted ribonucleic acid generates a base change through binding to an endogenous deaminase domain (e.g., adenosine deaminase or cytidine deaminase).

[0127]

[0128] In certain embodiments, a base editor system comprises a targeted 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 polynucleotide-programmable RNA-binding domain associates with the ribonucleic acid to bind to the region of the mRNA transcript, thereby causing a base change.

[0128]

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

[0129]

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

[0130]

[0131] In certain embodiments, the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0131]

[0132] Exemplary base editors that can be utilized to implement the methods of the invention include, for example, the following references and / or patent publications (each of which is incorporated by reference in its entirety): (a) WO 2015 / 089406 and its U.S. or international equivalents; (b) WO 2017 / 070632 and its U.S. or international equivalents; (c) WO 2017 / 070633 and its U.S. or international equivalents; (d) WO 2018 / 027078 and its U.S. or international equivalents; (e) WO 2018 / 071868 and its U.S. or international equivalents; (f) WO 2017 / 048390 and its U.S. or international equivalents; (f) WO 2018 / 119359 and its U.S. or international equivalents; (g) WO 2018 / 119354 and its U.S. or international equivalents; (h) WO 2018 / 031683 and its U.S. or international equivalents; (i) WO 2018 / 176009 and its U.S. or international equivalents; (j) WO 2018 / 021878 and its U.S. or international equivalents; (k) WO 2019 / 060746 and its U.S. or international equivalents; (l) WO 2020 / 160517 and its U.S. or international equivalents; (m) WO 2020 / 168132 and its U.S. or international equivalents; (n) WO 2020 / 028823 and its U.S. or international equivalents; (o) WO 2019 / 226953 and its U.S. or international equivalents; (p) WO 2019 / 005884 and its U.S. or international equivalents; (q) Komor, AC, Kim, YB, Packer, MS, Zuris, JA & Liu, DRProgrammable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature vol. 533, 420~ (2016); (r) Gaudelli, NM et al., Programmable base editing of AT to GC in genomic DNA without DNA cleavage. Nature 551, 464~ (2017); (s) any of the references listed herein that report or describe base editors known in the art; Examples of base editors include those described in

[0132]

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

[0133]

[0134] Some aspects of the present disclosure are based on the recognition that any of the base editors presented herein have the ability to effectively generate an intended mutation in a nucleic acid (e.g., a nucleic acid located on an episomal vector) without generating a significant number of unintended mutations. In some embodiments, the intended mutation is a mutation created by a specific base editor (specifically designed to result in the intended mutation) bound to a gRNA. In some embodiments, the intended mutation is a mutation that generates a stop codon, e.g., a premature stop codon, in the coding region of a gene. In some embodiments, the intended mutation is a mutation that removes a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters a regulatory sequence of a gene (e.g., a gene promoter or a gene repressor).

[0134]

[0135] In certain embodiments, the base change results in conversion of an amino acid codon to a premature stop codon, preferably upstream of the splice junction, thereby down-regulating expression of the gene. A "splice junction," as used herein, includes the region within a mature mRNA transcript or encoded polypeptide where the 3' end of a first exon is joined to the 5' end of a second exon. 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 nucleotides or amino acid residues (including all integers in between) on either side of the exact residue where the 3' end of one exon is joined to the 5' end of another exon. "Exon" refers to a nucleic acid sequence that is present in the mature form of an RNA molecule after a portion of a precursor RNA (intron) has been removed by cis-splicing or after two or more precursor RNA molecules have been ligated together by trans-splicing.

[0135]

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

[0136]

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

[0137]

[0138] In certain embodiments, the base change results in 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, and preferably the premature UAG stop codon is located near the 5' end of the gene.

[0138] vector

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

[0139]

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

[0140]

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

[0141]

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

[0142]

[0143] The term "expression cassette," as used herein, refers to a nucleic acid construct containing nucleic acid elements sufficient for expression of a polynucleotide molecule. Generally, an expression cassette includes a polynucleotide molecule operably linked to a promoter sequence.

[0143]

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

[0144]

[0145] Regulation of expression can be affected at the level of transcription, translation, splicing, message stability, etc. Generally, expression control elements that modulate transcription are located 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., within an intron). Expression control elements can be located near the transcribed sequence, at a distance from it (e.g., 1-10, 10-25, 25-50, 50-100, 100-500, or more nucleotides from the polynucleotide), or even at a greater distance. Nevertheless, due to length constraints on AAV vectors, expression control elements within AAV vectors are generally located within 1-1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0145]

[0146] Functionally, expression of an operably linked nucleic acid can be controlled, at least in part, by an element (e.g., a promoter), e.g., an element that modulates transcription of the nucleic acid and, optionally, translation of the transcript. A specific example of an expression control element is a promoter (usually located 5' to the nucleic acid sequence to be transcribed). A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0146]

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

[0147]

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

[0148]

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

[0149]

[0150] As used herein, the term "enhancer" may refer to a sequence located adjacent to a heterologous nucleic acid. Enhancer elements are typically located upstream of a promoter element, but can function even when located downstream 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, upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements generally increase expression of an operably linked nucleic acid conferred by a promoter element.

[0150]

[0151] Expression constructs may contain regulatory elements that serve to drive expression in specific cell or tissue types. Expression control elements (e.g., promoters) include elements that are active in specific tissues or cell types, and are referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are generally active in specific cells or tissues (e.g., liver). Expression control elements are generally active in specific cells, tissues, or organs because they are recognized by transcriptional activator proteins or other regulators of transcription that are specific to that cell, tissue, or organ type. Such regulatory elements are known to those skilled in the art (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al. (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0151]

[0152] Incorporation of tissue-specific regulatory elements into the expression construct confers at least partial tissue tropism to the expression of the heterologous nucleic acid encoding a protein or inhibitory RNA. Examples of promoters active in the liver include, among others, the transthyretin (TTR) gene promoter; the human alpha1-antitrypsin (hAAT) promoter; the apolipoprotein AI 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 an intronless scAAV); and the LSP1 promoter. Examples of enhancers active in the liver are apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0152]

[0153] Expression control elements also include ubiquitous or confluent promoter / enhancers capable of driving expression of a polynucleotide in many different cell types, including, but 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.

