mRNA regulon therapy for the treatment of haploinsufficiency disorders

Abstract

The composition and method for treating haploinsufficiency disorder by mRNA regulation are described herein.For example, the fusion protein is provided herein, comprising the RNA effector protein that targets the mRNA of the active allele of the gene associated with haploinsufficiency disorder and the regulon portion that stimulates and / or stabilizes said mRNA. TIFF2024534496000151.tif94128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 246,689, filed September 21, 2021, and U.S. Provisional Patent Application No. 63 / 352,530, filed June 15, 2022, which are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file entitled "44807-0408WO1.XML". The XML file was created on Sep. 20, 2022 and is 468,769 bytes in size. The data within the XML file is incorporated herein by reference in its entirety.

[0003] Technical Field Described herein are compositions and methods for treating haploinsufficiency disorders through mRNA modulation. [Background technology]

[0004] background Haploinsufficiency occurs when one gene allele is inactivated and the amount of gene product expressed from the remaining active allele is insufficient for proper gene function. Several disorders are associated with or caused by haploinsufficiency. An example of a haploinsufficiency disorder is Dravet syndrome.

[0005] Dravet syndrome is a rare, devastating form of intractable epilepsy that begins in infancy. Initially, patients experience long seizures. In the second year, another type of seizure begins to occur, which typically coincides with growth retardation, possibly due to repeated cerebral hypoxia. This leads to poor development of language and motor skills. Mutations in the SCNIA gene (encoding the voltage-gated sodium channel a subunit), SCNIB gene (encoding the voltage-gated sodium channel I subunit), SCN2A gene, SCN3A gene, SCN9A gene, GABRG2 gene (encoding the y-aminobutyric acid receptor y2 subunit), GABRD gene (encoding the y-aminobutyric acid receptor 11 subunit), and / or PCDH19 gene have been associated with Dravet syndrome.

[0006] SCN1A encodes the neuronal voltage-gated sodium channel NaV1.1, which is prominently expressed in inhibitory GABAergic neurons. Loss-of-function (LOF) mutations in SCN1A, including missense codons and premature stop codons (PTCs), are the most frequently identified causes of Dravet syndrome [1, 2].

[0007] LOF premature stop codons (PTCs) result from single-base mutations that convert a standard triplet nucleotide codon into one of three stop codons (TAG, TGA, or TAA). PTCs can be more deleterious than missense mutations because they result in a complete loss of protein expression [7]. There are 10 amino acid codons that are vulnerable to PTC conversion by single-base substitutions (arginine, tryptophan, tyrosine, cysteine, glutamic acid, lysine, glutamine, serine, leucine, and glycine). PTC mutations in SCN1A are a common class of mutations associated with Dravet syndrome (Figure 1), with over 180 known PTC mutations.

[0008] SCN1A LOF mutations result in hyperactivity of neuronal networks as a result of impaired inhibitory GABAergic neurotransmission. Frequent unprovoked seizures are one of the presenting features of Dravet syndrome, but patients also suffer from a series of comorbidities affecting the domains of cognition, mobility, speech, and behavior [3]. Dravet syndrome patients often have sleep and circadian rhythm disturbances, neurodevelopmental delay and intellectual disability, eye movement disorders, and psychomotor regression. Sudden unexpected death in epilepsy (SUDEP) is also prevalent in this population [4-6]. Due to the severity of these comorbidities, there is an urgent need for effective treatments that can address both the seizures and the series of comorbidities associated with Dravet syndrome. There are animal models available for Dravet syndrome that have been successfully applied in clinical trials.

[0009] Therapeutic options to repair SCN1A mutations are limited. Small molecules such as aminoglycosides [9], dipeptides

[10] , and oxadiazoles

[11] promote “read-through,” but this approach results in coding for a near-cognate amino acid

[12] , effectively resulting in a missense mutation instead of a PTC. Sodium channels such as SCN1A are poorly tolerant of missense mutations, which can lead to gain-of-function and loss-of-function neurological disorders [13, 14]. Furthermore, aminoglycosides are ototoxic and nephrotoxic

[15] , and the first-in-class oxadiazole (Ataluren) showed unexpectedly low efficacy in patient populations (ACT DMD Phase 3 Clinical Trial, NCT01826487; ACT CF, NCT02139306). Furthermore, most previous treatment initiatives for Dravet syndrome were aimed solely at reducing seizures. No true disease-modifying therapy exists for developmental epileptic encephalopathies (DEEs).

[0010] Recent and continuing advances in CRISPR / Cas9-mediated genome editing provide potentially permanent solutions to single-gene disorders. However, LOF missense mutations occur throughout the SCN1A coding region, making gene editing approaches extremely challenging [8]. Furthermore, features of this technology pose hurdles to its rapid use as a therapeutic [16, 17], including cell type-specific delivery, efficiency of homologous recombination, and frequency of on-target repair errors and off-target editing. Furthermore, at over 6 kb per transcript, the sodium channel coding region of SCN1A exceeds the capacity of AAV to accommodate, thus limiting the utility of AAV therapy for tissue-specific gene replacement, which is advancing at a fast pace. Thus, clinical options for SCN1A LOF associated with Dravet syndrome remain limited and the therapeutic pipeline is poor. The compositions and methods described herein address this and other deficiencies in the art. Summary of the Invention

[0011] overview The compositions and methods described herein have a wide range of applications and provide potential disease-modifying therapies for various haploinsufficiency disorders.The main advantage of this mRNA regulon approach is that it is 100% independent of the type and position of mutation by stabilizing wild-type (WT) allele mRNA.It is highly likely and easy to redesign these approaches for other indications.

