Systems for enhancing expression of target mRNA and uses thereof

Abstract

Provided herein are nucleic acid agents that are or comprise one or more nucleic acid molecules that together comprise (a) a complementary element that hybridizes to a target mRNA; and (b) a polyadenylic acid element. In some embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder associated with reduced protein expression by the mRNA, where the disorder is a haplodose insufficiency disorder.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 350,908 filed on June 10, 2022, U.S. Provisional Patent Application No. 63 / 407,964 filed on September 19, 2022, and U.S. Provisional Patent Application No. 63 / 434,367 filed on December 21, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in an XML file named 44807-0434WO1_ST26.XML. This XML file was created on June 7, 2023 and is 15501 bytes in size. The entire contents of the material in this XML file are incorporated herein by reference. Background Art

[0005] Although changes in gene expression are often thought to reflect programmed transcriptional variability, it is less well known that extensive regulation of messenger RNA expression also occurs during its translation. mRNA translation rate is a key feature that defines post-transcriptional regulation. All transcripts are translated at unique rates, and these rates can be controlled; significantly affecting the protein output of each mRNA molecule. Cells achieve translational regulation by recruiting specific positive or negative factors to mRNA through sequence and / or structural elements.

[0006] Human mRNA transfers genetic information from DNA to protein. Not only does mRNA accurately transfer genetic information, but it also imparts this information at the correct level. The amount of protein from mRNA can be determined by the mRNA translation rate, the time period over which translation occurs, and / or the stability of the mRNA transcript. A key feature that determines how long it takes for an mRNA to synthesize its protein is its 3' polyadenosine tail.

[0007] Almost all human mRNAs carry a poly-A tail at their 3' end. The average length of this tail in humans is about 200 nt. The poly-A tail acts as a major regulator of gene expression in the cytoplasm. Once the mRNA is in the cytoplasm, its poly-A tail is periodically removed by the deadenylase complex. When the poly-A tail is completely removed, translation of the mRNA stops and the mRNA is typically destroyed. Thus, the poly-A tail acts like a slow-burning fuse, determining how long a single mRNA will continue to synthesize protein. Summary of the invention

[0008] Targeting messenger RNA (mRNA) expression can provide therapeutic approaches to modulate mRNA translation and / or treat haploinsufficiency conditions. Human mRNA transfers genetic information from DNA to protein, wherein the amount of protein from the mRNA can be determined by the translation rate of the mRNA, the time period over which translation occurs, and / or the stability of the mRNA transcript.

[0009] This article describes a molecular therapeutic strategy that uses post-transcriptional regulation of target mRNA to enhance protein expression of target mRNA. Cells can recruit specific positive or negative factors to mRNA through sequence and / or structural elements to achieve translational regulation, and a key feature of the length of time that mRNA synthesizes its protein is its 3' poly-A tail. The poly-A tail is the main regulator of gene expression in the cytoplasm, where once the mRNA is present in the cytoplasm, its poly-A tail is regularly removed by the deadenylase complex. When the poly-A tail is completely removed, the mRNA stops translating, and the mRNA is usually destroyed. Therefore, the poly-A tail is like a slow-burning fuse, which determines the length of time that a single mRNA will continue to synthesize protein.

[0010] Provided herein are nucleic acid agents comprising: (a) a complementary element that hybridizes to a target mRNA; and (b) a polyadenylation region. In some embodiments, the complementary element hybridizes to the 3' untranslated region of the target mRNA. In some embodiments, the complementary element comprises RNA and / or DNA.

[0011] In some embodiments, the nucleic acid agent provided is a single nucleic acid molecule or comprises a single nucleic acid molecule, which comprises a complementary element (e.g., a complementary region) and a poly(A) element (e.g., a poly(A) region). In some such embodiments, the poly(A) region is located at the 5' end of the complementary region. In some embodiments, the poly(A) region is located at the 3' end of the complementary region. In some embodiments, the poly(A) region is located at the 3' end of the complementary region, and the nucleic acid molecule further comprises a 5' cap region located at the 5' end of the complementary region. In some embodiments, a nucleic acid molecule that can be used as a nucleic acid agent according to the present disclosure comprises a first poly(A) region located at the 5' end of the complementary region and a second poly(A) region located at the 3' end of the complementary region.

[0012] In some embodiments, the poly(A) element (e.g., poly(A) region) comprises about 20 nucleotides. In some embodiments, the poly(A) element (e.g., poly(A) region) comprises about 50 nucleotides. In some embodiments, the poly(A) element (e.g., poly(A) region) comprises about 75 nucleotides.

[0013] In some embodiments, the nucleic acid agent provided is a nucleic acid molecule containing a nucleotide modification or comprises a nucleic acid molecule containing a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A) or adenosine to inosine (A to I) modification. In some embodiments, the relevant nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU) or N4-acetylcytidine modification.

[0014] In some embodiments, the nucleic acid agent provided is a nucleic acid molecule encoded by a sequence or comprises a nucleic acid molecule encoded by a sequence, wherein the sequence comprises SEQ ID NO:2 or SEQ ID NO:3 or consists of SEQ ID NO:2 or SEQ ID NO:3, or has at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:2 or SEQ ID NO:3.

[0015] In some embodiments, the target mRNA is an mRNA encoding a polypeptide of interest (e.g., increased expression of a polypeptide provides an activity or benefit of interest to or in the cell, tissue, system, or organism in which it is expressed).

[0016] In some embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder associated with reduced protein expression by the mRNA. In some embodiments, the disorder is a haploinsufficiency disorder. In some embodiments, the target mRNA encodes a MeCP2 protein. In some embodiments, the complementary region comprises or consists of SEQ ID NO: 1.

[0017] In some embodiments, the target mRNA encodes a variant of a polypeptide of interest having reduced activity.

[0018] In some embodiments, the target mRNA can be exogenous to the cell, tissue, organism or other system of interest. For example, in some embodiments, the target mRNA can be a therapeutic mRNA and / or can be a transcript of a gene contained in a gene therapy agent or a cell therapy agent or otherwise provided by a gene therapy agent or a cell therapy agent.

[0019] In some specific embodiments, the target mRNA may encode a toxic polypeptide or a suicide polypeptide, for example, the expression of the toxic polypeptide or suicide polypeptide reduces the activity of cells expressing it or otherwise contacting it (e.g., by inducing or promoting cell death apoptosis or cell necrosis). In some specific such embodiments, such cells may be diseased cells (e.g., cancer cells); alternatively or additionally, in certain specific such embodiments, such cells may be cell therapy products (e.g., engineered T cells such as CAR-T cells, or engineered NK cells, or engineered B cell preparations, etc.).

[0020] Also provided herein is a recombinant expression system comprising a nucleic acid sequence encoding a nucleic acid, the nucleic acid being or comprising one or both of the following: (i) a complementary element (e.g., a complementary region) hybridized to a target mRNA and (ii) a polyadenylic acid element (e.g., a polyadenylic acid region). In some embodiments, the complementary region hybridizes to the 3' untranslated region of a target mRNA as described herein. In some embodiments, the complementary element comprises RNA and / or DNA. In some embodiments, the polyadenylic acid element is located at the 5' end of the complementary element. In some embodiments, the polyadenylic acid element is located at the 3' end of the complementary element. In some embodiments, the polyadenylic acid element is located at the 3' end of the complementary element, and wherein the nucleic acid agent further comprises a 5' cap element located at the 5' end of the complementary element. In some embodiments, the nucleic acid agent comprises a first polyadenylic acid element located at the 5' end of the complementary element and a second polyadenylic acid element located at the 3' end of the complementary element.

[0021] In some embodiments, the poly(A) element comprises about 20 nucleotides. In some embodiments, the poly(A) element comprises about 50 nucleotides. In some embodiments, the poly(A) element comprises about 75 nucleotides.

[0022] In some embodiments, the nucleic acid agent further comprises a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A) or adenosine to inosine (A to I) modification. In some embodiments, the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU) or N4-acetylcytidine modification.

[0023] In some embodiments, the recombinant expression system is encoded by a sequence comprising or consisting of SEQ ID NO:2 or SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:3.

[0024] In some embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder associated with reduced protein expression by the mRNA. In some embodiments, the disorder is a haploinsufficiency disorder. In some embodiments, the target mRNA encodes a MeCP2 protein. In some embodiments, the nucleic acid molecule comprises or consists of SEQ ID NO: 1.

[0025] Also provided herein is an expression vector comprising any of the recombinant expression systems described herein. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV), a lentiviral vector, or an adenoviral vector.

[0026] Also provided herein are pharmaceutical compositions comprising or delivering a nucleic acid agent as described herein (e.g., one or more nucleic acid molecules that together comprise (i) a complementation element; and / or (ii) a polyadenylation element); a recombinant expression system as described herein (e.g., an expression vector as described herein).

[0027] Also provided herein are methods for increasing expression of a protein (eg, increasing protein (ie, polypeptide) levels) encoded by an mRNA of interest as described herein.

[0028] In some embodiments, the present disclosure provides methods for treating a haploinsufficiency disorder in a subject, e.g., comprising administering to such a subject a therapeutically effective amount of any of the pharmaceutical compositions described herein. In some embodiments, the therapeutically effective amount of the pharmaceutical composition enhances target mRNA protein expression. In some embodiments, the haploinsufficiency disorder is selected from the group consisting of 5q syndrome, Adams-Oliver syndrome type 1, Adams-Oliver syndrome type 3, Adams-Oliver syndrome type 5, Adams-Oliver syndrome type 6, Alagille syndrome type 1, autoimmune lymphoproliferative syndrome type IA, autoimmune lymphoproliferative syndrome type V, autosomal dominant deafness-2A, brain malformation with or without urinary tract defects (BRMUTD), Carney complex type 1, CHARGE syndrome, cleidocranial dysplasia, Currarino syndrome, Denys-Drash / Frasier syndrome, developmental delay-intellectual disability-obesity and dysmorphic features (DIDOD), DiGeorge syndrome (TBXI-related), Dravet syndrome, Duane-radial syndrome (DIDOD), and raysyndrome), Ehlers-Danlos syndrome (classic type),Ehlers-Danlos syndrome (vascular type), Feingold syndrome 1, frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP) GRN-related, GLUT I deficiency syndrome, Greig polysyndactyly syndrome, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly type 3, holoprosencephaly type 4, holoprosencephaly type 5, Holt-Oram syndrome, hypoparathyroidism-sensorineural hearing loss-kidney disease (HDR), Kleefstra syndrome 1, venous malformation hyperostosis syndrome (Klippel-Trenaunay syndrome (AAGF-related), Leri-Weill achondroplasia, Marfan syndrome, mental retardation and distinctive facial features with or without cardiac defects (MRFACD), mental retardation, autosomal dominant 1, mental retardation, autosomal dominant 19, mental retardation, autosomal dominant 29, nail-patella syndrome (NPS), Phelan-McDermid syndrome, Pitt-Hopkins syndrome, primary pulmonary hypertension 1, Rett syndrome (congenital variant), Smith-Magenis syndrome (RAII-related), Sotos syndrome 1, Sotos syndrome 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAPI-related intellectual disability, Treacher Collins syndrome (Treacher Collins syndrome), Collins syndrome, tricho-nasal-phalangeal syndrome type 1, ulna-mammary syndrome, van der Woude syndrome 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the field to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0030] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A to 1C An exemplary schematic diagram depicting the design of RNA enhancement systems (red) provided by the present disclosure is shown. Three types of systems are shown, each of which has a region homologous to a gene of interest. The region is antisense to the mRNA of interest, and is typically antisense to a region in the 3' untranslated region of the mRNA of interest. Each system has a polyadenylated stretch (5' end of the antisense sequence ( Figure 1A ), the 3' end of the antisense sequence ( Figure 1B ) or both ( Figure 1C )). Each system can have unique nucleotide modifications at the 5' and 3' ends.

[0032] FIG. 2A to FIG. 2B Results of Western blots for MeCP2 protein are shown. Cells were transfected with different RNA enhancement systems that had the antisense region of MeCP2 mRNA. GAPDH is shown as a control ( Figure 2A ). Quantification of relative protein expression is shown in Figure 2B middle.

[0033] FIG. 3A to FIG. 3B An exemplary schematic diagram depicting the design of an RNA enhancement system (red) is shown. Two types of systems are shown, each with a region of homology to the gene of interest. Each system has a polyadenylated stretch, a 5' end of the antisense sequence ( Figure 3A , upper figure) or the 3' end of the antisense sequence and also contains a 5' cap ( Figure 3A , figure below). Figure 3B Results of Western blots for MeCP2 protein are shown. Cells were transfected with different RNA enhancer systems with the antisense region of MeCP2 mRNA. GAPDH is shown as a control.

[0034] Figure 4 Results are shown for MeCP2 expression in 3T3 cells incubated with enhancer RNA for 16 hours, where the results show uncapped short RNA and capped RNA.

[0035] Figure 5 Shown are the q-PCR results of SYNGAP1 expression in SH-SY5Y cells incubated with SYNGAP1 enhancing RNA for 16 h.