[0153]

[0154] Expression control elements can also effect expression in a regulatable manner, i.e., a signal or stimulus increases or decreases expression of an operably linked heterologous polynucleotide. Regulatable elements that increase expression of an operably linked polynucleotide in response to a signal or stimulus are also referred to as "inducible elements" (i.e., are induced by the signal). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. Generally, the amount of increase or decrease effected by such elements 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 metallothionine (MT) promoter, the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088), the tetracycline-repressible system (Gossen et al., Proc. Natl. cad. 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 regulatable control elements that may be useful in this context are elements that are regulated by specific physiological conditions (e.g., temperature, acute phase, development).

[0154]

[0155] Other examples of promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (a composite of the CMV enhancer, chicken beta-actin promoter (CBA), and rabbit beta-globin intron), and other constitutive promoters, NSE (neuron-specific enolase), synapsin, or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, SFFV promoter, Rous sarcoma virus (RSV) promoter, rat insulin promoter, TBG promoter, and other liver-specific promoters, desmin promoter and similar muscle-specific promoters, EF1-alpha promoter, synthetic promoters, hybrid promoters, promoters with multiple tissue specificities, and the like, all of which are well known and readily available to those skilled in the art. Other promoters may be derived from human origin or other species, including mouse.

[0155]

[0156] Expression control elements also include the native element(s) for the heterologous polynucleotide. When it is desired that expression of the heterologous polynucleotide mimic the native expression, the native control element (e.g., promoter) can be used. The native element can be used when the expression of the heterologous polynucleotide is regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. Other native expression control elements (e.g., introns, polyadenylation sites, Kozak consensus sequences, etc.) can also be used.

[0156]

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

[0157]

[0158] Thus, additional elements to 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 flanking the sequence (e.g., polyadenylation (polyA) sequences), such as one or more copies of AAV ITR sequences or introns.

[0158]

[0159] Additional elements include, for example, filler or stuffer polynucleotide sequences to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors generally tolerate DNA insertions ranging in size from about 4 kb to about 5.2 kb, or slightly larger. Therefore, for shorter sequences, a stuffer or filler is incorporated to adjust the length to or near the normal size of the viral genome sequence (the size tolerated when packaging the AAV vector into a viral particle). In certain embodiments, the filler / stuffer nucleic acid sequence is a non-translated (non-protein-coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence, when combined with the sequence (e.g., inserted into a vector), has a total length of about 3.0 to 5.5 kb, about 4.0 to 5.0 kb, or about 4.3 to 4.8 kb.

[0159]

[0160] As used herein, the term "gene delivery system" refers to any means for delivering a composition containing a nucleic acid sequence to a cell or tissue. For example, the gene delivery system may be a viral gene delivery system, such as an unmodified virus, a modified virus, or a VLP, to facilitate the delivery of a viral vector to a desired cell or tissue. The gene delivery system may also be a non-viral delivery system that does not contain viral coat proteins or does not form viral particles or VLPs, such as a liposome-based system, a polymer-based system, a protein-based system, a metal particle-based system, or a peptide cage system.

[0160]

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

[0161]

[0162] Retroviruses are enveloped, single-stranded RNA viruses containing 5' and 3' LTRs and a signal packaging sequence located just outside the LTRs. Different types of retroviral vectors can contain different amounts of viral genome. In certain embodiments, the retroviral vector is an HIV-based lentiviral vector, which 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 necessary, a stuffer sequence can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced by using the appropriate plasmids and cell lines, while supplying the viral proteins required for vector production in trans. (Bulcha et al. (2021) Sig. Transduct. Target Ther. 6:53).

[0162]

[0163] The term "recombinant" is used as a modifier of vector, such as recombinant AAV (rAAV) vector, and of sequences, such as recombinant polynucleotides and polypeptides, to mean that the composition has been manipulated (i.e., engineered) in a manner that does not normally occur in nature. Although the term "recombinant" is not always used herein when describing AAV vectors and sequences (e.g., polynucleotides), recombinant forms comprising polynucleotides are expressly included despite such omission.

[0163]

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

[0164]

[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 generally tolerate DNA insertions ranging in size from about 4 kb to about 5.2 kb. If necessary, a stuffer sequence may be used to increase the size and packaging efficiency of the rAAV nucleic acid. In various embodiments, the rAAV nucleic acid containing the stuffer is 4 to 5.2 kb, 3.0 to 5.5 kb, 4.0 to 5.0 kb, 4.3 to 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. The preferred stuffer sequence does not fall under the category of coding sequences, repetitive sequences, recombination sequences, or immunogenic sequences.

[0165]

[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 within AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITRs. In further embodiments, both a 5' and a 3' ITR are present and both ITRs are from the same serotype genome.

[0166]

[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 generated in which all three viral proteins are based on a particular serotype, or in which one, two, or all three viral proteins are based on different serotypes or variants thereof.

[0167]

[0168] Different serotypes exist within different virus species. Different serotypes may confer different activities, such as different cell or tissue tropism and the potential to elicit a host immune response. The term "serotype" broadly refers to both serologically distinct viruses and serologically indistinguishable viruses (which may exist within subgroups or variants of a given serotype). Serological distinctions can be determined based on the presence or absence of antibody cross-reactivity to one capsid compared to another. Such differences in cross-reactivity are typically due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0168]

[0169] As more naturally occurring virus isolates are discovered or capsid variants are generated, there may or may not be serological differences when compared to any of the currently existing serotypes. Thus, if a new virus does not have serological differences, it will be a subgroup or variant of the corresponding serotype.

[0169]

[0170] In certain embodiments, the AAV capsid is based on a VP1, VP2, or VP3 that has at least 80% sequence identity to the VP1, VP2, or VP3 of any 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 (capsid variants, e.g., amino acid insertions, additions, substitutions, and deletions, etc.). (See, e.g., U.S. Pat. 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 Application Publication No. 2013 / 0059732 and U.S. Pat. No. 9,169,299, which disclose LK01, LK02, and LK03; and U.S. Pat. No. 11,110,153, the disclosures of which are incorporated herein in their entireties).

[0170]

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

[0171]

[0172] In different embodiments, the rAAV capsid is at least 80%, at least 85%, or both relative to VP1, VP2, or VP3 of any 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 to VP1 of SEQ ID NO: 1 or SEQ ID NO: 2. , 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% identical.

[0172] [Table 3]

[0173] Recombinant AAV can be produced from different types of cell lines. In certain embodiments, human HEK293 cells are used (American Type Culture Collection Accession No. ATCC CRL1573). 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 in their entireties.