[0012] Thus, provided herein is a fusion protein comprising an RNA effector protein that targets the mRNA of an active allele of a gene associated with a haploinsufficiency disorder and a regulon portion that stimulates and / or stabilizes the mRNA.

[0013] In some embodiments, the RNA effector protein is a Cas effector protein selected from the group consisting of Cas9, Cas12, Cas13, and Cas14. In some embodiments, the RNA effector protein is Cas13b. In some embodiments, the Cas effector protein is a catalytically inactive Cas protein.

[0014] In some embodiments, the regulon portion is PABPC1 or NAT10.

[0015] In some embodiments, the fusion protein further comprises a linker and / or a spacer.

[0016] In some embodiments, the fusion protein further comprises a nuclear export signal and / or an epitope tag.

[0017] In some embodiments, the RNA effector protein is N-terminal to the regulon portion. In some embodiments, the RNA effector protein is C-terminal to the regulon portion.

[0018] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:48 or SEQ ID NO:49, or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:48 or SEQ ID NO:49.

[0019] Also provided herein is a polynucleotide that encodes any one of the fusion proteins described herein.Also provided herein is a vector that comprises any one of the polynucleotides described herein.Also provided herein is a cell that comprises any one of the vectors described herein.

[0020] Also provided herein is a system that includes: a fusion protein that includes an RNA effector protein that targets an mRNA of an active allele of a gene associated with a haploinsufficiency disorder and a regulon portion that stimulates and / or stabilizes the mRNA; and a gRNA that forms a complex with the RNA effector protein and includes a complementary region that hybridizes with the mRNA of the active allele.

[0021] In some embodiments, the RNA effector protein is a Cas effector protein selected from the group consisting of Cas9, Cas12, Cas13, and Cas14. In some embodiments, the Cas effector protein is Cas13b. In some embodiments, the Cas effector protein is a catalytically inactive Cas effector protein.

[0022] In some embodiments, the regulon portion is PABPC1 or NAT10.

[0023] In some embodiments, the fusion protein further comprises a linker and / or a spacer. In some embodiments, the fusion protein further comprises a nuclear export signal and / or an epitope tag.

[0024] In some embodiments, the RNA effector protein is N-terminal to the regulon portion. In some embodiments, the RNA effector protein is C-terminal to the regulon portion.

[0025] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:48 or SEQ ID NO:49, or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:48 or SEQ ID NO:49.

[0026] In some embodiments, the gRNA targets an mRNA encoding MeCP2, SCN1A, SYNGAP1, SHANK3, CHD2, or PTEN. In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof. In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:17, and combinations thereof.

[0027] In some embodiments, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding an amino acid selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and combinations thereof.

[0028] In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and combinations thereof.

[0029] In some embodiments, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding an amino acid selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and combinations thereof.

[0030] In some embodiments, the gRNA is selected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72.

[0031] Also provided herein is one or more polynucleotides encoding any one of the systems described herein.Also provided herein is one or more vectors comprising any one of the polynucleotides described herein.Also provided herein is a cell comprising any one of the vectors described herein.

[0032] Also provided herein is a complex comprising a fusion protein comprising an RNA effector protein that targets an mRNA of an active allele of a gene associated with a haploinsufficiency disorder and a regulon portion that stimulates and / or stabilizes the mRNA, the fusion protein linked to a gRNA that comprises a complementary region that hybridizes to the mRNA of the active allele.

[0033] In some embodiments, the RNA effector protein is dCas13b and the regulon portion is PABP1 or NAT10.

[0034] In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and combinations thereof. In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:17, and combinations thereof.

[0035] In some embodiments, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding an amino acid selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and combinations thereof.

[0036] In some embodiments, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and combinations thereof.

[0037] In some embodiments, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and combinations thereof.

[0038] Also provided herein is a complex comprising a fusion protein comprising an RNA effector protein that targets an mRNA of an active allele of a gene associated with a haploinsufficiency disorder and a regulon portion that stimulates and / or stabilizes the mRNA, wherein the fusion protein is bound to a gRNA and the mRNA.

[0039] In some embodiments, the RNA effector protein is dCas13b and the regulon portion is PABP1 or NAT10.

[0040] In some embodiments, the mRNA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof. In some embodiments, the mRNA encodes an amino acid selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, and combinations thereof.

[0041] In some embodiments, the mRNA is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and combinations thereof.

[0042] In some embodiments, the mRNA is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and combinations thereof.

[0043] In some embodiments, the mRNA encodes amino acids selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and combinations thereof.

[0044] Also provided herein is a pharmaceutical composition comprising any one of the fusion proteins or systems described herein. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0045] Also provided herein are pharmaceutical compositions comprising one or more nucleic acids encoding any one of the fusion proteins or systems described herein.

[0046] Also provided herein is a viral vector comprising one or more nucleic acids encoding any one of the fusion proteins or systems described herein. In some embodiments, the viral vector is an adeno-associated viral vector.

[0047] Also provided herein are nanoparticles or liposomes comprising any one of the fusion proteins or systems described herein, or one or more nucleic acids encoding any one of the fusion proteins or systems described herein.

[0048] Also provided herein is a method for stimulating or stabilizing mRNA, comprising contacting mRNA with any one of the fusion proteins or systems described herein.In some embodiments, the method is carried out in vitro, in vivo, or ex vivo.

[0049] Also provided herein is a method of treating or preventing a haploinsufficiency disorder in a subject, comprising the steps of: administering to a subject a fusion protein, or a nucleic acid encoding a fusion protein, comprising an RNA effector protein that targets an mRNA of an active allele of a gene associated with a haploinsufficiency disorder and a regulon portion that stimulates and / or stabilizes the mRNA; and a gRNA, or a nucleic acid encoding a gRNA, designed to form a complex with the RNA effector protein and comprising a complementary region designed to hybridize with the mRNA of the active allele.