[0036] Figure 6Results of qPCR in mouse liver are shown, indicating that MeCP2 mRNA increased more than two-fold in mice injected with the enhancer RNA.

[0037] Figure 7 Results for MeCP2 protein levels in mouse liver are shown, indicating up to two-fold increase in MeCP2 protein expression in mice injected with booster RNA. The booster RNA was mixed with lipid nanoparticles (LNPs) and injected into the tail vein. Each mouse was injected with 25 μg of booster RNA.

[0038] Figure 8 An exemplary schematic diagram describing the design of an exemplary RNA enhancing system is shown. The depicted enhancing RNA comprises a homologous region to a gene of interest (e.g., an antisense guide region). This region is antisense to the mRNA of interest, and is typically antisense to a region in the 3' untranslated region of the mRNA of interest. The system also comprises a polyadenylation segment (e.g., located at the 5' end of the antisense sequence and having a unique nucleotide modification at the 5' end).

[0039] Fig.9A and Fig. 9B The results of qPCR assays are shown, indicating that 24 h after transfection of Hek-293 cells with enhanced RNA levels, the β-catenin gene ( Fig.9A ) and PurA gene ( Fig. 9B ) mRNA levels were increased.

[0040] Fig. 10A and Fig. 10B Results of deep brain perfusion of SYNGAP1 enhancer RNA in wild-type animals are shown, demonstrating significant changes in SYNGAP1 levels in different brain regions 24 hours after injection. Two different types of enhancers (1-tailed PA-SG and 2-tailed PA-SG-PA) targeting the same region on the 3'UTR of SYNGAP1 were injected into the brains of wild-type mice. q-PCR ( Fig. 10A ) and Western blot ( Fig. 10B ) showed that SYNGAP1 levels increased in different brain regions. The results showed that SYNGAP1 levels increased by 30% to 40% in the hippocampus, cortex and midbrain, but there was no significant change in the cerebellum.

[0041] Fig.11 An exemplary schematic diagram showing the structure of the SYNGAP1 gene.

[0042] Fig.12Results of targeting SYNGAP1 compared to animals injected with scrambled RNA and non-operated animals are shown, where deep brain perfusion was performed in multiple animals under each condition. Results from RNA analysis and western blots show that SYNGAP1 levels were significantly altered in the hippocampus 48 hours after injection. DETAILED DESCRIPTION

[0043] The present disclosure provides that targeting messenger RNA (mRNA) expression can provide the discovery of the therapeutic method of regulating mRNA translation and / or treating haploinsufficient disease. The mRNA translation rate is a key feature that limits post-transcriptional regulation, in which all transcripts are translated at unique rates and these rates can be controlled, significantly affecting the protein output of each mRNA molecule. Cells can recruit specific positive or negative factors to mRNA through sequence and / or structural elements to achieve translational regulation.

[0044] Described herein are molecular therapeutic strategies that use post-transcriptional regulation of a target mRNA to enhance protein expression of a target mRNA.One skilled in the art reading this disclosure will appreciate that the provided technology will be useful in a variety of contexts.

[0045] To name just a few examples, in some embodiments, the systems provided can be designed and / or used to increase protein expression of a target mRNA endogenous to a host cell. Alternatively or additionally, in some embodiments, the systems provided can be designed and / or used to increase protein expression of an mRNA heterologous to a host cell (e.g., the system may have been introduced into such a host cell or may be, for example, a progeny of a transcription product and / or replication product of a nucleic acid that has been introduced into such a host cell). In some embodiments, the systems and methods provided can provide improved treatment for a disease (e.g., a haploinsufficient disorder) by using a nucleic acid agent (e.g., an "RNA enhancer") that contains a key positive mRNA regulatory factor (e.g., a polyadenylation region) that binds to and remains resident in the mRNA, thereby enhancing the expression of wild-type (WT) mRNA in a precise manner and restoring protein levels to normal.

[0046] In some embodiments, provided herein are nucleic acid agents comprising (a) complementary elements that hybridize to a target mRNA; and (b) a polyadenylation element. In some embodiments, provided herein are nucleic acid agents that are nucleic acid molecules or comprise nucleic acid molecules that comprise (a) a complementary region that hybridizes to a target mRNA; and (b) a polyadenylation region.

[0047] Also provided herein is a recombinant expression system comprising a nucleic acid sequence encoding a nucleic acid molecule comprising (i) a complementary element that hybridizes to a target mRNA and (ii) a polyadenylation element.

[0048] Various non-limiting aspects of these systems are described herein and may be used in any combination without limitation. Additional aspects of the individual components of these systems are known in the art.

[0049] Specific Definition

[0050] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the term "administering" generally refers to administering a composition to an object or system to realize the delivery of a composition or a medicament contained in the composition. Those of ordinary skill in the art will understand that various approaches that can be used to administer to an object (e.g., a person) in appropriate circumstances. For example, in some embodiments, administration can be administration through the eye, oral administration, parenteral administration, topical administration, etc. In some embodiments, administration can be bronchial (e.g., by bronchial instillation), cheek, dermis (which can be or include, for example, topical administration to one or more of dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a specific organ (e.g., intrahepatic), mucosa, nose, oral cavity, rectum, subcutaneous, sublingual, local, tracheal (e.g., by intratracheal instillation), vagina, vitreous, etc. administration. In some embodiments, administration can only involve a single dose. In some embodiments, administration can involve applying a fixed number of doses. In some embodiments, administration can involve intermittent (e.g., multiple doses separated in time) administration and / or periodic (e.g., individual doses separated by a common time period) administration. In some embodiments, administration can involve continuous administration (e.g., perfusion) for at least a selected time period.

[0052] As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components or parts with a reference substance. Typically, an "analog" shows significant structural similarity to a reference substance, such as a shared core or a shared consistent structure, but also differs in some discrete ways. In some embodiments, an analog is a substance that can be generated from a reference substance, for example, by chemically manipulating the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process substantially similar to the synthetic process for generating a reference substance (e.g., having a plurality of steps in common with the synthetic process for generating a reference substance). In some embodiments, an analog is or can be generated by performing a synthetic method different from the synthetic method for generating a reference substance.

[0053] If the presence, level, degree, type and / or form of one event or entity is related to the presence, level, degree, type and / or form of another event or entity, then the two events or entities are "associated" with each other, as the term is used herein. For example, a particular entity (e.g., a polypeptide, a gene marker, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder or condition if its presence, level and / or form is related to the incidence, susceptibility, severity, stage, etc. of the disease, disorder or condition (e.g., in a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly, such that they are physically close to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently associated, such as by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0054] As used herein, the term "binding" generally refers to non-covalent binding between two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts, including where the interacting entities or moieties are studied individually or in the context of a more complex system (e.g., simultaneously covalently or otherwise bound to a carrier entity and / or in a biological system or cell).

[0055] As used herein, "cell" may refer to a prokaryotic cell or a eukaryotic cell, optionally obtained from a subject or a commercial source.

[0056] As used herein, the phrase "characteristic sequence element" refers to a sequence element representing a characteristic portion of a polymer found in a polymer (e.g., in a polypeptide or nucleic acid). In some embodiments, the presence of a characteristic sequence element is related to the presence or level of a specific activity or property of a polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a specific polymer as a member (or not a member) of a specific family or group of such a polymer. A characteristic sequence element generally comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more monomers (e.g., monomers connected continuously). In some embodiments, a characteristic sequence element comprises at least the first and second extensions of continuous monomers separated by one or more spacer regions, and the length of the spacer region may vary or not vary in the polymer of the common sequence element.

[0057] As used herein, the term "comparable" refers to two or more agents, entities, situations, condition groups, etc., which may be different from each other, but are similar enough to allow comparisons between them, so that those skilled in the art will understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, the comparable condition groups, environments, individuals, or groups are characterized by a plurality of substantially identical features and one or a small amount of different features. Those of ordinary skill in the art will understand that, in the context of any given situation, two or more such agents, entities, situations, condition groups, etc. require what degree of consistency to be considered comparable. For example, those of ordinary skill in the art will understand that when there are features of substantially the same features of sufficient quantity and type, the collection of environments, individuals, or groups is comparable to each other to ensure a reasonable conclusion that the difference in the results or observed phenomena obtained under or using different environments, individuals, or groups is caused by or indicates the change of those change features.

[0058] As used herein, "delivery", "gene delivery", "gene transfection", "transduction" may refer to the introduction of exogenous polynucleotides into a host cell, regardless of the method used for introduction. These methods include various well-known techniques, such as vector-mediated gene transfection (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and technologies that facilitate "naked" polynucleotide delivery (e.g., electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides). In some embodiments, the introduced polynucleotide can be stably maintained in the host cell; in some embodiments, the introduced polynucleotide can be transiently maintained. Stable maintenance generally requires that the introduced polynucleotide contains a replication origin compatible with the host cell, or is integrated into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear chromosome or mitochondrial chromosome.

[0059] In some embodiments, the polynucleotide can be inserted into the host cell by a gene delivery system. Examples of gene delivery systems can include, but are not limited to, lipid-based systems (e.g., liposomes, micelles, lipid nanoparticles, lipid complexes), polymer-based systems (e.g., polymer particles (e.g., microparticles or nanoparticles)), metal-based systems (e.g., metal or metal oxide particles (e.g., microparticles or nanoparticles)), or bacteria and / or virus-based systems, or virus-like systems. In some embodiments, the delivery system can be or include one or more lipids, polypeptides, polysaccharides, metals, metal oxides, etc. Alternatively or additionally, in some embodiments, the delivery system can be or comprise one or more lipopolypeptides, lipopolysaccharides, liposomes, lipid nanoparticles, micelles, biocompatible polymers (including natural polymers and synthetic polymers); lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles and bacteria or viruses (e.g., baculoviruses, adenoviruses and retroviruses, bacteriophages), cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art, which have been described for use in various eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0060] As used herein, the term "designed" refers to an agent that (i) has a structure that is or is artificially selected; (ii) is produced by methods that require artificial intelligence; and / or (iii) is different from natural substances and other known agents.

[0061] As used herein, the terms "encoding" or "encoded" are used to describe the relationship between a nucleic acid and a polypeptide. For example, an mRNA whose sequence can be translated into a polypeptide (e.g., by the action of ribosomes) is said to "encode" the polypeptide. In addition, a nucleic acid (e.g., DNA or RNA), which can be used to generate such an mRNA through one or more replication steps (e.g., transcription, reverse transcription and / or other polymerization) and / or processing (e.g., splicing, capping, editing, etc.), is also said to "encode" the related polypeptide. The nucleic acid strand that encodes the polypeptide is called the "coding" strand; its complementary strand is the "antisense" strand.

[0062] As described herein, the term "exogenous" refers to a substance introduced into a cell, tissue, or organism (e.g., artificially) that originates from a source different from the cell, tissue, or organism into which it is introduced, or that does not naturally occur in such a cell, tissue, or organism. One skilled in the art will appreciate that in certain embodiments, such a material may still be considered "exogenous" to a cell, tissue, or organism (i.e., a descendant cell, tissue, or organism of the cell, tissue, or organism into which the material was originally introduced).

[0063] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed (and / or optionally processed, such as by one or more of splicing, capping, editing, etc.) into mRNA and / or the process by which mRNA is translated into peptides, polypeptides, or proteins. In some embodiments, for example, if the expressed polynucleotide is from or derived from genomic DNA, expression may include splicing and / or other processing or modification to produce mRNA (e.g., in eukaryotic cells). In some embodiments, the expression level of a gene can be determined, for example, by measuring the amount of mRNA or protein encoded thereby in a sample (e.g., a cell or tissue sample) expressing the gene; in some embodiments, the expression levels of multiple genes can be determined, for example, substantially simultaneously, such as to establish an expression profile for a particular sample.

[0064] As used herein, the term "nucleobase" refers to a portion of a nucleic acid that participates in hydrogen bonding a nucleic acid strand or sequence element to another complementary strand or sequence element in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase comprises a heteroaryl ring, wherein the ring atoms are nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, the nucleobase comprises a heterocycle, wherein the ring atoms are nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, the nucleobase is a "modified nucleobase", for example, a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C) and thymine (T). In some embodiments, the modified nucleobase is a substituted A, T, C, G or U. In some embodiments, the modified nucleobase is a substituted tautomer of A, T, C, G or U. In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine or thymine. In some embodiments, the modified nucleobase simulates the spatial arrangement, electronic properties or some other physicochemical properties of the nucleobase and retains the properties of hydrogen bonds that bind one nucleic acid strand to another nucleic acid strand in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of oligonucleotide duplexes. In some embodiments, as will be appreciated by those skilled in the art, the term "nucleobase" encompasses structural analogs, such as modified nucleobases and nucleobase analogs, that are used to replace natural or naturally occurring nucleotides. In some embodiments, a nucleobase is alternatively a substituted A, T, C, G, or U, or alternatively a substituted tautomer of A, T, C, G, or U. In some implementations, a "nucleobase" refers to a nucleobase unit (e.g., A, T, C, G, or U, as in an oligonucleotide or nucleic acid) in a nucleic acid.