[0174] In certain embodiments, AAV helper functions are introduced into host cells by transfecting them with an AAV helper construct prior to or simultaneously with transfection of the AAV expression vector. Host cells with AAV helper functions may be referred to as "helper cells" or "packaging helper cells." Thus, AAV helper constructs are sometimes used to complement deficiencies in AAV functions necessary for productive AAV transduction by at least transiently expressing the AAV rep and / or cap genes. AAV helper constructs often lack AAV ITRs and are unable to replicate or package themselves. These constructs may be in the form of, for example, a plasmid, phage, transposon, cosmid, virus, or virion. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products. Several other vectors encoding Rep and / or Cap expression products are known. Recombinant AAV can be produced, for example, as described in U.S. Pat. No. 9,408,904, and International Application Nos. PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are incorporated herein in their entireties.

[0175]

[0175] The term "isolated," when used as a modifier of a composition, means that the composition has been made by the hand of man or has been completely or at least partially separated from its naturally occurring 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 created by the hand of man, such as the combination of rAAV sequences or rAAV particles (packaging or encapsidating an AAV vector genome (vg)) with a pharmaceutical formulation. The term "isolated" also does not exclude alternative physical forms of the composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated), or derivatized forms, or forms expressed in a host cell and created by the hand of man.

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

[0178] Therapeutic Proteins

[0178] Episomal vectors can deliver a variety of different genes that can be expressed to provide proteins with desired activities, including genes that provide healthy copies of genes in subjects with genetic defects, or novel modified genes that may be useful in treating a disease or disorder, or novel genes that encode proteins that provide a beneficial effect.

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

[0180] In different embodiments, the gene is selected from the group consisting of 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), transfection factor (TGF ... The gene encoding transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin 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, noggin, sonic hedgehog, or tyrosine hydroxylase.

[0181] In different embodiments, the gene encodes thrombopoietin (TPO), interleukins (IL-1 through IL-36), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α or β, interferon α, β, or γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, or IgE, chimeric immunoglobulin, antibody, humanized antibody, single-chain antibody, T cell receptor, chimeric T cell receptor, single-chain T cell receptor, class I or class II MHC molecule. Antibodies and immunoglobulins can be provided to target, for example, cancer cells or other disease- or disorder-causing cells.

[0182] In different embodiments, the gene is selected from the group consisting of CFTR (cystic fibrosis transmembrane conductance regulator), clotting factors (factor XIII, factor IX (FIX), factor VIII (FVIII), factor X, factor VII, factor VIIa, or protein C), gain-of-function clotting factors, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters, and the like. 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, neurotrophic factors 3 and 4, brain-derived neurotrophic factor, glial-derived growth factor, transducing growth factor α and β, cytokines, α-interferon interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, 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)), immune modifiers peptides with tethering 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), LCA5 (LCA-leversillin), ornithine ketoacid aminotransferase (rotational 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), ACHM2, 3, and 4 (color blindness), 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 protein, or encoding one or more donor sequences used as a repair template for genome editing.

[0183] In different embodiments, the gene is selected from the group consisting of erythropoietin (EPO) for treating anemia; interferon-alpha, interferon-beta, and interferon-gamma for treating various immune diseases, viral infections, and cancer; interleukins (IL) and corresponding receptors, including any one of IL-1 through IL-36, for treating various inflammatory diseases or immune deficiencies; chemokines, including chemokine (C-X-C motif) ligand 5 (CXCL5), for treating immune disorders; 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; alpha-1-antitrypsin for treating emphysema or chronic obstructive pulmonary disease (COPD); mucopolysaccharidosis I (MPS ornithine transcarbamoylase (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 (Apo) for treating apolipoprotein (Apo) AI deficiency; low-density lipoprotein (LDL) for treating familial hypercholesterolemia (FH). Protein receptor (LDL-R); albumin for treating hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumarylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for treating homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl CoA dehydrogenase; propionyl CoA carboxylase;Encoding methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H protein; T protein; cystic fibrosis transmembrane conductance regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for treating Stargardt disease; or dystrophin;

[0184]

[0184] In a further embodiment, 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's disease, Parkinson's disease, Alzheimer's disease, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber's congenital amaurosis, and Stargardt disease. inhibitory nucleic acid

[0185] Episomal vectors can provide a variety of different genes encoding different inhibitory nucleic acids, such as short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), RNAi, ribozymes, and antisense RNA. In different embodiments, the inhibitory nucleic acid is selected from the group consisting of the huntingtin (HTT) gene, genes associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), androgen receptor on the X chromosome in spinal-bulbar muscular atrophy, human ataxin 1, 2, 3, and 7, Cav2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA binding protein, ataxin 8 inverse chain (ATXN8OS), serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxias (types 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; Myotonin-Protein Kinase (MT-PK) in Myotonic Dystrophy; Frataxin in Friedreich's Ataxia; Superoxide Dismutase 1 (SOD1) Gene Variants 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) in Hypercholesterolemia; HIV in HIV Infection Tat, transactivator of transcription gene of human immunodeficiency virus; HIV TAR, human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53 in acute renal failure after kidney transplantation or kidney injury with acute kidney injury or delayed graft function; protein kinase N3 (PKN3) in advanced, recurrent, or metastatic solid malignancies;LMP2 (also known as proteasome subunit beta 9 (PSMB9)) in metastatic melanoma; LMP7 (also known as proteasome subunit beta 8 (PSMB8)) in metastatic melanoma; MECL1 (also known as proteasome subunit beta 10 (PSMB10)) in metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein, apoptosis suppressor B-cell CLL / lymphoma (BCL-2) in solid tumors; and 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, beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor in 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; and keratin 6A in pachyonychia congenita. N17K mutant proteins; influenza A virus genome / gene sequences in influenza infections; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequences in SARS infections; respiratory syncytial virus genome / gene sequences in respiratory syncytial virus infections; Ebola virus genome / gene sequences in Ebola hemorrhagic fever infections; hepatitis B and C virus genome / gene sequences in hepatitis B and C infections; herpes simplex virus (HSV) genome / gene sequences in HSV infections; coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infections; silencing of pathogenic alleles of genes such as torsin A (TOR1A) in primary dystonia (allele-specific silencing) and silencing of specific pan-class I and HLA alleles in transplantation;and a polynucleotide repeat-type disease-associated gene or gene transcript selected from the group consisting of a mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP);

[0185] Gene editing

[0186] The episomal vector can provide a variety of different genes encoding a variety of different gene editing nucleic acids, such as ZFNs, TALENs, and CRISPR-Cas9, etc. In different embodiments, the gene editing nucleic acid edits the DNA of a subject to provide a therapeutic protein as provided above or to disrupt a gene as provided above.