[0050] In some embodiments, the RNA effector protein is a Cas effector protein selected from the group consisting of Cas9, Cas12, Cas13, and Cas14. In some embodiments, the Cas effector protein is Cas13b. In some embodiments, the Cas effector protein is a catalytically inactive Cas protein.

[0051] In some embodiments, the regulon portion is PABPC1 or NAT10.

[0052] In some embodiments, the fusion protein further comprises a linker and / or a spacer. In some embodiments, the fusion protein further comprises a nuclear export signal and / or an epitope tag.

[0053] In some embodiments, the RNA effector protein is N-terminal to the regulon portion. In some embodiments, the RNA effector protein is C-terminal to the regulon portion.

[0054] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:48 or SEQ ID NO:49, or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:48 or SEQ ID NO:49.

[0055] In some embodiments, the haploinsufficiency disorder is 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 malformations with or without urinary tract anomalies (BRMUTD), Carney complex type 1, CHARGE syndrome, cleidocranial dysplasia, Currarino syndrome, Dennis-Drash syndrome. / Frasier syndrome, Developmental delay, intellectual disability, obesity, and dysmorphic features (DIDOD), DiGeorge syndrome (TBXI-associated), Dravet syndrome, Duane-radial ray syndrome, Ehlers-Danlos syndrome (classic subtype), Ehlers-Danlos syndrome (vascular subtype), Feingold syndrome type 1, Frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP) (GRN-associated), GLUT1 deficiency syndrome, Greig acropolysyndactyly syndrome, Hereditary hemorrhagic telangiectasia type 1, Holoprosencephaly type 3, Holoprosencephaly type 4 type 5, Holoprosencephaly type 5, Holt-Oram syndrome, Hypoparathyroidism, Sensorineural deafness, Adrenal gland disease (HDR), Kleefstra syndrome type 1, Klippel-Trenaunay syndrome (AAGF-associated), Lery-Weil chondro-osseous dysplasia, Marfan syndrome, Mental retardation and distinctive facial features with or without heart defects (MRFACD), Mental retardation (autosomal dominant type 1), Mental retardation (autosomal dominant type 19), Mental retardation (autosomal dominant type 29), Nail-patellar syndrome (NPS), Phelan-McDermid syndrome , Pitt-Hopkins syndrome, primary pulmonary hypertension type 1, Rett syndrome (congenital), Smith-Magenis syndrome (RAII-associated), Sotos syndrome type 1, Sotos syndrome type 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAPI-associated intellectual disability, Treacher Collins syndrome, trichorhinophallic syndrome type I, ulno-mammary syndrome, van der Woude syndrome type 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C.

[0056] In some embodiments, the haploinsufficiency disorder is a CNS haploinsufficiency disorder. In some embodiments, the CNS haploinsufficiency disorder is selected from the group consisting of episodic ataxia, familial hemiplegic migraine, CDKL5 deficiency disorder, CHD2 myoclonic encephalopathy, familial focal epilepsy showing variable foci, FOXG1 syndrome, benign familial neonatal seizures, Rett syndrome, Dravet 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-associated intellectual disability, tuberous sclerosis complex, Lennox-Gastaut syndrome, FoxG1 syndrome, KCNQ2-associated epileptic encephalopathy, PCDH19-associated epilepsy, SLC6A1-associated myoclonic pretension epilepsy, STXBP1-associated epileptic encephalopathy, SYNGAP1 syndrome, and combinations thereof. In some embodiments, the haploinsufficiency disorder is Dravet syndrome or Rett syndrome.

[0057] In some embodiments, the subject has haploinsufficiency in a gene selected from the group consisting of AGGFI, ARHGAP31, BMPR2, CHD7, COL2A1, COL3A1, CTLA4, CTNNBI, DLL4, EHMTI, ELN, ENG, FAS, FBNI, FOXGI, GATA3, GLI3, GRN, IRF6, JAGI, KCNQ4, LMMIB, MBD5, MED13L, MITF, MNXI, MYCN, NFIA, NFIX, NOTCH1, NSDI, PAX3, PHIP, PRKARIA, RAil, RBPJ, RPS14, RUNX2, SALL4, SCNIA, SETBPI, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOXI0, SYNGAPI, TBXI, TBX3, TBX5, TCF4, TCOFI, TGIFI, TNXB, TRPSI, WTI, ZIC2, and combinations thereof. In some embodiments, the subject has haploinsufficiency in a gene 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.

[0058] In some embodiments, the subject has a mutation selected from the list in Table 4, and combinations thereof. In some embodiments, the subject has a mutation selected from the list in Table 6, and combinations thereof. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0059] In some embodiments, the fusion protein and gRNA are administered as part of a pharmaceutical composition.In some embodiments, the administering step comprises administering to the subject a viral vector comprising a nucleic acid sequence encoding the fusion protein and gRNA.In some embodiments, the administering step comprises administering to the subject a nanoparticle or liposome comprising the fusion protein and gRNA or the nucleic acid sequence encoding the fusion protein and gRNA.

[0060] Throughout this application, various aspects may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0061] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. For example, the term "a sample" includes multiple samples, including mixtures thereof.

[0062] The terms "determining," "measuring," "assessing," "evaluating," "analyzing," and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detecting). These terms can include quantitative, qualitative, or both quantitative and qualitative measurements. Evaluating can be relative or absolute. "Detecting the presence of" can include determining whether something is present or absent as well as measuring the amount of something present, depending on the context.