[0065] As used herein, the term "nucleoside" refers to a portion in which a nucleobase or modified nucleobase is covalently bound to a sugar or modified sugar. In some embodiments, a nucleoside is a natural nucleoside, for example, adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine or deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, for example, a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine and deoxycytidine. In some embodiments, a nucleoside is a modified nucleoside, for example, a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine and deoxycytidine. In some embodiments, a "nucleoside" refers to a nucleoside unit in an oligonucleotide or a nucleic acid.

[0066] As used herein, the term "nucleotide" refers to a nucleotide consisting of a nucleobase, a sugar, and one or more internucleotide bonds (e.g., phosphate bonds in natural DNA and RNA). Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, but it should be understood that natural and non-natural base analogs are also included. Naturally occurring sugars are pentose (five-carbon sugars) deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that in various embodiments, as will be apparent to those skilled in the art, natural and non-natural sugar analogs are also included. Nucleotides are linked by internucleotide bonds to form nucleic acids or polynucleotides. Various internucleotide bonds are known in the art (e.g., but not limited to, phosphates, phosphorothioates, boronate phosphates, etc.). Artificial nucleic acid includes PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, boranophosphate, methylphosphonate, phosphonoacetate, thiophosphonoacetate and other variants of the phosphate backbone of natural nucleic acid. In some embodiments, natural nucleotides include naturally occurring bases, sugars and internucleotide bonds. As used herein, the term "nucleotide" also encompasses structural analogs for replacing natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, "nucleotide" refers to a nucleotide unit in a polynucleotide.

[0067] As used herein, "nucleic acid" in its broadest sense refers to a compound and / or substance that is a polynucleotide chain, or a compound and / or substance that can be incorporated into a polynucleotide chain. In some embodiments, nucleic acid is a compound and / or substance that is a polynucleotide chain with a phosphodiester bond, or a compound and / or substance that can be incorporated into a polynucleotide chain with a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to a polynucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" is or comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization) or a combination thereof. In some embodiments, nucleic acid is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues or is composed of one or more natural nucleic acid residues. In some embodiments, nucleic acid is one or more nucleic acid residue analogs, comprises one or more nucleic acid residue analogs or is composed of one or more nucleic acid residue analogs. In some embodiments, nucleic acid analogs are different from nucleic acids in that: one or more residues, and in some embodiments, are linked together by means other than phosphodiester bonds. For example, in some embodiments, nucleic acid comprises one or more thiophosphate bonds and / or phosphoramidite bonds (e.g., 5'-N-phosphoramidite bonds) and does not comprise phosphodiester bonds. In some embodiments, nucleic acid includes one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine). In some embodiments, the nucleic acid is or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrole-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a protein.In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) based on polymerization of a complementary template, enzymatic synthesis in the absence of a complementary template, replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180 In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence encoding a polypeptide.

[0068] Those skilled in the art will appreciate that a variety of techniques including, for example, site-directed mutagenesis, polymerase chain reaction (PCR)-mediated mutagenesis, chemical mutagenesis, and the like, can be used to introduce modifications into a nucleic acid.

[0069] Conservative amino acid substitution refers to the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. Examples of such families are: amino acids with basic side chains (e.g., arginine, lysine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine), amino acids with β-branched side chains (e.g., isoleucine, threonine, and valine), and amino acids with aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine), and amino acids with aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine).

[0070] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition is suitable for administration to a human or animal subject by a specific route of administration (e.g., parenteral administration). In some embodiments, the active agent is present in a pharmaceutical composition in a unit dose suitable for administration in a treatment regimen, and the pharmaceutical composition shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0071] As used herein, the term "polypeptide" generally has the meaning of a polymer of at least three amino acids recognized in its field. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides with complete sequences as described herein, but also polypeptides representing functional fragments of these complete polypeptides (i.e., fragments retaining at least one activity). In addition, it will be understood by those of ordinary skill in the art that protein sequences generally tolerate some substitutions without destroying activity. Therefore, the related term "polypeptide" used herein encompasses any retention of activity and shares at least about 30% to 40% with another polypeptide of the same class, generally greater than about 50%, 60%, 70% or 80% overall sequence identity, and also generally contains at least one polypeptide with very high, generally greater than 90% or even 95%, 96%, 97%, 98% or 99% identity in one or more highly conserved regions, the polypeptide generally encompassing at least 3 to 4, generally up to 20 or more amino acids. The polypeptide may include L-amino acids, D-amino acids or both, and may include any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the protein may include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, and a biologically active portion thereof and / or a characteristic portion thereof.

[0072] As used herein, "prevention" when used in conjunction with the occurrence of a disease, disorder, and / or condition refers to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition. In some embodiments, prevention can be considered complete when the onset of a disease, disorder, or condition has been delayed for a predefined period of time.

[0073] As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which binding will occur. When other potential targets are present, a binding agent that interacts with a particular target is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the extent of binding between a binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0074] As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a specific or, in some embodiments, does not exhibit any symptoms or features of any disease, disorder, or condition. In some embodiments, the subject is a subject having one or more features of susceptibility or risk to a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is receiving and / or has received diagnosis and / or treatment.

[0075] As used herein, the term "treatment" refers to the administration of a therapy that partially or completely alleviates, improves, alleviates, inhibits one or more symptoms, features, and / or causes of a specific disease, disorder, and / or condition, delays its onset, reduces its severity, and / or reduces its incidence. In some embodiments, such treatment may be treatment for subjects that do not show signs of the relevant disease, disorder, and / or condition and / or for subjects that only show early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment for subjects that show one or more confirmed signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment for subjects that have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment for subjects that are known to have one or more susceptibility factors that are statistically associated with an increased risk of development of the relevant disease, disorder, and / or condition. Therefore, in some embodiments, treatment may be preventive; in some embodiments, treatment may be therapeutic.

[0076] As used herein in the context of molecules such as nucleic acids, proteins or small molecules, the term "variant" refers to a molecule that shows significant structural consistency with a reference molecule but is structurally different from a reference molecule, for example, the presence or absence of one or more chemical moieties or at the level of one or more chemical moieties compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered to be a "variant" of a reference molecule is based on the degree of structural consistency with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that has one or more such characteristic structural elements in common but is different from a reference molecule in at least one aspect. To name just a few examples, a polypeptide can have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to each other in a linear or three-dimensional space and / or contribute to a specific structural motif and / or biological function; a nucleic acid can have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to each other in a linear or three-dimensional space. In some embodiments, a variant polypeptide or variant nucleic acid may be different from a reference polypeptide or reference nucleic acid due to one or more differences in the sequence of amino acids or nucleotides and / or one or more differences in a chemical moiety (e.g., carbohydrates, lipids, phosphate groups) that is a covalent component of a polypeptide or nucleic acid (e.g., attached to the backbone of a polypeptide or nucleic acid). In some embodiments, a variant polypeptide or variant nucleic acid shows an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or variant nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or variant nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or variant nucleic acid lacks one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as a reference polypeptide or nucleic acid, but has a small sequence change at a specific position. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are replaced, inserted, or deleted compared to the reference.In some embodiments, the variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to a reference. Typically, the variant polypeptide or nucleic acid comprises a very small amount (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity) relative to a reference. In some embodiments, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to a reference, and in some embodiments, does not comprise additions or deletions. In some embodiments, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and generally less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0077] Nucleic acid reagents provided

[0078] The present disclosure provides certain nucleic acid agents as described herein, which can be used, for example, to enhance mRNA translation. In some embodiments, the provided nucleic acid agent is or comprises one or more nucleic acid molecules.

[0079] In some embodiments, a provided nucleic acid agent comprises (a) a complementary element connected to or otherwise bound to (b) a poly(A) element. In some embodiments, a provided nucleic acid agent consists of two or more different nucleic acid molecules that bind to each other, for example, by hybridization (e.g., by complementary binding elements on different molecules), the binding elements hybridize to each other and thereby bind the separated nucleic acid molecules to each other and form the provided nucleic acid agent, and / or by covalent bonding such as chemical bonds (e.g., by phosphodiester bonds or other internucleoside bonds, or by bonds generated, for example, by chemical reactions (e.g., by click chemistry). In many embodiments, a provided nucleic acid agent consists of a single nucleic acid molecule (e.g., a chain), wherein the complementary element and the poly(A) element are different regions of the same chain.

[0080] Thus, in some embodiments, provided herein are nucleic acid molecules comprising (a) a complementary region that hybridizes to a target mRNA; and (b) a polyadenylation region.

[0081] Also provided herein is a recombinant expression system comprising a nucleic acid sequence encoding one or both of a complementary element and a polyadenylic acid element as described herein. For example, in some embodiments, a recombinant expression system is provided that encodes an RNA molecule comprising (i) a complementary region that hybridizes to a target mRNA and (ii) a polyadenylic acid region.

[0082] In some embodiments, provided herein is an expression vector (e.g., a single vector or multiple vectors) comprising any one of the recombinant expression systems described herein. In some embodiments, the expression vector provided is a viral vector. In some embodiments, such a viral vector is an adeno-associated viral vector (AAV), a lentiviral vector, or an adenoviral vector.

[0083] In some embodiments, provided herein are cells comprising any one or more of the provided nucleic acid agents and / or recombinant expression systems (eg, one or more expression vectors described herein).

[0084] In some embodiments, a nucleic acid agent is provided that comprises about 50 to about 200 (e.g., about 50 to about 180, about 50 to about 160, about 50 to about 150, about 50 to about 140, about 50 to about 120, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 200, about 60 to about 180, about 60 to about 160, about 60 to about 150, about 60 to about 140, about 60 to about 120, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 60 to about 70, about 70 to about 200, about 70 to about 180, about 70 to about 160, about 70 to about 150, about 70 to about 140, about 70 to about 120, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 80 to about 200, about 80 to about 180, about 80 to about 160, about 80 to about 150, about 80 to about 140, about 80 to about 120 to about 120, about 80 to about 100, about 80 to about 90, about 90 to about 200, about 90 to about 180, about 90 to about 160, about 90 to about 150, about 90 to about 140, about 90 to about 120, about 90 to about 100, about 100 to about 200, about 100 to about 180, about 100 to about 160, about 100 to about 150, about 100 to about 140, about 100 to about 120, about 120 to about 200, about 120 to about 180 , about 120 to about 160, about 120 to about 150, about 120 to about 140, about 140 to about 200, about 140 to about 180, about 140 to about 160, about 140 to about 150, about 150 to about 200, about 150 to about 180, about 150 to about 160, about 160 to about 200, about 160 to about 180, or about 180 to about 200) nucleic acid residues (e.g., nucleotides), for example, as a single nucleic acid molecule, or as two or more different nucleic acid molecules bound to each other. In some embodiments, a provided nucleic acid agent can include about 100 to about 150 nucleic acid residues (e.g., nucleotides).

[0085] In some embodiments, the nucleic acid agent provided comprises or is composed of natural nucleic acid residues. In some embodiments, the nucleic acid agent provided comprises one or more nucleic acid residue analogs (e.g., one or more nucleotide analogs, such as one or more sugar and / or base analogs and / or one or more non-phosphodiester internucleoside bonds).