[0186] Delivery System

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

[0187]

[0188] Any RNA in the system, such as a guide RNA or a base editor-encoding mRNA, can be delivered in the form of RNA. Base editor-encoding mRNA can be produced using in vitro transcription. For example, a 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 a 3'UTR derived from a beta globin-polyA tail. The cassette can be used for transcription with T7 polymerase. Alternatively, a guide polynucleotide (e.g., a gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter followed by the sequence "GG" and the guide polynucleotide sequence. To enhance expression and reduce potential toxicity, the base editor-encoding sequence and / or guide nucleic acid can be modified to include one or more modified nucleosides, for example, using pseudo-U or 5-methyl-C.

[0188]

[0189] Nucleic acids encoding nucleobase editors according to the present disclosure can be administered to a subject or delivered to cells in vitro or in vivo by methods known in the art or as described herein. In one embodiment, the nucleobase editor can be delivered, for example, by 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.

[0189]

[0190] Nucleic acids encoding nucleobase editors can be delivered directly to cells as naked DNA or RNA, e.g., by transfection or electroporation, or can be conjugated to a molecule (e.g., N-acetylgalactosamine) that facilitates uptake by target cells. Nucleic acid vectors, such as those described herein, can also be used.

[0190]

[0191] The nucleic acid vector may include one or more sequences encoding a domain of the base editing system described herein. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted into or fused to) the protein-encoding sequence. As an example, the nucleic acid vector may include a Cas9 coding sequence including one or more nuclear localization sequences (e.g., a nuclear localization sequence derived from SV40) and an adenosine deaminase.

[0191] Non-viral methods

[0192] In certain embodiments, one or more nucleic acid molecules encoding the base editor system or components thereof of the invention are administered via a non-viral delivery system (including, for example, a system encapsulated in a lipid nanoparticle (LNP)).

[0192]

[0193] In certain embodiments, one or more nucleic acid molecules encoding the base editor systems or components thereof of the invention are delivered or administered using non-viral delivery systems, including, for example, chemical methods (such as non-viral vectors or extracellular vesicles) and physical methods (such as gene guns, electroporation, particle bombardment, the use of ultrasound, and magnetofection).

[0193]

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

[0194]

[0195] In certain embodiments, one or more nucleic acid molecules encoding the base editor systems of the invention or components thereof are delivered or administered within an AAV vector particle or other viral particle (which is further encapsulated with or complexed to a liposome, nanoparticle, lipid nanoparticle, polymer, microparticle, microcapsule, micelle, or extracellular vesicle).

[0195]

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

[0196]

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

[0197]

[0198] In light 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, etc.

[0198]

[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.

[0199] Lipid-Based Delivery Systems

[0200] Lipid-based delivery systems are well known in the art, and in light of this disclosure, any suitable lipid-based delivery system known to one 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.

[0200]

[0201] "Lipid nanoparticle" or "LNP" refers to lipid-based vesicles useful for the delivery of AAV and non-viral vectors, having nanoscale dimensions, i.e., about 10 nm to about 1000 nm, or about 50 to about 500 nm, or about 75 to about 127 nm. Without being bound by theory, it is believed that LNPs provide polynucleotides, expression cassettes, AAV vectors, or non-viral vectors while shielding them partially or completely from the immune system. Shielding allows for delivery of the polynucleotide, expression cassette, AAV vector, or non-viral vector to tissues or cells while avoiding eliciting a substantial immune response against the polynucleotide, expression cassette, AAV vector, or non-viral vector in vivo. Shielding may also allow for repeated administration of the polynucleotide, expression vector, AAV vector, or non-viral vector without eliciting a substantial immune response against the polynucleotide, expression cassette, AAV vector, or non-viral vector in vivo (e.g., in a human subject). Shielding may also improve or increase the efficiency of delivery of the polynucleotide, expression cassette, AAV vector, or non-viral vector in vivo.

[0201]

[0202] The pI (isoelectric point) of AAV is in the pH range of about 6 to about 6.5. Therefore, the surface of AAV carries a slight negative charge. Therefore, it can be beneficial for the LNP to contain a cationic lipid, such as an amino lipid. Exemplary amino lipids are 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, as well as U.S. Patent Application Publication Nos. 6,111,159, 6,112,159, and 6,113,159. and 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 entireties.

[0202]

[0203] The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include lipids and their salts having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino group (e.g., an alkylamino group or a dialkylamino group). 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. The cationic lipid may be a titratable cationic lipid. In certain embodiments, the cationic lipid comprises a protonatable tertiary amine group (e.g., a pH-titratable group); a C18 alkyl chain (each alkyl chain independently having 0 to 3 (e.g., 0, 1, 2, or 3) double bonds); and an ether, ester, or ketal bond located between the head group and the alkyl chain.

[0203]

[0204] Cationic lipids include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (g-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K)). , 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA (also known as MC2)), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-M-C3-DMA (also known as MC3)), salts thereof, and mixtures thereof. Other cationic lipids 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-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, y-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0204]

[0205] Still further cationic lipids include, but are not limited to, 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC ... -morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-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-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleyloxy)propane) N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)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-carboxyamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-di Examples of suitable methyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS), and disubstituted spermine (DSS), or mixtures thereof, may be mentioned.

[0205]

[0206] Several commercially available preparations of cationic lipids are available, such as LIPOFECTIN® (containing DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECT AMINE® (containing DOSPA and DOPE, available from GIBCO / BRL).

[0206]

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

[0207]

[0208] Sterols can impart fluidity to LNPs. As used herein, "sterol" refers to any naturally occurring sterol of plant origin (phytosterols) or animal origin (zoosterols), 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 preparations, most commonly cholesterol. Plant sterols can include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156, the disclosure of which is incorporated herein in its entirety. In certain embodiments, the sterol can be present in an amount of about 5% by weight of the LNP to about 50% by weight of the lipid nanoparticles or about 10% by weight of the LNP to about 25% by weight of the LNP.

[0208]

[0209] LNPs can include neutral lipids. Neutral lipids can include any lipid species that exist in either an uncharged form or a neutral zwitterionic form at physiological pH. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations of particle size and required stability, among other factors. In certain embodiments, the neutral lipid component can be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0209]

[0210] Lipids with various acyl chain groups of varying chain length and degree of saturation are available or can be isolated or synthesized by well-known techniques. In certain embodiments, lipids containing saturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In another group of embodiments, lipids containing mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. Additionally, lipids with 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 with other head groups, such as serine or inositol.