[0063] As used herein, a number followed by the term "about" refers to the number plus or minus 10% of the number. A range followed by the term "about" refers to a range from a value minus 10% of the minimum value to a value plus 10% of the maximum value.

[0064] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0065] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0066] The patent or application contains at least one drawing executed in color. Copies of the patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0067] [Figure 1] Figure 1 is a schematic showing SCN1A topology and premature termination codons (PTCs). Each of the 182 SCN1A PTCs is indicated by a color. [Diagram 2] Figure 2 is a schematic diagram depicting a tethered functional assay. A functional protein fused to a tethered protein binds to an mRNA of interest and enhances or stabilizes the mRNA. [Diagram 3]Figure 3A is a schematic diagram showing the experimental approach applied in this assay. Briefly, PABPC1 was fused to dCas13b tethering protein and guided to the 3'UTR of luciferase gene via a designed guide RNA (gRNA) to promote its translation. Figure 3B shows firefly luciferase evaluation, which shows that PAB-tethered luciferase targeting was increased approximately 2-fold compared to non-targeting in HEK-293 cells. [Figure 4] FIG. 4 shows a map of plasmid pJC1211, which encodes the Cas13b-PABPC1 fusion protein. [Diagram 5] 5A-5AB show the sequence and characteristics of plasmid pJC1211 (SEQ ID NO: 50). The nucleic acid sequence is shown in both the 5'→3' and reverse complement (3'→5') strands. [Figure 6] FIG. 6 is a map of plasmid pJC1212, which encodes a luciferase-targeting Cas13b gRNA. [Figure 7] 7A-7G show the sequence and characteristics of plasmid pJC1212 (SEQ ID NO: 52). The nucleic acid sequence is shown in both the 5'→3' and reverse complement (3'→5') strands. [Figure 8] FIG. 8 is a map of plasmid pJC1213, which encodes a luciferase-targeting Cas13b gRNA. [Figure 9] 9A-9F show the sequence and characteristics of plasmid pJC1213 (SEQ ID NO: 53). [Figure 10] FIG. 10 is a map of Addgene plasmid 103854 (empty vector compared to pJC1212 and pJC1213). [Figure 11] 11A-11H show the sequence and characteristics of Addgene plasmid 103854 (empty vector compared to pJC1212 and pJC1213) (SEQ ID NO: 54). [Figure 12]Figures 12A-12G show that the fusion protein approach promotes mRNA expression in multiple cell types. Figure 12A is an exemplary schematic depicting the tethered mRNA amplifier approach. Briefly, PABPC1 is fused to dCas13b. The fusion is directed to a specific mRNA via a guide RNA (gRNA) targeting the 3'UTR. In HEK293 cells, the tethered mRNA amplifier stimulates the expression of a gRNA-dependent luciferase reporter (Figure 12B) and endogenous MeCP2 mRNA (Figure 12C) using luciferase activity assays or Western blot, respectively. The red bars in the corresponding schematics represent the approximate locations of the two gRNAs used in each experiment. The effect of the tethered mRNA amplifier requires PABPC1 (Figure 12D). The tethered mRNA amplifier also enhances MeCP2 mRNA levels (Figure 12E). Similar stimulatory effects on MeCP2 can be observed in SH-SY5Y and HepG2 cells (Figure 12F). Figure 12G shows that the stimulatory effects vary depending on the location of the gRNA along the 3'UTR of MeCP2 (p values: *<0.05, **<0.005, ***<0.0005). [Figure 13] Figures 13A-13D show that the fusion proteins promote expression of transcripts associated with haploinsufficiency disorders; the tethered mRNA amplifier targeted SYNGAP1 (Figure 13A), SHANK3 (Figure 13B), PTEN (Figure 13C), and CHD2 (Figure 13D) mRNAs in a gRNA-dependent manner. Protein and mRNA analysis results in SH-SY5Y cells are shown. All protein assays were performed with four different biological replicates, and for RNA analysis, at least two biological replicates were used. (p-values: *<0.05, **<0.005, ***<0.0005). [Figure 14]Figures 14A-14F show the function of the minimal fusion protein in cells. Figure 14A shows an exemplary schematic of PABPC1 and its functional motifs. RRM1-4 are RNA recognition motifs. MLLE is the Mademoiselle domain. Figure 14B shows an AlphaFold predicted model of dCas13b fusion with the MLLE domain (amino acids 545-636) of PABPC1. Figure 14C shows a predicted model of full-length PABPC1 - all residues other than amino acids 545-636 are hidden after prediction. Figure 14D shows a predicted model of full-length dCas13b itself. Figure 14E shows a PyMol alignment of Figures 14A-14D. Figure 14F shows a Western blot comparing the tethering of full-length PABPC1 to the MLLE domain alone for targeting MeCP2 transcripts in HEK293 cells. [Figure 15] Figure 15 is an exemplary schematic diagram showing the outcome of a haploinsufficiency disorder when one copy of a gene is mutated but the other is normal. This mutation ultimately reduces protein expression by half, causing the disease state. The mRNA amplifier targets the messenger RNA from the normal gene, theoretically doubling protein expression to normal amounts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description The compositions and methods described herein have a wide range of applications and provide potential disease-modifying therapy for various haploinsufficiency disorders.Haploinsufficiency occurs when one gene allele is inactivated and the amount of gene product expressed from the remaining active allele is insufficient for proper gene function.Some disorders are associated with or caused by haploinsufficiency.An example of haploinsufficiency disorder is Dravet syndrome.