[0086] In some embodiments, the nucleic acid agent provided comprises a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A), or adenosine to inosine (A to I) modification. In some embodiments, the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. In some embodiments, the nucleotide modification is 1,2'-O-dimethyladenosine (m1Am), 1,2'-O-dimethylguanosine (m1Gm), 1,2'-O-dimethylinosine (m1Im), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Y), 1-methyladenosine (m1A), 1-methylguanosine (m1G), 1-methylinosine (m1I), 1-methylpseudouridine (m1Y), 2,8-dimethyladenosine (m2,8A), 2-geranylthiouridine (ges2U), 2-lysine (k2C), 2-methyladenosine (m2A), 2-methyl Thiomethylenethio-N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6-threonylaminoformyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalylaminoformyladenosine (ms2hn6A), 2-methylthio-N6-isopentenyl adenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylaminoformyladenosine (ms2t6A), 2-selenouridine (se2U), 2-thio-2 '-O-methyluridine (s2Um), 2'-O-thiocytidine (s2C), 2-thiouridine (s2U), 2'-O-methyladenosine (Am), 2'-O-methylcytidine (Cm), 2'-O-methylguanosine (Gm), 2'-O-methylinosine (Im), 2'-O-methylpseudouridine (Ym), 2'-O-methyluridine (Um), 2'-O-methyluridine 5-hydroxyacetate (mcmo5Um), 2'-O-riboadenosine (phosphate) (Ar(p)), 2'-O-riboguanosine (phosphate) (Gr(p)), 3,2'-O-dimethyluridine (m3Um), 3- (3-amino-3-carboxypropyl)-5,6-dihydrouridine (acp3D), 3-(3-amino-3-carboxypropyl) pseudouridine (acp3Y), 3-(3-amino-3-carboxypropyl) uridine (acp3U), 3-methylcytidine (m3C), 3-methylpseudouridine (m3Y), 3-methyluridine (m3U), 4-demethylwyoside (imG-14), 4-thiouridine (s4U), 5,2'-O-dimethylcytidine (m5Cm), 5,2'-O-dimethyluridine (m5Um), 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm5Um),5-(Carboxyhydroxymethyl)uridine methyl ester (mchm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 5-(isopentenylaminomethyl)uridine (inm5U), 5-aminomethyl-2-geranylthiouridine (nm5ges2U), 5-aminomethyl-2-seleno-uridine (nm5se2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nchm5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2'-O-methyluridine (nc m5Um), 5-carboxymethylaminomethyluridine (ncm5U), 5-carboxyhydroxymethyluridine (chm5U), 5-carboxymethyl-2-thiouridine (cm5s2U), 5-carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5-carboxymethylaminomethyl-2-seleno-uridine (cmnm5se2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyluridine (cm5U), 5-cyanomethyluridine (cnm5U), 5-formyl-2'-O-methylcytidine (f5Cm), 5-formylcytidine uridine (f5C), 5-hydroxycytidine (ho5C), 5-hydroxymethylcytidine (hm5C), 5-hydroxyuridine (ho5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (m5s2U), 5-methylaminomethyl-2-geranylthiouridine (mnm5ges2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyluridine (mnm5U), 5- Methylcytidine (m5C), 5-methyldihydrouridine (m5D), 5-methyluridine (m5U), 5-taurinemethyl-2-thiouridine (tm5s2U), 5-taurinemethyluridine (tm5U), 7-aminocarboxypropyl-demethyl wyoside (yW-86), 7-aminocarboxypropyl wyoside (yW-72), 7-aminocarboxypropyl wyoside methyl ester (yW-58), 7-aminomethyl-7-deazaguanosine (preQ1tRNA), 7-cyano-7-deazaguanosine (preQ0tRNA), 7-methylguanosine (m7G), 8-methyladenosine (m8A), N2,2'-O-dimethylguanosine (m2Gm), N2,7,2'-O-trimethylguanosine (m2,7Gm),N2,7-dimethylguanosine (m2,7G), N2,N2,2'-O-trimethylguanosine (m2,2Gm), N2,N2,7-trimethylguanosine (m2,2,7G), N2,N2-dimethylguanosine (m2,2G), N2-methylguanosine (m2G), N4,2'-O-dimethylcytidine (m4Cm), N4,N4,2'-O-trimethylcytidine (m4,4Cm), N4,N4-dimethylcytidine (m4,4C), N4-acetyl-2'-O-methylcytidine (ac4Cm), N4- Acetylcytidine (ac4C), N4-methylcytidine (m4C), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m6,6Am), N6,N6-dimethyladenosine (m6,6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A), N6-formyladenosine (f6A), N6-glycylaminoformyladenosine (g6A), N6-hydroxymethyladenosine (hm6A), N6-hydroxynorvalylaminoformyladenosine (hn 6A), N6-isopentenyl adenosine (i6A), N6-methyl-N6-threonylaminoformyl adenosine (m6t6A), N6-methyl adenosine (m6A), N6-threonylaminoformyl adenosine (t6A), Agmatidine (C+), archaeosine (G+), cyclic N6-threonylaminoformyl adenosine (ct6A), dihydrouridine (D), epoxy nucleosides (oQtRNA), galactosyl nucleosides (galQtRNA), glutamyl-nucleosides (gluQtRNA), hydroxy-N6-threonyl Aminoformyl adenosine (ht6A), hydroxy wyosine (OHyW), inosine (I), isowyosine (imG2), mannosyl-nucleoside (manQtRNA), methylated unmodified hydroxy wyosine (OHyWy), methyl wyosine (mimG), peroxy wyosine (o2yW), pseudouridine (Y), nucleoside (QtRNA), unmodified hydroxy wyosine (OHyWx), uridine 5-hydroxyacetic acid (cmo5U), uridine 5-hydroxyacetic acid methyl ester (mcmo5U), wyosine (yW) or wyosine (imG) modification.

[0087] Alternatively or additionally, in some embodiments, the nucleic acid agent (and / or its elements) provided is a single-stranded nucleic acid or comprises a single-stranded nucleic acid. In some embodiments, the nucleic acid agent (and / or its elements) provided is or comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, for example, LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization) or a combination thereof (e.g., "hybrid"). In some embodiments, the nucleic acid agent provided is a single RNA strand. In some embodiments, the nucleic acid agent provided is a single DNA strand. In some embodiments, the nucleic acid agent provided is a single strand comprising one or more or all modified nucleotides.

[0088] In some embodiments, a nucleic acid agent can comprise a complementary element and a polyadenylation element that hybridizes to a target mRNA (see, e.g., Figure 1A , Figure 1B , Figure 1C , Figure 3A and / or Figure 8 One or more of the exemplary agents shown in ).

[0089] Complementary elements

[0090] As described herein, the nucleic acid agents provided generally include "complementary elements" (e.g., "complementary regions") designed and / or constructed based on nucleobase complementarity. In some embodiments, the complementary element includes a nucleic acid sequence complementary to an RNA (e.g., a target mRNA). In some embodiments, the complementary element can hybridize with a target mRNA. In some embodiments, the complementary element hybridizes with the 3' untranslated region of the target mRNA. In some embodiments, the complementary element can include about 30 (e.g., about 15, about 20, about 25, about 35, about 40, about 45, or about 50) nucleotides. In some embodiments, the complementary element includes RNA. In some embodiments, the complementary element includes DNA. In some embodiments, the complementary element includes DNA and RNA.

[0091] In some embodiments, the complementary element comprises a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A), or adenosine to inosine (A to I) modification. In some embodiments, the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. In some embodiments, the nucleotide modification is 1,2'-O-dimethyladenosine (m1Am), 1,2'-O-dimethylguanosine (m1Gm), 1,2'-O-dimethylinosine (m1Im), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Y), 1-methyladenosine (m1A), 1-methylguanosine (m1G), 1-methylinosine (m1I), 1-methylpseudouridine (m1Y), 2,8-dimethyladenosine (m2,8A), 2-geranylthiouridine (ges2U), 2-lysine (k2C), 2-methyladenosine (m2A), 2-methylthio ... -N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6-threonylaminoformyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalylaminoformyladenosine (ms2hn6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylaminoformyladenosine (ms2t6A), 2-seleno-uridine (se2U), 2-thio-2'-O-methyluridine (s2 Um), 2-thiocytidine (s2C), 2-thiouridine (s2U), 2'-O-methyladenosine (Am), 2'-O-methylcytidine (Cm), 2'-O-methylguanosine (Gm), 2'-O-methylinosine (Im), 2'-O-methylpseudouridine (Ym), 2'-O-methyluridine (Um), 2'-O-methyluridine 5-hydroxyacetate (mcmo5Um), 2'-O-riboadenosine (phosphate) (Ar(p)), 2'-O-riboguanosine (phosphate) (Gr(p)), 3,2'-O-dimethyluridine (m3Um), 3-(3-amino-3-carboxypropyl)-5,6 -dihydrouridine (acp3D), 3-(3-amino-3-carboxypropyl) pseudouridine (acp3Y), 3-(3-amino-3-carboxypropyl) uridine (acp3U), 3-methylcytidine (m3C), 3-methyl pseudouridine (m3Y), 3-methyl uridine (m3U), 4-demethyl wyoside (imG-14), 4-thiouridine (s4U), 5,2'-O-dimethylcytidine (m5Cm), 5,2'-O-dimethyl uridine (m5Um), 5-(carboxyhydroxymethyl)-2'-O-methyl uridine methyl ester (mchm5Um), 5-(carboxyhydroxymethyl) uridine methyl ester (mchm5U),5-(Isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(Isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 5-(Isopentenylaminomethyl)uridine (inm5U), 5-aminomethyl-2-geranylthiouridine (nm5ges2U), 5-aminomethyl-2-seleno-uridine (nm5se2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nchm5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carbamoylmethyluridine (ncm5 U), 5-carboxyhydroxymethyluridine (chm5U), 5-carboxymethyl-2-thiouridine (cm5s2U), 5-carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5-carboxymethylaminomethyl-2-seleno-uridine (cmnm5se2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyluridine (cm5U), 5-cyanomethyluridine (cnm5U), 5-formyl-2'-O-methylcytidine (f5Cm), 5-formylcytidine (f5C), 5-hydroxycytidine (ho5C), 5-Hydroxymethylcytidine (hm5C), 5-hydroxyuridine (ho5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (m5s2U), 5-methylaminomethyl-2-geranylthiouridine (mnm5ges2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyluridine (mnm5U), 5-methylcytidine (m5C), 5-methyldihydrouridine ( m5D), 5-methyluridine (m5U), 5-taurine methyl-2-thiouridine (tm5s2U), 5-taurine methyluridine (tm5U), 7-aminocarboxypropyl-demethyl wyoside (yW-86), 7-aminocarboxypropyl wyoside (yW-72), 7-aminocarboxypropyl wyoside methyl ester (yW-58), 7-aminomethyl-7-deazaguanosine (preQ1tRNA), 7-cyano-7-deazaguanosine (preQ0tRNA), 7-methylguanosine (m7G), 8-methyladenosine (m8A), N2,2'-O-dimethylguanosine (m2Gm), N2,7,2'-O-trimethylguanosine (m2,7Gm), N2,7-dimethylguanosine (m2,7G),N2,N2,2'-O-trimethylguanosine (m2,2Gm), N2,N2,7-trimethylguanosine (m2,2,7G), N2,N2-dimethylguanosine (m2,2G), N2-methylguanosine (m2G), N4,2'-O-dimethylcytidine (m4Cm), N4,N4,2'-O-trimethylcytidine (m4,4Cm), N4,N4-dimethylcytidine (m4,4C), N4-acetyl-2'-O-methylcytidine (ac4Cm), N4-acetylcytidine (ac4C), N4- Methylcytidine (m4C), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m6,6Am), N6,N6-dimethyladenosine (m6,6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A), N6-formyladenosine (f6A), N6-glycylaminoformyladenosine (g6A), N6-hydroxymethyladenosine (hm6A), N6-hydroxynorvalylaminoformyladenosine (hn6A), N6-isopentyl adenosine (i6A), N6-methyl-N6-threonylaminoformyl adenosine (m6t6A), N6-methyladenosine (m6A), N6-threonylaminoformyl adenosine (t6A), Agmatidine (C+), archaeosine (G+), cyclic N6-threonylaminoformyl adenosine (ct6A), dihydrouridine (D), epoxynucleosides (oQtRNA), galactosyl nucleosides (galQtRNA), glutamyl-nucleosides (gluQtRNA), hydroxy-N6-threonylaminoformyl adenosine (gluQtRNA), acyladenosine (ht6A), hydroxywybutosine (OHyW), inosine (I), isowybutosine (imG2), mannosyl-nucleoside (manQtRNA), methylated unmodified hydroxywybutosine (OHyWy), methylwybutosine (mimG), peroxywybutosine (o2yW), pseudouridine (Y), nucleoside (QtRNA), unmodified hydroxywybutosine (OHyWx), uridine 5-hydroxyacetic acid (cmo5U), uridine 5-hydroxyacetic acid methyl ester (mcmo5U), wybutosine (yW) or wybutosine (imG) modification. 、

[0092] In some embodiments, the target mRNA is an mRNA encoding a polypeptide of interest (e.g., increased expression of a polypeptide provides an activity or benefit of interest to or in the cell, tissue, system, or organism in which it is expressed).

[0093] In some embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder associated with reduced protein expression by the mRNA. In some embodiments, such a disorder is a haploinsufficiency disorder. In some embodiments, the target mRNA encodes a MeCP2 protein. In some embodiments, the nucleic acid agent is or comprises a nucleic acid molecule comprising or consisting of SEQ ID NO: 1.

[0094] SEQ ID NO: 1 - Targeting the complementary region of MeCP2 3'UTR

[0095] CCACAGGCTAAAATGTATATGCCCAAAGA

[0096] In some embodiments, the target mRNA encodes a variant of a polypeptide of interest with reduced activity. In some embodiments, the target mRNA may be exogenous to a cell, tissue, organism, or other system of interest. For example, in some embodiments, the target mRNA may be a therapeutic mRNA and / or may be a transcript of a gene contained in a gene therapy agent or a cell therapy agent or otherwise provided. In some embodiments, the target mRNA may encode a toxic polypeptide or a suicide polypeptide, such as a toxic polypeptide or a suicide polypeptide, and the expression of the polypeptide may reduce the activity of the cell expressing it or otherwise contacting it (e.g., by inducing or promoting cell death apoptosis or cell necrosis). In some specific embodiments, such cells may be diseased cells (e.g., cancer cells); alternatively or additionally, in certain specific embodiments, such cells may be cell therapy products (e.g., engineered T cells such as CAR-T cells, or engineered NK cells, or engineered B cell preparations, etc.).