[0210]

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

[0211]

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

[0212]

[0213] LNP preparations can be combined with additional components, non-limiting examples of which include polyethylene glycol (PEG) and sterols.

[0213]

[0214] The term "PEG" refers to polyethylene glycol, a linear, water-soluble polymer composed of repeating ethylene PEG units with two terminal hydroxyl groups. PEGs are classified by their 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. PEGs are commercially available from Sigma Chemical Co. and other companies, and include, for example, the following functionalized PEGs: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH), monomethoxypolyethylene glycol tresylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM).

[0214]

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

[0215]

[0216] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397, the disclosures of which are incorporated herein in their entirety. Useful PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "tethering" lipid moieties for fixing the PEG moiety to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20) described in U.S. Patent No. 5,820,873, the disclosure of which is incorporated herein in its entirety, PEG-modified dialkylamine, and PEG-modified l,2-diacyloxypropan-3-amine. In certain embodiments, the PEG-modified lipid can be PEG-modified diacylglycerol and dialkylglycerol. In certain embodiments, PEG can be in an amount of about 0.5% by weight to about 20% by weight of the LNP, or about 5% by weight to about 15% by weight of the LNP.

[0216]

[0217] Furthermore, the LNPs may be PEG-modified and sterol-modified LNPs. The LNPs combined with additional components may be the same LNP or different LNPs. In other words, the same LNP may be PEG-modified and sterol-modified, or a first LNP may be PEG-modified and a second LNP may be sterol-modified. Optionally, the first and second modified LNPs may be combined.

[0217]

[0218] In certain embodiments, LNPs may 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 prior to encapsulation. In certain embodiments, LNP-encapsulated nucleic acids, expression vectors, AAV vectors, or non-viral vectors may have a size in the range of about 10 nm to 500 nm.

[0218] Polymer-Based Systems

[0219] Polymer-based delivery systems are well known in the art, and in light of this 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 within the polymer matrix of the polymer nanoparticle, or adsorbed or conjugated to the surface of the nanoparticle. Examples of polymers commonly used for gene delivery include, for example, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI), chitosan, dendrimers, polyanhydrides, polycaprolactone, and polymethacrylate.

[0219]

[0220] The 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 less.

[0220] Protein-based systems

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

[0221]

[0222] CPPs are short peptides (6-30 amino acid residues) that have the potential to penetrate cells and deliver therapeutic molecules. Most CPPs are composed primarily of arginine and lysine residues and are cationic and hydrophilic; however, CPPs can also be amphipathic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., Tat, HIV-1 protein) or obtained synthetically (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996-2006). Examples of CPPs include cationic CPPs (highly positively charged) (e.g., Tat peptide, penetratin, protamine, poly-L-lysine, polyarginine, etc.); amphipathic CPPs (chimeric or fusion peptides constructed from different sources and containing both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin-7 (Bac7), proline-rich peptides (PPR), SAP (VRLPPP)3, TP10, pep-1, MPG, etc.); membrane-directed CPPs (exhibiting both hydrophobic and amphipathic properties simultaneously and containing both large aromatic and small residues) (e.g., gH625, SPIONs-PEG-CPP NP, etc.); and hydrophobic CPPs (containing only nonpolar motifs or residues) (e.g., SG3, PFVYLI, pep-7, fibroblast growth factor (FGF), etc.).

[0222]

[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 less.

[0223] Peptide cage system

[0224] Peptide cage-based delivery systems are well known in the art, and in light of this disclosure, any suitable peptide cage-based delivery system known to those of skill in the art can be used in the present invention. Generally, any proteinaceous material that can assemble into a cage-like structure to form a confined internal environment can be used. Several different types of "shell" proteins can be assembled and loaded with different types of materials. For example, protein cages comprising shells of viral coat protein(s) (e.g., derived from the protein coat of cowpea chlorotic mottle virus (CCMV)) that encapsulate non-viral materials, as well as protein cages formed from non-viral proteins, have been described (see, e.g., U.S. Pat. Nos. 6,180,389 and 6,984,386; U.S. Patent Application Publication Nos. 20040028694 and 20090035389, the disclosures of which are incorporated herein in their entireties). A peptide cage may comprise a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an internal cavity that is naturally accessible to solvent or can be made so by varying solvent concentration, pH, or equilibrium ratio).

[0224]

[0225] Examples of protein cages derived from non-viral proteins include ferritins and apoferritins from both eukaryotic and prokaryotic species, e.g., 12- and 24-subunit ferritins; and protein cages formed from heat shock proteins (HSPs), e.g., the class of 24-subunit heat shock proteins that form the inner core space, the small HSP of Methanococcus jannaschii, the 12-mer Dsp HSP of Escherichia coli, MrgA protein, etc. As will be appreciated by those skilled in the art, the monomers of the protein cage can be naturally occurring or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0225]

[0226] The protein cages may have different core 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 less.

[0226] Administration and Treatment

[0227] The present invention can be used for human and veterinary medical applications. Accordingly, suitable subjects include mammals such as humans, as well as non-human mammals. The term "subject" refers to animals, generally mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), livestock (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, juveniles, and adults.

[0227]

[0228] As used herein, the term "administering" or "administration" refers to providing one or more compositions described herein to a patient or subject. By way of example, and without limitation, administration, e.g., injection, of a composition may be performed by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes may be employed. Parenteral administration may be performed, for example, by bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.

[0228]

[0229] The terms "treatment," "treating," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms "treatment," "treating," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have appeared and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors) prior to the onset of symptoms. Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0229]

[0230] An "effective amount" or "sufficient amount" for treating (e.g., ameliorating or providing a therapeutic benefit or improvement) is generally effective to achieve a measurable response to one, more, or all adverse symptoms, consequences, or complications of a disease, e.g., one or more adverse symptoms, disorders, illnesses, pathologies, or complications caused by or associated with the disease (wherein a decrease, reduction, inhibition, suppression, limitation, or control of the progression or worsening of the disease would be a satisfactory result).

[0230]

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

[0231]

[0232] An effective or sufficient amount need not be effective in every single subject treated, or in the majority of subjects treated within a given group or population. An effective or sufficient amount means effective or sufficient in a particular subject, rather than a group or the general population. As is typical of such methods, some subjects will respond more, less, or not at all to a given treatment method or use thereof.