[0069] Targeting messenger RNA metabolism in the treatment of haploinsufficiency offers a novel therapeutic window. However, changes in gene expression are commonly viewed as reflecting programmed transcriptional variability. It is less common that large-scale regulation of messenger RNA expression also occurs during translation. For example, in early oocytes, large changes in protein expression occur via coordinated activation / inactivation of maternally derived mRNAs. Similarly, translationally-quiescent transcripts are activated upon synaptic stimulation in neurons. A striking discrepancy between mRNA and protein levels is also observed in somatic cells, revealing the widespread distribution and physiological importance of posttranscriptional regulons.

[0070] Transcript stability and translation rate are key features that define post-transcriptional regulons. Every transcript degrades and is translated at a specific rate. mRNA stability and translation are often proportional to gene function; housekeeping genes typically result in long-lived mRNAs that persist in cells for more than 24 hours, whereas transiently expressed genes produce short-lived transcripts that persist for a few minutes or so. In part, cells achieve this very wide range of transcript stability and expression through sequence and / or structural elements that recruit specific factors that act positively or negatively. The compositions and methods described herein utilize, among other things, this mRNA / post-transcriptional regulon as a novel disease-modifying treatment for haploinsufficiency disorders, such as Dravet syndrome.

[0071] mRNA modulating therapeutic substances A novel molecular therapeutic strategy using post-transcriptional regulation of mRNA, e.g., SCN1A mRNA, to overcome haploinsufficiency in genetic brain disorders is described herein. The theory of this technology is based on what is called the "tethered function approach" or "tethered mRNA amplifier approach". In brief, this technology tethers a specific factor to the 3' untranslated region (3'UTR) of a target mRNA, thereby altering its stability and / or translation rate. This approach is commonly used to determine the function of RNA-binding proteins [18, 19].

[0072] Proteins and protein complexes that regulate mRNA metabolism have two activities. They specifically bind to mRNA and then induce some function, i.e. regulate mRNA splicing, transport, localization, translation, or stability. These two activities can often be present in different proteins in a complex, or in different regions of a single polypeptide. In the vast majority of cases involving mRNA, the individual RNA binding activity and function are different. In these cases, the effective approach to therapeutic modalities is realized by uncoupling the two activities from each other. Essentially, the functional activity (without the natural RNA binding activity) can be linked to new mRNA via the intrinsic RNA binding activity.

[0073] In the tethered functional assay (e.g., as shown in FIG. 2), a chimeric protein in which protein X is tethered to a tethering polypeptide is expressed in vivo to measure the effect of protein X on mRNA metabolism (see FIG. 2). The tethering protein is an RNA-binding protein that recognizes an RNA tag sequence with high specificity and affinity. The effect of the fusion protein on mRNA metabolism is measured by co-expressing the chimera with an mRNA reporter (e.g., lacZ or luciferase) in which the tag RNA sequence is embedded. The effect of the fusion protein on mRNA metabolism is evaluated by conventional means (e.g., Western blot, Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), etc.). However, this assay has only been utilized in model organisms / cell cultures to evaluate the contribution of RNA-binding proteins to post-transcriptional regulation of mRNA. A novel therapeutic modality based on tethering for disease modification, e.g., for modification of Dravet syndrome, is described herein.

[0074] Tethered functional assays were developed as a tool to dissect the function of unknown RNA-binding proteins in the post-transcriptional control of mRNA. The devised systems utilized exogenous RNA-binding activities, often of bacteriophage origin (MS2 coat protein and lambda N-peptide being the most common). An obvious limitation of this approach was that the corresponding RNA-binding elements had to be cloned and expressed in a reporter mRNA. Novel protein / RNA bindings were used to "tether" the activity to the mRNA and test its function. Crucially, the ability to link a functional activity to any mRNA of interest could be very effective in altering the expression pattern of that mRNA. This approach could then be exploited in the treatment of haploinsufficiency.

[0075] CRISPR-based research tools have established a system capable of selectively recognizing RNA molecules. This novel RNA targeting system has made it possible to detect and manipulate specific RNAs using various approaches, such as RNA knockdown, site-specific RNA editing, RNA localization, and the destruction of toxic RNAs that lead to human neurodegenerative disorders [20, 21].

[0076] In some cases, the mRNA regulon therapy described herein utilizes CRISPR-based RNA binding approach to provide precise and advanced technology to target genetic disorders at the transcriptional level.RNA targeting CRISPR-Cas13 has RNA strand specificity and binds with high affinity.This allows efficient and precise delivery to tether known factors that promote mRNA expression to improve haploinsufficiency by appropriately utilizing the mRNA-specific targeting ability of Cas13b (Figure 15).

[0077] Haploinsufficiency disorders and genes In some cases, haploinsufficiency disorders include 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 malformations with or without urinary tract anomalies (BRMUTD), Carney complex type 1, CHARGE syndrome, cleidocranial dysplasia, Clarino syndrome, and Dennis-Drash syndrome. Group / Frasier syndrome, developmental delay, intellectual disability, obesity, and dysmorphic features (DIDOD), DiGeorge syndrome (TBXI-related), Dravet syndrome, Duane-radial ray syndrome, Ehlers-Danlos syndrome (classic type), Ehlers-Danlos syndrome (vascular type), Feingold syndrome type 1, frontotemporal lobar degeneration with TDP43 inclusions (FTLD-TDP) (GRN-related), GLUT1 deficiency syndrome, Greig acropolysyndactyly syndrome, hereditary hemorrhagic telangiectasia type 1, holoprosencephaly type 3, holoprosencephaly cystic encephaly type 4, holoprosencephaly type 5, Holt-Oram syndrome, hypoparathyroidism, sensorineural hearing loss, adrenal gland disease (HDR), Kleefstra syndrome type 1, Klippel-Trenaunay syndrome (AAGF-associated), Lery-Weil chondro-osseous dysplasia, Marfan syndrome, mental retardation and distinctive facial features with or without heart defects (MRFACD), mental retardation (autosomal dominant type 1), mental retardation (autosomal dominant type 19), mental retardation (autosomal dominant type 29), nail-patellar syndrome (NPS), Phelan-McDermis syndrome The haploinsufficiency disorder is selected from: Dravet syndrome, Pitt-Hopkins syndrome, primary pulmonary hypertension type 1, Rett syndrome (congenital), Smith-Magenis syndrome (RAII-associated), Sotos syndrome type 1, Sotos syndrome type 2, Stickler syndrome type I, supravalvular aortic stenosis, SYNGAPI-associated intellectual disability, Treacher Collins syndrome, trichorhinophallic syndrome type I, ulno-mammary syndrome, van der Woude syndrome type 1, Waardenburg syndrome type 1, Waardenburg syndrome type 2A, and Waardenburg syndrome type 4C. In some cases, the haploinsufficiency disorder is Dravet syndrome.