[0097] Polyadenylation element

[0098] A "poly(A) region" comprised in a nucleic acid agent as described and / or utilized herein is typically or comprises a chain of adenosine residues.

[0099] In some embodiments, the nucleic acid agent is arranged and constructed such that the polyadenylation element is located 5' to the complementary element. For example, in some embodiments, the nucleic acid agent is or comprises a nucleic acid molecule comprising a polyadenylation region located 5' to the complementary region.

[0100] In some embodiments, the nucleic acid agent is arranged and constructed such that the polyadenylation element is located 3' to the complementary element. For example, in some embodiments, the nucleic acid agent is or comprises a nucleic acid molecule comprising a polyadenylation region located 3' to the complementary region.

[0101] In some embodiments, the nucleic acid agent is arranged and constructed so that the polyadenylation element is located at the 5' end of the complementary element, and the nucleic acid agent further comprises a cap element located at the 5' end of the complementary element. For example, in some embodiments, the nucleic acid agent is a nucleic acid molecule or comprises a nucleic acid molecule that comprises a polyadenylation region located at the 3' end of the complementary region and further comprises a 5' cap region located at the 5' end of the complementary region.

[0102] In some embodiments, the nucleic acid agent is arranged and constructed such that the first polyadenylation element is located at the 5' end of the complementary element and the second polyadenylation region is located at the 5' end of the complementary region. For example, in some embodiments, the nucleic acid agent is a nucleic acid molecule or comprises a nucleic acid molecule comprising a first polyadenylation region located at the 5' end of the complementary region and a second polyadenylation region located at the 3' end of the complementary region.

[0103] Those skilled in the art will be familiar with the term "poly (A) tail", which generally refers to a stretch of adenosine residues located at the end of an mRNA. In the wild-type case, the poly (A) tail is located at the 3' end of the mRNA and is synthesized after transcription on an mRNA containing a poly (A) signal sequence. The term "poly (A) signal sequence" or "poly (A) signal" is generally used to refer to a sequence that triggers endonuclease cleavage of an mRNA and adds an adenosine sequence to the 3' end of the cleaved mRNA. Non-limiting examples of poly (A) signals include: bovine growth hormone (bGH) poly (A) signal, human growth hormone (hGH) poly (A) signal. Other examples of poly (A) signal sequences are known in the art.

[0104] The poly (A) tail or poly (A) element (e.g., poly (A) region) can function by binding to poly (A) binding protein (PABP). PABP is a highly conserved RNA binding protein in eukaryotes. The protein has four N-terminal RNA recognition motif (RRM) domains, which bind to poly (A) RNA with nanomolar affinity. RRM is followed by a proline-rich linker and a C-terminal MLLE domain. The MLLE domain recognizes a peptide motif called poly (A) interaction motif 2 (PAM2), which is present in many PABP partner proteins that regulate mRNA metabolism (stability and translation). The presence of PABP on mRNA is known to stimulate their activity, enhance translation and mRNA stability. In some embodiments, the poly (A) element (e.g., the poly (A) region of the nucleic acid molecule provided) in the nucleic acid agent provided can function by recruiting translation stimulators (e.g., PABC1). In some embodiments, the poly(A) element (e.g., a poly(A) region) can be resistant to deadenylation; without wishing to be bound by any particular theory, we propose that the poly(A) element (e.g., a poly(A) region) can therefore maintain the target mRNA in a positive translation state.

[0105] A poly(A) tail or poly(A) element (e.g., a poly(A) region) can be added to the 3' end of most early eukaryotic messenger RNAs (mRNAs) in a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction driven by a poly(A) signal sequence. The term "polyadenylation" refers to the covalent attachment of a poly(A) moiety or a modified variant thereof to the 3' end of an mRNA molecule.

[0106] In some embodiments, the gene delivery vector provided and / or utilized according to the present disclosure may include a sequence encoding a poly(A) region adjacent to a sequence encoding a complementary region. In some embodiments, the gene delivery vector may include a poly(T) sequence adjacent to a sequence encoding a complementary region, wherein the poly(T) sequence encodes a poly(A) region. In some embodiments, the gene delivery vector may include a sequence including a poly(A) region at the end of a separated nucleic acid encoding a complementary region.

[0107] In some embodiments, the poly(A) element (e.g., a poly(A) region) comprises about 20 to about 80 (e.g., about 25 to about 80, about 30 to about 80, about 35 to about 80, about 40 to about 80, about 45 to about 80, about 50 to about 80, about 55 to about 80, about 60 to about 80, about 65 to about 80, about 70 to about 80, about 75 to about 80, about 20 to about 75, about 25 to about 75, about 30 to about 75, about 35 to about 75, about 40 to about 75, about 45 to about 80, about 50 to about 80, about 55 to about 80, about 60 to about 80, about 65 to about 80, about 70 to about 80, about 75 to about 80, about 20 to about 75, about 25 to about 75, about 30 to about 75, about 35 to about 75, about 40 to about 75, about 45 to about 80 5 to about 75, about 50 to about 75, about 55 to about 75, about 60 to about 75, about 65 to about 75, about 70 to about 75, about 20 to about 70, about 25 to about 70, about 30 to about 70, about 35 to about 70, about 40 to about 70, about 45 to about 70, about 50 to about 70, about 55 to about 70, about 60 to about 70, about 65 to about 70, about 20 to about 65, about 25 to about 65, about 30 to about 70, about 35 to about 70, about 40 to about 70, about 45 to about 70, about 50 to about 70, about 55 to about 70, about 60 to about 70, about 65 to about 70, about 20 to about 65, about 25 to about 65, about 30 to about 65, about 35 to about 70, about 40 to about 6 5, about 45 to about 65, about 50 to about 65, about 55 to about 65, about 60 to about 65, about 20 to about 60, about 25 to about 60, about 30 to about 60, about 35 to about 60, about 40 to about 60, about 45 to about 60, about 50 to about 60, about 55 to about 60, about 20 to about 55, about 25 to about 55, about 30 to about 55, about 35 to about 55, about 40 to about 55, about 45 to about 55, about 50 to about 55, about 20 to about 50, about 2 In some embodiments, the poly(A) element (e.g., a poly(A) region) comprises about 20 nucleotides. In some embodiments, the poly(A) element (e.g., a poly(A) region) comprises about 50 nucleotides. In some embodiments, the poly(A) element (e.g., a poly(A) region) comprises about 50 nucleotides. In some embodiments, a poly(A) element (eg, a poly(A) region) comprises about 75 nucleotides.

[0108] In some embodiments, a poly(A) element (eg, a poly(A) region) may comprise non-adenine nucleotides.

[0109] In some embodiments, the polyadenylic acid element (e.g., a polyadenylic acid region in a nucleic acid molecule) in a nucleic acid agent provided and / or utilized according to the present disclosure comprises a nucleotide modification. In some embodiments, the polyadenylic acid region comprises a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A), or adenosine to inosine (A to I) modification. In some embodiments, the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU), or N4-acetylcytidine. In some embodiments, the nucleotide modification is 1,2'-O-dimethyladenosine (m1Am), 1,2'-O-dimethylguanosine (m1Gm), 1,2'-O-dimethylinosine (m1Im), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Y), 1-methyladenosine (m1A), 1-methylguanosine (m1G), 1-methylinosine (m1I), 1-methylpseudouridine (m1Y), 2,8-dimethyladenosine (m2,8A), 2-geranylthiouridine (ges2U), 2-lysine (k2C), 2- methyladenosine (m2A), 2-methylthiomethylthio-N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6-threonylaminoformyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalylaminoformyladenosine (ms2hn6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylaminoformyladenosine (m s2t6A), 2-selenouridine (se2U), 2-thio-2'-O-methyluridine (s2Um), 2-thiocytidine (s2C), 2-thiouridine (s2U), 2'-O-methyladenosine (Am), 2'-O-methylcytidine (Cm), 2'-O-methylguanosine (Gm), 2'-O-methylinosine (Im), 2'-O-methylpseudouridine (Ym), 2'-O-methyluridine (Um), 2'-O-methyluridine 5-hydroxyacetate (mcmo5Um), 2'-O-riboadenosine (phosphate) (Ar(p)), 2'-O -riboguanosine (phosphate) (Gr(p)), 3,2'-O-dimethyluridine (m3Um), 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine (acp3D), 3-(3-amino-3-carboxypropyl)pseudouridine (acp3Y), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 3-methylcytidine (m3C), 3-methylpseudouridine (m3Y), 3-methyluridine (m3U), 4-demethylwyoside (imG-14), 4-thiouridine (s4U), 5,2'-O-dimethylcytidine (m5Cm),5,2'-O-dimethyluridine (m5Um), 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm5Um), 5-(carboxyhydroxymethyl)uridine methyl ester (mchm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 5-(isopentenylaminomethyl)uridine (inm5U), 5-aminomethyl-2-geranylthiouridine (nm5ges2U), 5-aminomethyl-2-seleno-uridine (nm5se2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nch m5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carbamoylmethyluridine (ncm5U), 5-carboxyhydroxymethyluridine (chm5U), 5-carboxymethyl-2-thiouridine (cm5s2U), 5-carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5-carboxymethylaminomethyl-2-seleno-uridine (cmnm5se2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyl Uridine (cm5U), 5-cyanomethyluridine (cnm5U), 5-formyl-2'-O-methylcytidine (f5Cm), 5-formylcytidine (f5C), 5-hydroxycytidine (ho5C), 5-hydroxymethylcytidine (hm5C), 5-hydroxyuridine (ho5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (m5s2U), 5-methylaminomethyl-2-geranylthiouridine (mnm5ges2U), 5-methylaminomethyl-2-selenouridine glycoside (mnm5se2U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl uridine (mnm5U), 5-methylcytidine (m5C), 5-methyldihydrouridine (m5D), 5-methyluridine (m5U), 5-taurine methyl-2-thiouridine (tm5s2U), 5-taurine methyl uridine (tm5U), 7-aminocarboxypropyl-demethyl wyoside (yW-86), 7-aminocarboxypropyl wyoside (yW-72), 7-aminocarboxypropyl wyoside methyl ester (yW-58), 7-aminomethyl-7-deazaguanosine (preQ1tRNA), 7-cyano-7-deazaguanosine (preQ0tRNA), 7-methylguanosine (m7G),8-methyladenosine (m8A), N2,2'-O-dimethylguanosine (m2Gm), N2,7,2'-O-trimethylguanosine (m2,7Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,2'-O-trimethylguanosine (m2,2Gm), N2,N2,7-trimethylguanosine (m2,2,7G), N2,N2-dimethylguanosine (m2,2G), N2-methylguanosine (m2G), N4,2'-O-dimethylcytidine (m4Cm), N4,N4,2'-O-trimethylcytidine (m4,4Cm), N4, N4-dimethylcytidine (m4,4C), N4-acetyl-2'-O-methylcytidine (ac4Cm), N4-acetylcytidine (ac4C), N4-methylcytidine (m4C), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m6,6Am), N6,N6-dimethyladenosine (m6,6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A), N6-formyladenosine (f6A), N6-glycylaminoformyladenosine (g6A), N6-hydroxymethyl adenosine (hm6A), N6-hydroxynorvalylaminoformyladenosine (hn6A), N6-isopentenyl adenosine (i6A), N6-methyl-N6-threonylaminoformyladenosine (m6t6A), N6-methyladenosine (m6A), N6-threonylaminoformyladenosine (t6A), agmatidine (C+), archaeosine (G+), cyclic N6-threonylaminoformyladenosine (ct6A), dihydrouridine (D), epoxynucleosides (oQtRNA), galactosylnucleosides (galQtRNA), glutamyl-nucleosides (gluQtRNA), tRNA), hydroxy-N6-threonylaminoformyl adenosine (ht6A), hydroxy wyobutosine (OHyW), inosine (I), isowyobutosine (imG2), mannosyl-nucleoside (manQtRNA), methylated unmodified hydroxy wyobutosine (OHyWy), methyl wyobutosine (mimG), peroxy wyobutosine (o2yW), pseudouridine (Y), nucleoside (QtRNA), unmodified hydroxy wyobutosine (OHyWx), uridine 5-hydroxyacetic acid (cmo5U), uridine 5-hydroxyacetic acid methyl ester (mcmo5U), wyobutosine (yW) or wyobutosine (imG) modification. ,

[0110] 5' cap element

[0111] In some embodiments, the nucleic acid agent (eg, which is or comprises a nucleic acid molecule) further comprises a 5' cap element.