[0232]

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

[0233]

[0234] The order and timing of administering one or more gene editing agents and the episomal vector 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.

[0234]

[0235] The treatment dosage of the episomal vector may vary depending on the type, onset, progression, severity, frequency, duration, or probability of the disease or disorder being treated, the desired clinical endpoint, previous or concurrent treatments, the subject's general health, age, sex, race, or immunological competence, and other factors understood by those skilled in the art. The dosage, number, frequency, or duration may be proportionally increased or decreased as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy, and the subject's condition.

[0235]

[0236] The dosage to achieve a therapeutic effect, e.g., the dosage of episomal vector per kilogram of body weight (mg / kg), will again vary based on several factors, including the route of administration, the level of gene expression required to achieve a therapeutic effect, the specific disease or disorder being treated, the host's immune response to the DNA, the host's immune response to the transgene expression product, and the stability of the expressed protein, peptide, or nucleic acid. Based on the guidance provided herein, one of skill in the art will be able to determine an appropriate episomal vector dosage range for treating a patient with a particular disease or disorder.

[0236]

[0237] The overall level of gene expression can vary depending on the use of episomal vectors. 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%, or at least 100% of the normal expression of the corresponding protein of interest.

[0237]

[0238] The dosage of an editing agent to achieve modulation of expression, e.g., the dosage per kilogram of 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 expression of the gene or mRNA is increased or decreased, or the stability of the guide RNA. Based on the guidance provided herein, one of skill in the art will be able to determine the appropriate dosage range of the editing agent for treating a patient with a particular disease or disorder.

[0238]

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

[0239]

[0240] Modulation of expression by the one or more editing agents can occur within one month of administration of one or more nucleic acid molecules encoding one or more gene editing agents, for example, 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 administration of one or more nucleic acid molecules encoding one or more editing agents.

[0240] Illustrative Diseases and Disorders

[0241] Diseases and disorders that can be treated include pulmonary 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 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 These include: mucopolysaccharidosis (Hurler disease and variants, Hunter syndrome, Sanfilippo types A, B, C, D, Morquio disease types A and B, Maroteaux-Lamy syndrome, 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 disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), and diseases of solid organs (e.g., brain, liver, kidney, heart).

[0241]

[0242] Synucleinopathies can be treated by the methods of the present invention. Synucleinopathies are neurodegenerative diseases or disorders characterized by neuronal and / or glial cell inclusions. Pathologically, synucleinopathies can be divided into two major disease groups: Lewy body diseases or disorders and multiple system atrophy (MSA). Lewy body diseases and disorders are characterized by α-synuclein aggregation 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).

[0242]

[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 characterized by the abnormal accumulation of misfolded α-synuclein protein aggregates in various regions of the brain. Dopaminergic neuronal loss 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).

[0243]

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

[0244]

[0245] Treatable blood clotting disorders include hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any of the following clotting 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 gamma-carboxylase deficiency.

[0245]

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

[0246]

[0247] Other treatable diseases and disorders include proliferative diseases (e.g., cancer, tumors, and dysplasia), Crigler-Najjar syndrome, and metabolic diseases (e.g., metabolic diseases of the liver, etc.); Friedreich's ataxia; infectious diseases; viral diseases induced by, for example, hepatitis B or C virus, HIV, herpes, and retroviruses; genetic diseases (e.g., cystic fibrosis, dystroglycanopathy, etc.), myopathies (e.g., Duchenne myomyopathies or dystrophies, myotubular myopathies, etc.), and inflammatory diseases (e.g., inflammatory bowel disease ... , sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubular myopathy, etc.), motor neuron diseases (e.g., spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy, or Charcot-Marie-Tooth disease, etc.); arthritis; severe combined immunodeficiency (e.g., RS-SCID, ADA-SCID, or X-SCID, etc.); Wiskott-Aldrich syndrome; X-linked thrombocytopenia; X-linked congenital neutropenia; chronic granulomatous disease; coagulation factor deficiency cardiovascular diseases (e.g., restenosis, ischemia, dyslipidemia, and homozygous familial hypercholesterolemia); ophthalmic or visual diseases (e.g., 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 following retinal vein occlusion, optic neuropathy, non-arteritic ischemic optic neuropathy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, achromatopsia, and Stargardt's disease; lysosomal storage diseases (e.g., Sanfilippo syndrome); hyperbilirubinemia (e.g., CNI or II, or Gilbert's syndrome); glycogen storage diseases (e.g., GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or fatal congenital glycogen storage disease of the heart).

[0247]

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

[0248]

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

[0249]

[0250] Although several different aspects and embodiments of the present invention have been described throughout this application, those skilled in the art will be able to make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention.

[0250] Embodiment Embodiment A

[0251] 1. A method of regulating expression of a gene located on an episomal vector in a subject in need thereof, comprising the step of administering to the subject one or more gene editing agents that modify a region of the gene, thereby regulating 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 via modification of the region, or an amino acid codon that can be converted to a premature stop codon via modification of the region.

[0251]

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

[0252]

[0253] 3. The method of 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.

[0253]

[0254] 4. The method of any one of the above embodiments, wherein 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 a gene and is capable of incorporating into and modifying the region of a gene.

[0254]

[0255] 5. The method of any one of the above embodiments, wherein the one or more gene editing agents are encoded by one or more nucleic acid molecules administered to the subject, preferably wherein the one or more gene editing agents are encoded by an RNA molecule, particularly an mRNA molecule, administered to the subject.

[0255]

[0256] 6. The method of embodiment 5, wherein the one or more nucleic acid molecules, such as one or more mRNA molecules, are administered to the subject together with a liposome, lipid nanoparticle (LNP), peptide cage, or polymer nanoparticle.

[0256]

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

[0257]

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

[0258]

[0259] 9. The method of embodiment 7 or 8, wherein the base editor system comprises a ribonucleic acid complementary to a region of a 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 associates with the ribonucleic acid to bind to the region of the gene, thereby causing the base change.

[0259]

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

[0260]

[0261] 11. The method of 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.

[0261]

[0262] 12. The method of 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.

[0262]

[0263] 13. The method of any one of embodiments 9 to 12, wherein (i) the cytidine deaminase domain is selected from the group consisting of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, 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 the group consisting of adenosine deaminase 1 (ADA1) and ADA2.