[0078] In some cases, 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, LMMIB, MBD5, MED13L, MITF, MNXI, MYCN, NFIA, NFIX, NOTCH1, NSDI, PAX3, PHIP, PRKARIA, RAil, RBPJ, RPS14, RUNX2, SALL4, SCNIA, SETBPI, SHANK3, SHH, SHOX, SLC2A1 / GLUT1, SOXI0, SYNGAPI, TBXI, TBX3, TBX5, TCF4, TCOFI, TGIFI, TNXB, TRPSI, WTI, ZIC2, and combinations thereof. In some cases, the haploinsufficient gene is SCNIA.

[0079] In some cases, the combination of the haploinsufficiency disorder and the haploinsufficiency gene combination is a combination shown in Table 1.

[0080] Table 1 Haploinsufficiency disorders and genes TIFF2024534496000002.tif152156TIFF2024534496000003.tif152156

[0081] CNS haploinsufficiency disorders In some cases, the haploinsufficiency disorder is a CNS haploinsufficiency disorder. In some cases, the haploinsufficient disorder is selected from the group consisting of episodic ataxia, familial hemiplegic migraine, CDKL5 deficiency disorder, CHD2 myoclonic encephalopathy, familial focal epilepsy showing variable foci, FOXG1 syndrome, benign familial neonatal seizures, Rett syndrome, Dravet 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-associated intellectual disability, tuberous sclerosis complex, Lennox-Gastaut syndrome, FoxG1 syndrome, KCNQ2-associated epileptic encephalopathy, PCDH19-associated epilepsy, SLC6A1-associated myoclonic pretension epilepsy, STXBP1-associated epileptic encephalopathy, SYNGAP1 syndrome, and combinations thereof.

[0082] In some cases, 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.

[0083] In some cases, the combination of the haploinsufficiency disorder and the haploinsufficiency gene combination is a combination shown in Table 2.

[0084] Table 2. CNS haploinsufficiency disorders and genes TIFF2024534496000004.tif115156

[0085] SCN1A SCN1A (NCBI Gene ID: 6323; reference sequence NG_011906.1) encodes the voltage-gated sodium channel alpha subunit 1. Its transcripts and protein isoforms are shown in Table 3.

[0086] FIG. 1 is a schematic showing SCN1A topology and premature termination codons (PTCs).

[0087] Table 3. SCN1A transcripts and isoforms TIFF2024534496000005.tif178156

[0088] SCN1A mutations associated with Dravet syndrome are shown in Table 4.

[0089] Table 4. SCN1A mutations associated with Dravet syndrome TIFF2024534496000006.tif43156TIFF2024534496000007.tif236156TIFF2024 534496000008.tif237156TIFF2024534496000009.tif236156TIFF20245344960 00010.tif237156TIFF2024534496000011.tif237156TIFF2024534496000012.t if237156TIFF2024534496000013.tif237156TIFF2024534496000014.tif180156

[0090] MECP2 MECP2 (NCBI gene ID: 4204; reference sequence NG_007107.3) encodes methylated CpG binding protein 2. Its transcripts and protein isoforms are shown in Table 5.

[0091] Table 5. MECP2 transcripts and isoforms TIFF2024534496000015.tif122156

[0092] MECP2 mutations associated with Rett disease are shown in Table 6.

[0093] Table 6. MECP2 mutations associated with Rett disease TIFF2024534496000016.tif99156TIFF2024534496000017.tif138156

[0094] Fusion proteins Described herein are non-natural or engineered fusion proteins comprising RNA effector proteins and regulatory moieties, as well as variants and mutants thereof.

[0095] In some cases, the RNA effector protein amino acid sequence is N-terminal to the regulatory moiety amino acid sequence. In some cases, the RNA effector protein amino acid sequence is C-terminal to the regulatory protein amino acid sequence. In some cases, the RNA effector protein amino acid sequence is inserted into the regulatory protein amino acid sequence. In some cases, the regulatory protein amino acid sequence is inserted into the RNA effector protein amino acid sequence.

[0096] In some cases, the fusion protein includes a linker and / or spacer between the RNA effector protein and the regulatory moiety.

[0097] In some cases, the fusion protein further comprises a nuclear export signal. In some cases, the nuclear export signal is derived from the HIV Rev protein (LPPLERLTL, SEQ ID NO: 51). In some cases, the nuclear export signal is between the RNA effector protein amino acid sequence and the regulatory moiety amino acid sequence.

[0098] In some cases, the RNA effector protein is dCas13b and the regulon portion is PABPC1. Thus, in some cases, the fusion protein comprises or consists of SEQ ID NO: 48. In some cases, the fusion protein comprises or consists of SEQ ID NO: 49.

[0099] In some cases, the RNA effector protein is dCas13b and the regulon portion is NAT10.