[0112] As used herein, the term "5' cap element" generally refers to a modified nucleotide on the 5' end of a nucleic acid molecule. In some embodiments, the 5' cap element may include a nucleotide modification. In some embodiments, the nucleotide modification is N1-methyladenosine (m1A), N6-methyladenosine (m6A) or adenosine to inosine (A to I) modification. In some embodiments, the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU) or N4-acetylcytidine. In some embodiments, the 5' cap region comprises a 5' cap structure of a eukaryotic organism. In some embodiments, the 5' cap region may include a G(5')ppp(5')G sequence. In some embodiments, the nucleotide modification is 1,2'-O-dimethyladenosine (m1Am), 1,2'-O-dimethylguanosine (m1Gm), 1,2'-O-dimethylinosine (m1Im), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (m1acp3Y), 1-methyladenosine (m1A), 1-methylguanosine (m1G), 1-methylinosine (m1I), 1-methylpseudouridine (m1Y), 2,8-dimethyladenosine (m2,8A), 2-geranylthiouridine (ges2U), 2-lysine (k2C), 2-methyladenosine (m2A), 2-methylthiomethylthio-N6-isopentenyl-adenosine (msms2i6A), 2-methylthio-cyclic-N6-threonylaminoformyladenosine (ms2ct6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)-adenosine (ms2io6A), 2-methylthio-N6-hydroxynorvalylaminoformyladenosine (ms2hn6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio 2-thio-2'-O-methyluridine (s2Um), 2-thiocytidine (s2C), 2-thiouridine (s2U), 2'-O-methyladenosine (Am), 2'-O-methylcytidine (Cm), 2'-O-methylguanosine (Gm), 2'-O-methylinosine (Im), 2'-O-methylpseudouridine (Ym), 2'-O-methyluridine (Um), 2'-O-methyluridine 5-hydroxyacetate (mcmo5Um), 2' -O-riboadenosine (phosphate) (Ar(p)), 2'-O-riboguanosine (phosphate) (Gr(p)), 3,2'-O-dimethyluridine (m3Um), 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine (acp3D), 3-(3-amino-3-carboxypropyl)pseudouridine (acp3Y), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 3-methylcytidine (m3C), 3-methylpseudouridine (m3Y), 3-methyluridine (m3U), 4-demethylwyoside (imG-14),4-thiouridine (s4U), 5,2'-O-dimethylcytidine (m5Cm), 5,2'-O-dimethyluridine (m5Um), 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester (mchm5Um), 5-(carboxyhydroxymethyl)uridine methyl ester (mchm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 5-(isopentenylaminomethyl)uridine (inm5U), 5-aminomethyl-2-geranylthiouridine (nm5ges2U), 5-aminomethyl-2-seleno-uridine (nm5se2U), 5-aminomethyl-2-thiouridine (nm5s2 U), 5-aminomethyluridine (nm5U), 5-carbamoylhydroxymethyluridine (nchm5U), 5-carbamoylmethyl-2-thiouridine (ncm5s2U), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carbamoylmethyluridine (ncm5U), 5-carboxyhydroxymethyluridine (chm5U), 5-carboxymethyl-2-thiouridine (cm5s2U), 5-carboxymethylaminomethyl-2-geranylthiouridine (cmnm5ges2U), 5-carboxymethylaminomethyl-2-seleno-uridine (cmnm5se2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-carboxymethylaminomethyl-2'-O-methyluridine ( cmnm5Um), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethyluridine (cm5U), 5-cyanomethyluridine (cnm5U), 5-formyl-2'-O-methylcytidine (f5Cm), 5-formylcytidine (f5C), 5-hydroxycytidine (ho5C), 5-hydroxymethylcytidine (hm5C), 5-hydroxyuridine (ho5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxyuridine (mo5U), 5-methyl-2-thiouridine (m5s2U), 5-methylaminomethyl -2-geranylthiouridine (mnm5ges2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyluridine (mnm5U), 5-methylcytidine (m5C), 5-methyldihydrouridine (m5D), 5-methyluridine (m5U), 5-taurinemethyl-2-thiouridine (tm5s2U), 5-taurinemethyluridine (tm5U), 7-aminocarboxypropyl-demethyl wyoside (yW-86), 7-aminocarboxypropyl wyoside (yW-72), 7-aminocarboxypropyl wyoside methyl ester (yW-58), 7-aminomethyl-7-deazaguanosine (preQ1tRNA),7-cyano-7-deazaguanosine (preQ0tRNA), 7-methylguanosine (m7G), 8-methyladenosine (m8A), N2,2'-O-dimethylguanosine (m2Gm), N2,7,2'-O-trimethylguanosine (m2,7Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,2'-O-trimethylguanosine (m2,2Gm), N2,N2,7-trimethylguanosine (m2,2,7G), N2,N2-dimethylguanosine (m2,2G), N2-methylguanosine (m2G), N4,2'-O-dimethylcytidine (m4Cm), N4 ,N4,2'-O-trimethylcytidine (m4,4Cm), N4,N4-dimethylcytidine (m4,4C), N4-acetyl-2'-O-methylcytidine (ac4Cm), N4-acetylcytidine (ac4C), N4-methylcytidine (m4C), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m6,6Am), N6,N6-dimethyladenosine (m6,6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), N6-acetyladenosine (ac6A), N6-formyladenosine (f6A), N6-glycyl Carbamoyl adenosine (g6A), N6-hydroxymethyladenosine (hm6A), N6-hydroxynorvalylcarbamoyl adenosine (hn6A), N6-isopentenyl adenosine (i6A), N6-methyl-N6-threonylcarbamoyl adenosine (m6t6A), N6-methyladenosine (m6A), N6-threonylcarbamoyl adenosine (t6A), Agmatidine (C+), archaeosine (G+), cyclic N6-threonylcarbamoyl adenosine (ct6A), dihydrouridine (D), epoxy nucleosides (oQtRNA), galactosyl nucleosides (galQtRNA), glutamine acyl-nucleoside (gluQtRNA), hydroxy-N6-threonylaminoformyl adenosine (ht6A), hydroxy wyoside (OHyW), inosine (I), isowyoside (imG2), mannosyl-nucleoside (manQtRNA), methylated unmodified hydroxy wyoside (OHyWy), methyl wyoside (mimG), peroxy wyoside (o2yW), pseudouridine (Y), nucleoside (QtRNA), unmodified hydroxy wyoside (OHyWx), uridine 5-hydroxyacetic acid (cmo5U), uridine 5-hydroxyacetic acid methyl ester (mcmo5U), wyoside (yW) or wyoside (imG) modification. 、

[0113] In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure comprises a polyadenylic acid element at the 3' end of the complementary element, and further comprises a 5' cap element at the 5' end of the complementary region. In some embodiments, the nucleic acid agent may comprise a polyadenylic acid element at the 3' end of the complementary element, and may further comprise a 5' cap element at the 5' end of the complementary element. In some or all of the aforementioned embodiments, the nucleic acid agent increases protein production of the target mRNA. In some such embodiments, the target mRNA is an mRNA encoding a polypeptide whose expression is increased. In certain specific such embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder, and the disorder is associated with reduced protein expression by the mRNA. In some embodiments, the disorder is a haploinsufficient disorder.

[0114] In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure is a nucleic acid molecule or comprises a nucleic acid molecule, which comprises a polyadenylic acid region at the 3' end of the complementary region, and further comprises a 5' cap element at the 5' end of the complementary region. In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure is a nucleic acid molecule or comprises a nucleic acid molecule, which comprises a polyadenylic acid element at the 3' end of the complementary element, and further comprises a 5' cap element at the 5' end of the complementary region. In some or all of the aforementioned embodiments, the nucleic acid agent increases protein production of the target mRNA. In some such embodiments, the target mRNA is an mRNA encoding a polypeptide whose expression is increased. In certain specific such embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder, and the disorder is associated with reduced protein expression by mRNA. In some embodiments, the disorder is a haploinsufficient disorder.

[0115] Coding agent

[0116] In some embodiments, a provided nucleic acid agent is or comprises one or more nucleic acid molecules that can be encoded by a nucleic acid sequence.

[0117] In certain embodiments, a molecular nucleic acid agent useful according to the present disclosure and / or one or more components thereof (e.g., nucleic acid molecules) is encoded by a sequence (e.g., in an encoding agent) comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 2, or the sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0118] SEQ ID NO:2—Plasmid for nucleic acid molecules having a polyadenylation region located 3' to the complementary region (pJC1294)

[0119]

[0120] SEQ ID NO:3—Plasmid for nucleic acid molecules having polyadenylation regions located at the 3' and 5' ends of the complementary region (pJC1295)

[0121]

[0122] In some embodiments, a nucleic acid agent provided and / or utilized in accordance with the present disclosure is or comprises: (a) a nucleic acid molecule that is or comprises a complementary element, such as a complementary region, that hybridizes to a target mRNA (i.e., having a target site therein); and (b) a nucleic acid molecule that is or comprises a polyadenylation element, wherein in some embodiments, the complementary element and the polyadenylation element are contained in the same nucleic acid molecule (e.g., as a region thereof).

[0123] In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase target mRNA expression. In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the target mRNA in the object and / or one or more cells or tissues (e.g., organs) thereof. In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the target mRNA of people (e.g., one or more cells or tissues (e.g., organs) of people). In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the target mRNA of mice (e.g., one or more cells or tissues (e.g., organs) of mice).

[0124] In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the protein expression of the target mRNA. In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the protein expression of the target mRNA of the object. In some embodiments, the nucleic acid agent provided and / or utilized according to the present disclosure can increase the protein expression of the target mRNA of a person (e.g., one or more cells or tissues (e.g., organs) of a person).

[0125] In some embodiments, a nucleic acid agent provided and / or utilized according to the present disclosure can increase a target mRNA in a mouse (eg, in one or more cells or tissues (eg, organs) of a mouse).

[0126] In some specific embodiments, the target mRNA encodes MeCP2 protein. In some embodiments, the nucleic acid agent provided and / or used according to the present invention comprises SEQ ID NO: 4 or SEQ ID NO: 5 or consists of SEQ ID NO: 4 or SEQ ID NO: 5, wherein the target mRNA encodes human or mouse MeCP2 protein. In some embodiments, the nucleic acid agent provided and / or used according to the present invention comprises a nucleic acid molecule or consists of a nucleic acid molecule, which is SEQ ID NO: 6 or SEQ ID NO: 7, or one or more components of SEQ ID NO: 6 or SEQ ID NO: 7 or comprises one or more components of SEQ ID NO: 6 or SEQ ID NO: 7, wherein the target mRNA encodes mouse MeCP2 protein. In some embodiments, the target mRNA encodes SYNGAP1 protein. In some embodiments, the nucleic acid agent provided and / or used according to the present invention comprises a nucleic acid or consists of a nucleic acid, which is or comprises one or more components of SEQ ID NO: 8 or SEQ ID NO: 9, wherein the target mRNA encodes human SYNGAP1 protein.

[0127] SEQ ID NO:4—Mouse / Human Pan Enhancer RNA (G1-2 tail)

[0128] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCCCACTTTAAAACAAGCGCAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0129] SEQ ID NO:5—Mouse / Human Pan Enhancer RNA (G1-1 tail)

[0130] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGCCCACTTTAAAACAAGCGCAGGT

[0131] SEQ ID NO:6-mouse specific enhancer RNA (G2-2 tail)

[0132] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATGGAAATACAGCACCAGCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0133] SEQ ID NO:7- Mouse specific enhancer RNA (G2-1 tail)

[0134] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGGAAATACAGCACCAGCAG

[0135] SEQ ID NO:8—Human Syngap1 guide RNA (Syng-tail)

[0136] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACACCCTGAAGTTGAAAGTTTGGAGGTGCCA

[0137] SEQ ID NO:9—Human Syngap1 guide RNA (Syng-2 tail)

[0138] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0139] In some embodiments, provided herein are recombinant expression systems comprising a nucleic acid sequence encoding a nucleic acid molecule comprising one or both of (i) a complementary element that hybridizes to a target mRNA and (ii) a polyadenylation element.

[0140] In some embodiments, provided herein is an expression vector comprising a recombinant expression system as described herein. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral vector (AAV), a lentiviral vector or an adenoviral vector.

[0141] In some embodiments, provided herein are pharmaceutical compositions comprising or delivering (e.g., by expression) a nucleic acid agent, or an element thereof, e.g., a nucleic acid molecule, e.g., an RNA molecule, which is or comprises one or both of: i) a complementary element that hybridizes to a target mRNA (i.e., having a site therein) and (ii) a polyadenylation element. In some embodiments, provided pharmaceutical compositions comprise or deliver a recombinant expression system (e.g., an expression vector) as described herein.

[0142] Therapeutic applications

[0143] Provided herein are techniques for increasing the translation of a target mRNA. Thus, in some embodiments, techniques are provided for increasing the level of a polypeptide of interest encoded by such a target mRNA.

[0144] Those skilled in the art who read this disclosure will appreciate the various applications provided thereby, including various therapeutic applications.

[0145] In some embodiments, provided technology achieves increased expression of a polypeptide that is beneficial for treating a condition in a subject, for example, by administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising or delivering a nucleic acid agent as described herein).