[0263]

[0264] 14. The method of embodiment 7 or 8, wherein the base editor system comprises a ribonucleic acid complementary to a region of an 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 associates with the ribonucleic acid to bind to the region of the mRNA transcript, thereby causing the base change.

[0264]

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

[0265]

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

[0266]

[0267] 17. (i) the cytidine deaminase domain is selected from the group consisting of an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; an activation-induced cytidine deaminase (AID), such as activation-induced cytidine deaminase (AICDA); cytosine deaminase 1 (CDA1) or CDA2; or a cytosine deaminase acting on tRNA (CDAT); and (ii) The method of any one of embodiments 14 to 16, wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0267]

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

[0268]

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

[0269]

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

[0270]

[0271] 21. The method of any one of embodiments 7 to 20, wherein the base change causes conversion of an amino acid codon to a premature stop codon, preferably upstream of a splice junction, thereby downregulating expression of the gene.

[0271]

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

[0272]

[0273] 23. The method of any one of embodiments 7 to 20, wherein the base change causes conversion of a premature stop codon to an amino acid codon, thereby upregulating expression of the gene.

[0273]

[0274] 24. The method of embodiment 23, wherein the base change results in 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, and preferably the premature UAG stop codon is located near the 5' end of the gene.

[0274]

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

[0275]

[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.

[0276]

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

[0277]

[0278] 28. The method of any one of the above, wherein 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, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease.

[0278] Embodiment B

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

[0279]

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

[0280]

[0281] 3. The method of embodiment 1, wherein the alteration 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.

[0281]

[0282] 4. The method of embodiment 2, wherein the region of the mRNA transcript comprises a premature stop codon that is convertible to an amino acid codon via a base change, or an amino acid codon that is convertible to a premature stop codon via a base change.

[0282]

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

[0283]

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

[0284]

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

[0285]

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

[0286]

[0287] 9. The method of any one of embodiments 6 to 8, wherein the targeted ribonucleic acid causes the base change through binding to an endogenous adenosine deaminase domain.

[0287]

[0288] 10. The method of embodiment 9, wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, and ADAR3.

[0288]

[0289] 11. The method of 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 a base editor, wherein the polynucleotide-programmable RNA-binding domain cooperates with the targeted ribonucleic acid to cause the base change.

[0289]

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

[0290]

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

[0291]

[0292] 14. (i) the cytidine deaminase domain is selected from the group consisting of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, 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 method of any one of embodiments 11 to 13, wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

[0292]

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

[0293]

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

[0294]

[0295] 17. The method of embodiment 16, wherein the targeting ribonucleic acid and / or one or more nucleic acid molecules, such as one or more mRNA molecules, are administered to the subject together with lipid nanoparticles (LNPs), peptide cages, or polymer nanoparticles.

[0295]

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

[0296]

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

[0297]

[0298] 20. The method of any one of embodiments 2 to 17, wherein the base change causes conversion of a premature stop codon to an amino acid codon, thereby upregulating expression of the gene.

[0298]

[0299] 21. The method of embodiment 20, wherein the base change results in 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, and preferably the premature UAG stop codon is located near the 5' end of the gene.

[0299]

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

[0300]

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

[0301]

[0302] 24. The method of any one of the above 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.

[0302]

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

[0303]

[0304] 26. The method of any one of the above embodiments, wherein 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, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber congenital amaurosis, and Stargardt disease. [Example]

[0304]

[0305] Examples are presented to further illustrate different aspects of the present invention and methodologies for practicing the invention, but the present invention is not limited thereto.

[0305] Example 1. In Vivo Administration of Base Editors to Reduce Expression Levels

[0306] Mice (Balb / c; Jackson Laboratory) were intravenously administered 5e10vg / mouse and 5e9vg / mouse of recombinant AAV (rAAV) particles (expressing human factor IX (FIX)), resulting in consistent serum levels of approximately 50,000ng / ml and 5,000ng / ml of FIX expression, respectively, 4 weeks after administration. The expression cassette is packaged into AAV viral particles by encapsidation within the AAV capsid. Viral particles are typically produced using a triple transfection protocol.

[0306]

[0307] Five weeks after administration, a cytosine base editor (CBE) and gRNA targeting the FIX exon 1 glutamine codon were administered to convert the CAG codon to a premature stop codon, UAG. The CBE mRNA and gRNA were formulated into a single LNP for administration. Mice were divided into five groups and administered 0.25 mpk, 0.5 mpk, 1.0 mpk, or 2.0 mpk mRNA / gRNA LNPs, or 2.0 mpk control mRNA / gRNA LNPs. The first plasma sample was collected by serial sampling from mice one day before intravenous administration. Samples were collected from all groups at 4 hours, 24 hours, 72 hours, and one week after administration. A total of 80 μL of whole blood was collected by retroorbital bleeding. The blood was transferred to lithium heparin tubes and centrifuged at 9,800 x g for 10 minutes at 2-5°C. After collection, samples are frozen and stored at or below -70°C for further analysis. Transgenic hFIX protein in plasma is measured by ELISA.

[0307] Example 2. In Vivo Administration of Base Editors to Increase Expression Levels

[0308] Mice (Balb / c; Jackson Laboratory) are intravenously administered a non-expressing variant of recombinant AAV (rAAV) particles expressing human factor IX (FIX). This variant contains a UAG premature termination codon (PTC) in FIX exon 1. The expression cassette is packaged into AAV viral particles by encapsidation within the AAV capsid. Viral particles are typically generated using a triple transfection protocol.

[0308]

[0309] Five weeks after administration, mice were administered an adenine base editor (ABE) and gRNA targeting the FIX exon 1 PTC codon to convert the UAG PTC codon to a glutamine CAG codon. The ABE mRNA and gRNA were formulated into a single LNP for administration. Mice were divided into five groups and administered 0.25 mpk, 0.5 mpk, 1.0 mpk, or 2.0 mpk mRNA / gRNA LNPs, or 2.0 mpk control mRNA / gRNA LNPs. The first plasma sample was collected by serial sampling from mice one day before intravenous administration. Samples were collected from all groups at 4 hours, 24 hours, 72 hours, and one week after administration. A total of 80 μL of whole blood was collected by retroorbital bleeding. The blood was transferred to lithium heparin tubes and centrifuged at 9,800 × g for 10 minutes at 2–5°C. After collection, samples are frozen and stored at or below -70°C for further analysis. Transgenic hFIX protein in plasma is measured by ELISA.