[0100] In some cases, the RNA effector protein and / or regulatory portion is the functionally active portion of the effector protein and / or regulatory portion.Thus, for example, when the RNA effector protein is C-terminal to the regulatory portion, one or more N-terminal amino acids (e.g., N-terminal methionine) of the regulatory portion can be replaced or deleted.Similarly, when the regulatory portion is C-terminal to the RNA effector protein, one or more N-terminal amino acids (e.g., N-terminal methionine) of the RNA effector portion can be replaced or deleted.

[0101] Also described herein are polynucleotide sequences encoding the fusion proteins described herein, vectors containing the polynucleotide sequences, and cells containing and / or expressing the vectors.

[0102] In some cases, the nucleic acid sequence is codon optimized.

[0103] In some cases, the vector comprises a promoter sequence that drives expression of the fusion protein and / or the gRNA.

[0104] RNA effector proteins The fusion proteins described herein include an RNA effector protein. In some cases, the RNA effector protein is a Cas effector protein or a variant or mutant thereof.

[0105] In some cases, the RNA effector protein is a Cas9 effector protein (e.g., SEQ ID NO: 46) or a mutant or variant thereof. In some cases, the RNA effector protein is a catalytically inactive Cas9 effector protein, e.g., a Cas9 effector protein with removed cleavage activity (e.g., dCas9; e.g., SEQ ID NO: 46 with mutations D10A and H840A).

[0106] In some cases, the RNA effector protein is a Cas12 effector protein, such as Cas12a (Cpf1), for example, LbCas12a, or a mutant or variant thereof, for example, a mutant or variant with cleavage activity removed. In some cases, the RNA effector protein is a Cas12b effector protein, such as AapCas12b or AacCas12b, for example, a mutant or variant with cleavage activity removed.

[0107] In some cases, the RNA effector protein is a Cas14 effector protein or a mutant or variant thereof.See, e.g., Harrington et al., "Programmed DNA Destruction by Miniature CRISPR-Cas13 Enzymes," Science 362(6146):839-42 (2018).See also Karvelis et al., "PAM Recognition by Miniature CRISPR-Cas12f Nucleases Triggers Programmable Double-stranded DNA Target Cleavage," Nucleic acids Res 48(9):5016-23 (2020).

[0108] Type VI CRISPR-Cas systems include a programmable single-effector RNA-guided RNase Cas13. See, e.g., Cox et al., "RNA Editing with CRISPR-Cas13," Science 358(6366):1019-27 (2017). The Cas13 family includes at least four known subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c, and Cas13d.

[0109] In some cases, the RNA effector protein is a Cas13 effector protein, such as Cas13a, Cas13b, Cas13c, or Cas13d. In some cases, the RNA effector protein is a Cas13b effector protein. See, e.g., Smargon et al. (2017), "Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28," Molecular Cell 65, 618-630 (Feb. 16, 2017). See also, Smargon et al., "RNA-Targeting CRISPR Systems from Metagenomic Discovery to Transcriptome Engineering," Nat Cell Biol 22(2):143-50 (2020).

[0110] In some cases, the RNA effector protein is a catalytically inactive RNA effector protein, such as a Cas13 effector protein with deleted cleavage activity (dCas13), such as dCas13b (SEQ ID NO: 47).

[0111] In some cases, the RNA effector protein has at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO:47.

[0112] In some cases, the Cas13 effector protein is Cas13bt. See, e.g., Kannan et al., "Compact RNA Editors with Small Cas13 Proteins," Nature Biotechnology 18:499-560 (2021). In some cases, the Cas13 effector protein is a catalytically inactive Cas13bt effector protein (dCas13bt). In some cases, the Cas13 effector protein is Cas13bt with mutations corresponding to H133A and H1058 of dCas13bt.

[0113] In some cases, the Cas13 effector protein is a Cas13 effector protein described in WO2018170333A1 (e.g., Table 1A of WO2018170333A1). In some cases, the Cas13 is a catalytically inactive mutant of Cas13 described in WO2018170333A1 (e.g., a mutant of Cas13 shown in Table 1A of WO2018170333A1).

[0114] Regulon part The fusion protein described herein comprises a regulatory portion. In some cases, the regulatory portion stimulates and / or stabilizes mRNA, for example, the mRNA of the active allele of the gene associated with haplotype insufficiency disorder. In some cases, the regulatory portion stimulates the activity of mRNA. In some cases, the regulatory portion stabilizes mRNA.

[0115] In some cases, the regulatory moiety can be a translation stimulator protein. In some cases, the regulatory moiety can be a wild-type protein. In some cases, the regulatory moiety can be a truncated variant of the wild-type protein.

[0116] PABPC1 In some cases, the regulatory moiety is PABPC1. Polyadenylate-binding protein 1 (PABPC1) is a highly conserved RNA-binding protein in eukaryotes. The protein has four N-terminal RNA recognition motif (RRM) domains that bind poly(A) RNA with nanomolar affinity [23, 24]. The RRMs are followed by a proline-rich linker and a C-terminal MLLE domain. The MLLE domain recognizes a peptide motif called poly(A)-interacting motif 2 (PAM2), which is present in several PABPC partner proteins that regulate mRNA metabolism (stability and translation). The presence of PABPC1 on mRNA is known to stimulate their activity to enhance translation and mRNA stability

[19] .

[0117] Thus, in some cases, the regulatory portion comprises or consists of SEQ ID NO:42 or SEQ ID NO:43.

[0118] In some cases, the regulatory portion comprises or consists of a polypeptide sequence having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:42 or SEQ ID NO:43.