[0146] In some embodiments, the treatment methods provided include the step of administering to a subject a composition comprising or delivering a nucleic acid agent in which a complementary element hybridizes with a target mRNA encoding an endogenous protein. In some embodiments, the treatment methods provided include the step of administering to a subject a composition comprising or delivering a nucleic acid agent in which a complementary element hybridizes with a target mRNA encoding an exogenous protein.

[0147] In some embodiments, the increased protein expression achieved in accordance with the present disclosure provides a protein that is beneficial to a cell or tissue (e.g., an organ) of interest in a subject. In some embodiments, the increased protein expression achieved in accordance with the present disclosure provides a protein that is harmful to an undesirable cell or tissue (e.g., an organ) in a subject. To cite just one such example, in some embodiments, the provided techniques achieve and / or increase expression of a protein (e.g., a suicide protein) that is toxic to an undesirable cell (e.g., a cancer cell) and / or a cell therapy product (e.g., an engineered cell).

[0148] In some embodiments, provided herein are methods of treating or preventing a disorder in a subject by administering to the subject any one of the recombinant expression systems or any one of the expression vectors described herein.

[0149] In some embodiments, the condition treated is a haploinsufficiency condition. In some embodiments, the condition treated is cancer. In some embodiments, the cancer is a B-ALL tumor, liver cancer, or breast cancer.

[0150] In some embodiments, compositions are provided herein, which contain and / or deliver nucleic acid agent molecules (e.g., nucleic acid molecules) for increasing protein expression of target mRNA. In some embodiments, compositions are provided herein, which contain and / or deliver nucleic acid agents (e.g., nucleic acid molecules) for increasing expression of target mRNA. In some embodiments, such nucleic acid agents (e.g., nucleic acid molecules) include polyadenylic acid elements at the 5' end of complementary elements. In some embodiments, such nucleic acid agents (e.g., nucleic acid molecules) include polyadenylic acid elements at the 3' end of complementary elements. In some embodiments, such nucleic acid agents (e.g., nucleic acid molecules) include polyadenylic acid elements at the 3' end of complementary elements, and wherein the 5' cap element at the 5' end of complementary elements is also included. In some embodiments, such nucleic acid agents (e.g., nucleic acid molecules) include the first polyadenylic acid element at the 5' end of complementary elements and the second polyadenylic acid element at the 3' end of complementary elements. In some embodiments, such a nucleic acid agent (e.g., a nucleic acid molecule) may include a polyadenylic acid element at the 3' end of a complementary element and a second polyadenylic acid element at the 5' end of a complementary element, wherein the nucleic acid molecule increases the protein expression of the target mRNA. In some embodiments, such a nucleic acid agent is a nucleic acid molecule or includes a nucleic acid molecule (e.g., an RNA molecule), which may, for example, include a polyadenylic acid element at the 3' end of a complementary element, and in some embodiments, may also include a 5' cap element at the 5' end of a complementary element, wherein the nucleic acid molecule increases the protein expression of the target mRNA. In some embodiments, such a nucleic acid agent (e.g., a nucleic acid molecule) includes a polyadenylic acid element at the 5' end of a complementary element, wherein the nucleic acid agent increases the expression of the target mRNA. In some embodiments, such a nucleic acid agent (e.g., a nucleic acid molecule) includes a polyadenylic acid element at the 3' end of a complementary element, wherein the nucleic acid agent increases the expression of the target mRNA. In some embodiments, such a nucleic acid agent (e.g., a nucleic acid molecule) comprises a polyadenylic acid element at the 5' end of a complementary element and a second polyadenylic acid element at the 3' end of the complementary element, wherein the nucleic acid agent increases target mRNA expression. In some specific such embodiments, the target mRNA is an mRNA of an active allele of a gene associated with a disorder associated with reduced protein expression by the mRNA. In some embodiments, the disorder is a haploinsufficient disorder.

[0151] In some embodiments, provided herein are compositions comprising and / or delivering nucleic acid agents as described herein for treating haploinsufficient conditions. In some embodiments, provided herein are methods of using provided nucleic acid agents (e.g., nucleic acid molecules) for treating haploinsufficient conditions. In some embodiments, provided herein are methods of using nucleic acid agents (e.g., nucleic acid molecules) in the preparation of a medicament for treating haploinsufficient conditions.

[0152] Haploinsufficiency occurs when one allele of a gene is inactivated and the amount of gene product expressed from the remaining active allele is insufficient for proper gene function. Many disorders are associated with or caused by haploinsufficiency.

[0153] In some embodiments, the haploinsufficiency disorder is selected from 5q syndrome, Adams-Oliver syndrome type 1, Adams-Oliver syndrome type 3, Adams-Oliver syndrome type 5, Adams-Oliver syndrome type 6, Alagille syndrome type 1, autoimmune lymphoproliferative syndrome type IA, autoimmune lymphoproliferative syndrome type V, autosomal dominant deafness-2A, brain malformation with or without urinary tract defects (BRMUTD), Carney complex type 1, CHARGE syndrome, cleidocranial dysplasia, Currarino syndrome, Denys-Drash / Frasier syndrome, developmental delay-intellectual disability-obesity and dysmorphic features (DIDOD), DiGeorge syndrome (TBXI-related), Dravet syndrome, Duane-radial ray syndrome, syndrome), Ehlers-Danlos syndrome (classic), Ehlers-Danlos syndrome (vascular), Feingold syndrome type 1, frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP) GRN-related, GLUT I deficiency syndrome, Greig polysyndactyly syndrome, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly type 3, holoprosencephaly type 4, holoprosencephaly type 5, Holt-Oram syndrome, hypoparathyroidism-sensorineural hearing loss-kidney disease (HDR), Kleefstra syndrome 1, Klippel-Trenaunay syndrome syndrome) (AAGF-related), Leri-Weill achondroplasia, Marfan syndrome, mental retardation and distinctive facial features with or without cardiac defects (MRFACD), mental retardation, autosomal dominant 1, mental retardation, autosomal dominant 19, mental retardation, autosomal dominant 29, nail-patella syndrome (NPS), Phelan-McDermid syndrome, Pitt-Hopkins syndrome, primary pulmonary hypertension 1, Rett syndrome (congenital variant),Smith-Magenis syndrome (RAII-associated), Sotos syndrome 1, Sotos syndrome 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAPI-associated intellectual disability, Treacher Collins syndrome, trichotillo-naso-phalangeal syndrome type I, ulna-mammary syndrome, van der Woude syndrome 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C.

[0154] In some embodiments, the haploinsufficient gene is selected from AGGFI, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, CTLA4, CTNNBI, DLL4, EHMTI, ELN, ENG, FAS, FBNI, FOXGI, GATA3, GLI3, GRN, IRF6, JAGI, KCNQ4, LMXIB, MBD5, MED13L, MITF, MNXI, MYCN, NFIA, NFIX, NOTCH1, NSDI, PAX3, PHIP, PRKARIA, RAil, RBPJ, RPS14, RUNX2, SALL4, SCNIA, SETBPI, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOXIO, SYNGAPI, TBXI, TBX3, TBX5, TCF4, TCOFI, TGIFI, TNXB, TRPSI, WTI, ZIC2, and combinations thereof. In some embodiments, the haploinsufficient gene can be a CTNNBI gene. In some embodiments, the haploinsufficient gene can be a PURA gene.

[0155] In some embodiments, the target mRNA is, for example, an mRNA of an active allele of a tumor suppressor gene associated with a haploinsufficiency disorder. In some embodiments, the tumor suppressor gene is selected from the group consisting of CD19, CD22, C / EBP-α, HER2, CDKN1A, TP53, VHL, CEBPA, INTS6, HIC1, PTEN, CDH1, VEZT, CPYSL3, NKX3-1, PAWR, sFHIT, DIRAS1, KLF4, WT1, and MAS1.

[0156] In some embodiments, the combination of a haploinsufficiency disorder and a haploinsufficiency gene is a combination shown in Table 1.

[0157] Table 1 Haploinsufficiency disorders and genes

[0158]

[0159]

[0160]

[0161] CNS haploinsufficiency disorders

[0162] In some embodiments, the haploinsufficiency disorder is a CNS haploinsufficiency disorder. In some embodiments, the haploinsufficiency disorder is selected from the group consisting of episodic ataxia, familial hemiplegic migraine, CDKL5 deficiency disorder, CHD2 myoclonic encephalopathy, familial focal epilepsy with variable origin, FOXG1 syndrome, benign familial neonatal seizures, Rett syndrome, Dravat syndrome, SCN2A-epileptic encephalopathy, SCN2A-developmental encephalopathy, SCN8A-epileptic encephalopathy, SC8A familial infantile epilepsy, early infantile epileptic encephalopathy, myoclonic-atonic epilepsy, early infantile epileptic encephalopathy, SYNGAP1-related intellectual disability, tuberous sclerosis, Lennox-Gastaut syndrome, FoxG1 syndrome, KCNQ2-related epileptic encephalopathy, PCDH19-related epilepsy, SLC6A1-related myoclonic-atonic epilepsy, STXBP1-related epileptic encephalopathy, SYNGAP1 syndrome, and combinations thereof.

[0163] In some embodiments, the haploinsufficient gene is selected from the group consisting of SCN1A, SCN2A, SCN8A, SCN12A5, SPTAN1, CDKL5, CHD2, FOXG1, KCNQ2, PCDH19, SLC6A1, STXBP1, SYNGAP1, CACNA1A, DEPDC5, MECP2, TSC1, TSC2, and combinations thereof.

[0164] In some embodiments, the combination of a haploinsufficiency disorder and a haploinsufficiency gene is a combination shown in Table 2.

[0165] Table 2 CNS haploinsufficiency disorders and genes

[0166] Haploinsufficiency Haploinsufficient gene Episodic ataxia, familial hemiplegic migraine CACNA1A CDKL5 Deficiency Disorders CDKL5 CHD2 Myoclonic Encephalopathy CHD2 Lennox-Gastaut syndrome CHD2 Familial focal epilepsy with variable origin DEPDC5 FOXG1 syndrome FOXG1 Benign familial neonatal seizures KCNQ2 Rett syndrome MECP2 Dravet syndrome SCN1A SCN2A-Epileptic Encephalopathy SCN2A SCN2A-Developmental Encephalopathy SCN2A SCN8A-Epileptic Encephalopathy SCN8A SCN8A familial infantile epilepsy SCN8A Early Infantile Epileptic Encephalopathy SLC12A5 Myoclonic-atonic epilepsy SLC6A1 Early Infantile Epileptic Encephalopathy SPTAN1 SYNGAP1-related intellectual disability SYNGAP1 Tuberous sclerosis TSC1 Tuberous sclerosis TSC2 KCNQ2-related epileptic encephalopathy KCNQ2 SLC6A1-related myoclonic-instability epilepsy SLC6A1 STXBP1-related epileptic encephalopathy STXBP1 SYNGAP1 syndrome SYNGAP1

[0167] Example

[0168] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0169] Example 1 - Construct preparation and in vitro synthesis of MeCP2 mRNA

[0170] To prepare a construct targeting the polyadenylation region + short RNA of MeCP2 3'UTR, the best short RNA targeting MeCP2 3'UTR (CCACAGGCTAAAAATGTATATGCCCAAAGA (SEQ ID NO: 1)) was screened and selected, and 50 adenosine (A) nucleotides were added to the 3' end or 5' end of the sequence ( Figures 1A to 1C ). Oligonucleotides with different combinations of polyadenylation regions and short targeting RNAs were designed and synthesized by IDT (Integrated DNA Technologies). The synthesized oligonucleotides were cloned into pJc315 (bluescript-SK+) as gene blocks using the Gibson assembly strategy. A construct with a polyadenylation region at the 3' end of the sequence (pJC1294) and a construct with polyadenylation regions at the 3' and 5' ends, respectively (pJC1295) were selected for in vitro transcription (IVT assay) and further studies. For IVT, pJc1294 and pJC1295 were digested with SacI restriction enzyme and gel purified. Transcription of the linearized plasmid was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolab) according to the production protocol. The RNA product was purified using the Monarch RNA Cleanup Kit (New England Biolab) for cell transfection.

[0171] Example 2 - Cell preparation and RNA transfection

[0172] The human embryonic kidney 293 cell line was grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS. The cells were incubated at 37°C, 5% CO2, and their medium was changed every 48 to 72 hours. To transfect cells with small mRNAs, Hek-293 cells at approximately 70% confluency were split 1:2 one day before transfection. Immediately prior to transfection, the cells were trypsinized, pelleted, and the pellet resuspended in fresh medium and diluted to 500,000 cells / mL. The cells were cultured with 1% 4% PBS according to the manufacturer's instructions. - mRNA transfection kit (Mirus) was used to transfect the cell suspension. The transfection complex was added to the cell suspension in a 12-well plate and incubated for 6 hours at 37°C, 5% CO2. Whole cell lysate and total RNA were extracted with RIPA buffer and Trizol / chloroform, respectively.

[0173] Western blot analysis was performed on total cell extracts using MeCP2(D4F3) diluted 1:1000 Rabbit mAb 3456 cell signaling antibody showed MeCP2 protein levels. The results obtained were quantified and normalized to the level of GAPDH protein ( FIG. 2A to FIG. 2B ).