[0309] Example 3. ADAR-based RNA editing of AAV-delivered transgenes

[0310] Editing mRNA transcripts to allow for premature stop codons allows for 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 by introducing a G-to-A point mutation in the TGG codon for tryptophan at amino acid position 118 of the FIX sequence (SEQ ID NO: 53), converting it to a premature or premature stop codon (Figure 1A). This point mutation completely abolished FIX40 protein expression in Huh7 cell transfection experiments, as demonstrated by WES™ automated capillary-based immunoassay analysis (ProteinSimple, Bio-Techne) (Figure 1B) and enzyme-linked immunosorbent assay (ELISA) (Figure 1C).

[0310]

[0311] The ability of endogenous ADARs to perform A-to-I editing on FIX40_W118STOP mRNA was tested in Huh7 cells. Briefly, cotransfection experiments of the FIX40_W118STOP construct and a plasmid carrying a trigger RNA achieved FIX expression after 48 hours, reaching 30–45% of the FIX level obtained from the wild-type FIX expression construct, depending on the FIX construct and trigger RNA concentrations, as detected by WES (Figure 1B) and ELISA (Figure 1C). The trigger RNA was driven by pol3 from a human U6 promoter plasmid. The trigger RNA was 200 nucleotides long, self-circularized, and either perfectly 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, scattered mismatches may contribute to reduced bystander (off-target) editing. Collectively, these results demonstrate that ADAR-based RNA editing in vitro can drive transgene expression.

[0311]

[0312] To clarify 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 by copy number revealed that the introduction of the STOP codon reduced FIX40 mRNA levels by approximately 35% compared to mRNA obtained from the wild-type FIX expression construct (Figure 2, lane 1 vs. lane 6). Without wishing to be bound by any theory, we believe that the reduction in FIX40 mRNA levels is driven by nonsense-mediated decay. Furthermore, comparing protein levels and mRNA abundance in base-edited samples revealed a strong correlation between both measurements (Figure 1B and Figure 1C vs. Figure 2, lanes 2–5). Finally, Sanger sequencing of the mRNA revealed that all of the mRNA molecules were edited from TAG to TGG at W118 (not shown), demonstrating that no bystander editing had occurred, consistent with the high correlation observed between protein and mRNA levels.

[0312] conclusion

[0313] The results indicate that endogenous ADARs can be used as ON switches to control the expression of exogenously delivered transgenes, which is applicable to gene therapy. These in vitro experiments demonstrate that trigger RNA specifically targets ADARs, editing STOP codons in all mRNA molecules produced from the delivered transgene, allowing protein expression.

[0313] Example 4. Mouse In Vivo Test

[0314] To demonstrate that the ADAR-mediated base editing system works in animals, we designed an in vivo mouse study using recombinant AAV (rAAV) particles to deliver a FIX expression cassette payload. Mice were administered intravenously (tail vein) with either AAV-encapsidated FIX40 or AAV-encapsidated FIX40_W118STOP (Table 4). Low and high doses of rAAV were tested to ensure that FIX40_W118STOP mRNA levels were not limiting. [Table 4]

[0314]

[0315] Plasma FIX40 expression was measured by ELISA on days 0, 7, and 14 in animals dosed at 5e9 or 5e10 vg / animal. No FIX40 expression was detected in animals dosed with FIX40_W118STOP (Table 5; BQL: below detection limit). Expression of the control FIX40 construct showed the expected dose- and time-dependent increase from days 7 to 14. Values ​​are expressed as the mean ng / ml of three animals per group. [Table 5]

[0315]

[0316] After establishing a baseline level of FIX40 expression for each group, trigger RNA is delivered via LNP. Based on in vitro results, addition of circular trigger RNA is expected to recruit endogenous ADARs and edit the W118STOP codon to a TGG tryptophan codon. Plasma FIX40 expression levels are measured daily for 4 weeks to determine editing efficiency and persistence. At the end of the study, animals are sacrificed and liver RNA is extracted to determine FIX40 RNA levels and editing efficiency.

[0316] Example 5. In vivo studies to determine editing efficiency and durability of FIX40W118STOP induction by ADAR-cadRNAis complexes

[0317] In follow-up studies, animals will receive rAAV vectors on day 0 and LNP on day 14. FIX40 levels will be measured daily to determine the ability of trigger cadRNAis and endogenous or exogenous ADARs to edit FIX40W118STOP mRNA to allow protein expression. [Table 6]

[0317]

[0318] Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adaptations, 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 for regulating the expression of a gene located in an episomal vector in a subject in need thereof, comprising administering to the subject an editing agent that causes a change in a region of the mRNA transcript of the gene, thereby regulating the expression of the gene.

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

3. The method of claim 1, wherein the alteration 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.

4. The method of claim 2, wherein the region of the mRNA transcript comprises a premature stop codon that can be converted into an amino acid codon via the base change or an amino acid codon that can be converted into a premature stop codon via the base change.

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

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

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

8. 7. The method of claim 6, wherein the targeting ribonucleic acid is circular.

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

10. 10. The method of claim 9, wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, and ADAR3.

11. 11. The method of any one of claims 6 to 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 a base editor, wherein the polynucleotide-programmable RNA-binding domain cooperates with the targeted ribonucleic acid to cause the base change.

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

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

14. (i) the cytidine deaminase domain is selected from the group consisting of the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, 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 method of any one of claims 11 to 13, wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).

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

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

17. 17. The method of claim 16, wherein the targeted ribonucleic acid and / or the one or more nucleic acid molecules, such as one or more mRNA molecules, are administered to the subject together with a lipid nanoparticle (LNP), a peptide cage, or a polymer nanoparticle.

18. 18. The method of any one of claims 2 to 17, wherein the base change causes conversion of an amino acid codon to a premature stop codon, preferably upstream of a splice junction, thereby downregulating expression of the gene.

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

20. 18. The method of any one of claims 2 to 17, wherein the base change causes conversion of a premature stop codon to an amino acid codon, thereby upregulating expression of the gene.

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

22. The method of any one of claims 1 to 21, 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 of any one of claims 1 to 22, further comprising administering the episomal vector containing the gene to the subject.

24. The method of any one of claims 1 to 23, 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. 25. The method of claim 24, wherein the episomal vector is an AAV vector.

26. 26. The method of any one of claims 1 to 25, wherein 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, synucleinopathy, epilepsy, neuropathic pain, wet macular degeneration, Usher syndrome type 1F, Usher syndrome type 1B, glaucoma, Leber's congenital amaurosis, and Stargardt disease.

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