[0174] RNA extracts were subjected to cDNA synthesis and qRT-PCR was performed using PowerUp SYBER Green Master Mix (provided by biosystem) and a primer set designed for MeCP2 mRNA.

[0175] Example 3 - Lipid Nanoparticle (LNP) Synthesis and Characterization and In Vivo mRNA Synthesis

[0176] Enhancer RNAs compatible with the 3'UTR of MeCP2 mRNA in mice have been developed based on targeting mRNA enhancer 3 ( Figure 1C ) and cloned into a T7 promoter plasmid for IVT. The 5'-end modified synthetic enhancer RNA was sent for packaging into LNPs for in vivo injection.

[0177] By in 1.5

[0178] The organic phase containing lipids was added directly to the aqueous phase containing RNA in a ml microcentrifuge tube to synthesize LNPs. To prepare the organic phase, a mixture of Dlin-MC3 DMA, cholesterol (Sigma-Aldrich), DMG-PEG 2000 (Avanti) and auxiliary lipids (18PG) (Avanti) was dissolved in ethanol. To prepare the aqueous phase, the corresponding RNA was prepared in 25mM magnesium acetate buffer (pH 4.0, Fisher). The storage temperature of all RNAs was -80 ° C, and all RNAs were allowed to thaw on ice before use. For larger-scale LNP production, a syringe pump was used to mix the prepared aqueous phase and ethanol phase in a FNC device at a ratio of 3: 1. The resulting LNP was dialyzed with DI water at 4 ° C for 24 h in a 100,000 MWCO box (Fisher) and stored at 4 ° C before injection.

[0179] LNP was administered to each mouse by intravenous injection at a predetermined dose (25 μg RNA per mouse). After 24 h, the mice were sacrificed and liver tissue was isolated ( Figure 6 to Figure 7 ).

[0180] Example 4 - Construct preparation and in vitro CTNNB1 and PurA mRNA synthesis

[0181] Enhancer RNA targeting the 3'UTR of CTNNB1 (β-catenin) and Pur-α gene was prepared as described in Example 1, wherein the best short RNA with the highest guiding score was selected and 50 adenosine (A) nucleotides were added to the 5' and 3' ends of the sequence. The 5' end was also modified with a cap analog to prevent RNA digestion ( Figure 1C Oligonucleotides were designed and synthesized by IDT (Integrated DNA Technologies). The synthesized oligonucleotides were used as gene blocks for cloning in pJc 315 (bluescript-SK+) using the Gibson assembly strategy and used as templates for IVT (following the protocol described herein).

[0182] The human embryonic kidney 293 cell line was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. The cells were incubated at 37°C, 5% CO2, and their medium was changed every 48 to 72 hours. To transfect cells with small mRNAs, Hek-293 cells at approximately 70% confluency were split 1:2 one day before transfection. Immediately prior to transfection, the cells were trypsinized, pelleted, and the pellet resuspended in fresh medium and diluted to 500,000 cells / mL. The cells were cultured with 1% 4% PBS according to the manufacturer's instructions. - mRNA transfection kit (Mirus) was used to transfect the cell suspension. The transfection complex was added to the cell suspension in a 12-well plate and incubated for 6 hours at 37°C, 5% CO2. Total cell lysate and total RNA were extracted with RIPA buffer and Trizol / chloroform, respectively.

[0183] Human embryonic kidney 293 cell line was grown and transfected with enhanced RNA overnight, and the cells were harvested the next day for RNA extraction and qPCR. The results of qPCR showed that when CTNNB1 or PurA mRNA was exposed to enhanced RNA overnight in culture, CTNNB1 or PurA mRNA levels increased up to 3-fold ( FIG. 9A to FIG. 9B ).

[0184] Example 5 - Deep brain perfusion experiment: 24 hours and 48 hours

[0185] The most effective enhancers throughout the 3'UTR of SYNGAP1 have been selected to enhance protein expression. Two differently designed enhancer RNAs PA-SG and PA-SG-PA have been selected for in vivo studies. Using in vitro transcription technology (IVT), the designed enhancers were synthesized and purified, and then encapsulated into LNPs. Deep brain perfusion technology was used to deliver LNPs to specific brain regions of mice and study their in vivo efficacy. This technology involves a titanium catheter that is placed in the curved part of the hippocampus and enables repeated local injections without repeating the surgical procedure. Wild-type animals were injected with 25 micrograms of enhancer RNA, as well as scrambled RNA as a control. The experiment was repeated twice. In the first attempt, the injected animals were killed 24 hours after the injection ( Fig. 10A and Fig. 10B ), and in the second experiment, animals were sacrificed 48 h after injection ( Fig.12 The whole brain was dissected into four major regions: hippocampus, cortex, midbrain and cerebellum, and analyzed separately. The results of both experiments showed that Syngap1 was enhanced at both protein and RNA levels.

Claims

1. A nucleic acid agent, comprising: (a) a complementary element that hybridizes to a target mRNA; and (b) Polyadenylation element. 2 . The nucleic acid element of claim 1 , wherein the complementary element hybridizes to a 3′ untranslated element of the target mRNA.

3. The nucleic acid agent according to claim 1 or 2, wherein the complementary element comprises RNA or DNA. 4 . The nucleic acid agent according to claim 1 , wherein the polyadenylation element is located at the 5′ end of the complementary element. 5 . The nucleic acid agent according to claim 1 , wherein the polyadenylation element is located at the 3′ end of the complementary element. 6 . The nucleic acid agent according to claim 5 , wherein the polyadenylation element is located at the 3′ end of the complementary element, and the nucleic acid agent further comprises a 5′ cap element located at the 5′ end of the complementary element. 7 . The nucleic acid agent according to claim 1 , wherein the nucleic acid agent comprises a first polyadenylation element located at the 5′ end of the complementary element and a second polyadenylation element located at the 3′ end of the complementary element.

8. The nucleic acid agent according to any one of claims 1 to 7, wherein the poly(A) element comprises about 20 nucleotides.

9. The nucleic acid agent according to any one of claims 1 to 7, wherein the poly(A) element comprises about 50 nucleotides.

10. The nucleic acid agent according to any one of claims 1 to 7, wherein the poly(A) element comprises about 75 nucleotides. The nucleic acid agent according to any one of claims 1 to 10, wherein the nucleic acid agent further comprises a nucleotide modification.

12. The nucleic acid agent according to claim 11, wherein the nucleotide modification is N 1 -methyladenosine (m 1 A) N 6 -methyladenosine (m 6 A) or adenosine to inosine (A to I) modification.

13. The nucleic acid agent according to claim 11, wherein the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU) or N4-acetylcytidine modification.

14. The nucleic acid agent according to any one of claims 1 to 13, wherein the nucleic acid agent is encoded by a sequence comprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

3. 15 . The nucleic acid agent according to claim 1 , wherein the target mRNA is an mRNA of an active allele of a gene associated with a disorder, the disorder being associated with reduced protein expression by the mRNA.

16. The nucleic acid agent of claim 15, wherein the disorder is a haploinsufficiency disorder. The nucleic acid agent according to claim 15 or 16, wherein the target mRNA encodes MeCP2 protein. 18 . The nucleic acid agent according to claim 17 , wherein the complementary element comprises or consists of SEQ ID NO:

1.

19. A recombinant expression system comprising a nucleic acid sequence encoding a nucleic acid agent comprising (i) a complementary element that hybridizes to a target mRNA and (ii) a polyadenylation element.

20. The recombinant expression system of claim 19, wherein the complementing element hybridizes to a 3' untranslated element of the target mRNA.

21. The recombinant expression system of claim 19 or 20, wherein the complementing elements comprise RNA or DNA.

22. The recombinant expression system according to any one of claims 19 to 21, wherein the polyadenylation element is located at the 5' end of the complementation element.

23. The recombinant expression system according to any one of claims 19 to 22, wherein the polyadenylation element is located at the 3' end of the complementation element.

24. The recombinant expression system of claim 23, wherein the polyadenylation element is located at the 3' end of the complementary element, and wherein the RNA molecule further comprises a 5' cap element located at the 5' end of the complementary element.

25. The recombinant expression system according to any one of claims 19 to 24, wherein the nucleic acid molecule comprises a first polyadenylation element located at the 5' end of the complementary element and a second polyadenylation element located at the 3' end of the complementary element.

26. The recombinant expression system of any one of claims 19 to 25, wherein the polyadenylation element comprises about 20 nucleotides.

27. The recombinant expression system of any one of claims 19 to 25, wherein the polyadenylation element comprises about 50 nucleotides.

28. The recombinant expression system of any one of claims 19 to 25, wherein the polyadenylation element comprises about 75 nucleotides.

29. The recombinant expression system of any one of claims 19 to 28, wherein the nucleic acid agent further comprises a nucleotide modification.

30. The recombinant expression system of claim 29, wherein the nucleotide modification is N 1 -methyladenosine (m 1 A) N 6 -methyladenosine (m 6 A) or adenosine to inosine (A to I) modification.

31. The recombinant expression system of claim 29, wherein the nucleotide modification is pseudouracil, M1-pseudouracil, 5-methoxyuridine (5moU) or N4-acetylcytidine modification.

32. A recombinant expression system according to any one of claims 19 to 31, wherein the recombinant expression system is encoded by a sequence comprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO:

3.

33. The recombinant expression system according to any one of claims 19 to 32, wherein the target mRNA is an mRNA of an active allele of a gene associated with a disorder, the disorder being associated with reduced expression of a protein by the mRNA.

34. The recombinant expression system of claim 33, wherein the disorder is a haploinsufficiency disorder.

35. The recombinant expression system of claim 33 or 34, wherein the target mRNA encodes MeCP2 protein.

36. The recombinant expression system of claim 35, wherein the nucleic acid agent is or comprises a nucleic acid molecule comprising or consisting of SEQ ID NO:

1.

37. An expression vector comprising the recombinant expression system according to any one of claims 19 to 36.

38. The expression vector of claim 37, wherein the expression vector is a viral vector.

39. The expression vector of claim 38, wherein the viral vector is an adeno-associated viral vector (AAV), a lentiviral vector or an adenoviral vector.

40. A pharmaceutical composition comprising or delivering the nucleic acid agent of any one of claims 1 to 18, the recombinant expression system of any one of claims 19 to 36, or the expression vector of any one of claims 37 to 39.

41. A method for treating a haploinsufficiency disorder in a subject, the method comprising: Administering a therapeutically effective amount of the pharmaceutical composition of claim 40 to the subject.

42. The method of claim 41, wherein the therapeutically effective amount of the pharmaceutical composition enhances protein expression of the target mRNA.

43. The method of claim 41 or 42, wherein the haploinsufficiency disorder is selected from the group consisting of: 5q syndrome, Adams-Oliver syndrome type 1, Adams-Oliver syndrome type 3, Adams-Oliver syndrome type 5, Adams-Oliver syndrome type 6, Alagille syndrome type 1, autoimmune lymphoproliferative syndrome type IA, autoimmune lymphoproliferative syndrome type V, autosomal dominant deafness-2A, brain malformation with or without urinary tract defect (BRMUTD), Carney complex Type 1, CHARGE syndrome, cleidocranial dysplasia, Currarino syndrome, Denis–Draser syndrome / Fraser syndrome, developmental delay, intellectual disability, obesity, and dysmorphic features (DIDOD), DiGeorge syndrome (TBXI-related), Dravet syndrome, Duane radial syndrome, Ehlers–Danlos syndrome (classic), Ehlers–Danlos syndrome (vascular), Feingold syndrome 1, frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP) GRN-related, GLUT I deficiency syndrome, Greig polysyndactyly, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly type 3, holoprosencephaly type 4, holoprosencephaly type 5, Holt-Oram syndrome, hypoparathyroidism-sensorineural hearing loss-renal disease (HDR), Kleefstra syndrome 1, venous malformation hyperostosis syndrome (AAGF-related), Leri-Weill achondroplasia, Marfan syndrome, mental retardation and distinctive facial features with or without heart defects (MRFACD), mental retardation-autosomal dominant 1, mental retardation-autosomal dominant 19. Mental retardation - autosomal dominant 29. Nail-patella syndrome (NPS), Phelan-McDermid syndrome, Pitt-Hopkins syndrome, primary pulmonary hypertension 1, Rett syndrome (congenital variant), Smith-Magennis syndrome (RAII-related), Sotos syndrome 1, Sotos syndrome 2, Stickler syndrome type I, supravalvular aortic stenosis, Syngapi-related intellectual disability, Treacher Collins syndrome, tricho-nose-phalangeal syndrome type I, ulna-mammary syndrome, van der Ward syndrome 1, Wasdenburg syndrome type 1, Wasdenburg syndrome type 2A, and Wasdenburg syndrome type 4C.

44. The method of any one of claims 41 to 43, wherein the subject is a mammal.

45. The method of claim 44, wherein the subject is a human.

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