[0119] In some cases, the regulatory portion comprises a wild-type PABPC1 protein. In some cases, the regulatory portion comprises a truncated variant of the wild-type PABPC1 protein. In some embodiments, the regulatory portion comprises an RRM domain and an MLLE domain. In some cases, the regulatory portion comprises an MLLE domain.

[0120] NAT10 In some cases, the regulatory moiety is NAT10. RNA cytidine acetyltransferase NAT10 is a highly conserved enzyme that catalyzes the conversion of cytidine to N4-acetylcytidine (ac4C)

[25] . Posttranscriptional ribonucleoside modifications resulting in the epitranscriptome add an additional layer of regulatory complexity to RNA structure and function. It has been established that N4-acetylcytidine mRNA modification has a strong stimulatory effect on mRNA stabilization and translation in human cells

[25] .

[0121] Thus, in some cases, the regulatory portion comprises or consists of SEQ ID NO: 44 or SEQ ID NO: 45. In some cases, the regulatory portion comprises or consists of a polypeptide sequence having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45.

[0122] Pharmaceutical compositions and methods of treatment Pharmaceutical Compositions Also described herein are pharmaceutical compositions comprising the fusion proteins described herein, pharmaceutical compositions comprising a nucleic acid encoding the fusion proteins described herein, and pharmaceutical compositions comprising a vector comprising a nucleic acid encoding the fusion proteins described herein.

[0123] In some cases, the pharmaceutical composition further comprises a gRNA, e.g., as described herein. In some cases, the pharmaceutical composition further comprises a nucleic acid encoding a gRNA, e.g., as described herein. In some cases, the pharmaceutical composition further comprises a vector comprising a nucleic acid encoding a gRNA, e.g., as described herein.

[0124] In some cases, the pharmaceutical composition is formulated for gene therapy, for example, as described herein.

[0125] Fusion proteins In some cases, the pharmaceutical compositions described herein include a fusion protein, such as a fusion protein described herein. In some cases, the pharmaceutical compositions described herein include a nucleic acid encoding a fusion protein, such as a fusion protein described herein. In some cases, the pharmaceutical compositions include a nucleic acid encoding a fusion protein, such as a fusion protein described herein.

[0126] gRNA In some cases, e.g., when the fusion protein comprises a Cas effector protein, e.g., as described herein, the pharmaceutical composition further comprises a guide RNA (gRNA) that forms a complex with the Cas protein and comprises a complementary region designed to hybridize to a nucleic acid, e.g., an mRNA of an active allele of a gene associated with a haploinsufficiency disorder, e.g., the 3'UTR of an mRNA of an active allele of a gene associated with a haploinsufficiency disorder.

[0127] In some cases, gRNA is designed to hybridize to the 3'UTR of mRNA. In some cases, gRNA can hybridize to the 3' end of the 3'UTR transcript. In some cases, gRNA can hybridize to a position within the 3'UTR transcript.

[0128] In some cases, the gRNA comprises a CRISPR RNA (crRNA). In some cases, the gRNA comprises a trans-activating CRISPR RNA (tracrRNA). In some cases, the gRNA is a single guide RNA (sgRNA). In some cases, the gRNA does not comprise a tracrRNA.

[0129] In some cases, the gRNA is designed to form a complex with Cas13b and includes a complementary region designed to hybridize to the mRNA of an active allele of a gene associated with a haploinsufficiency disorder, e.g., the 3'UTR of the mRNA of an active allele of a gene associated with a haploinsufficiency disorder.

[0130] In some cases, the Cas13b is dCas13b (SEQ ID NO: 47).

[0131] In some cases, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and combinations thereof. In some cases, the gRNA comprises a complementary region designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, and combinations thereof.

[0132] In some cases, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding an amino acid selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and combinations thereof. In some cases, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding SEQ ID NO: 21.

[0133] In some cases, the gRNA comprises a region of complementarity designed to hybridize to an mRNA selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, and combinations thereof. In some cases, the gRNA comprises a region of complementarity designed to hybridize to SEQ ID NO: 26.

[0134] In some cases, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and combinations thereof. In some cases, the gRNA comprises a complementary region designed to hybridize to an mRNA encoding SEQ ID NO: 37.

[0135] In some cases, the gRNA targets the mRNA encoding Mecp2. In some cases, the gRNA targets the mRNA encoding SynGAP. In some cases, the gRNA targets the mRNA encoding SHANK3. In some cases, the gRNA targets the mRNA encoding PTEN. In some cases, the gRNA targets the mRNA encoding CHD2. In some cases, the gRNA comprises or consists of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.

[0136] A list of gRNAs targeting the 3'UTR of genes of interest is provided in Table 7.

[0137] (Table 7) List of guide RNAs targeting the 3'UTR of genes of interest. TIFF2024534496000018.tif123156

[0138] Also provided herein are polynucleotide sequences encoding the gRNAs described herein, vectors comprising polynucleotide sequences encoding the gRNAs described herein, and cells comprising vectors encoding the gRNAs described herein.

[0139] In some cases, the polynucleotide encoding the fusion protein and the polynucleotide encoding the gRNA are on the same vector. In some cases, the polynucleotide encoding the fusion protein and the polynucleotide encoding the gRNA are on different vectors.

[0140] Gene Therapy Nucleic acids described herein, for example, nucleic acids encoding fusion proteins and / or gRNAs described herein, can be incorporated into gene constructs used as part of gene therapy protocols.Thus, targeted expression vectors are also provided herein for in vivo transfection and expression of polynucleotides encoding fusion proteins and / or gRNAs described herein.Expression constructs containing such components can be administered in any effective carrier, for example, in any formulation or composition that can effectively deliver component genes to cells in vivo.Approaches include viral vectors, including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1, or insert genes into recombinant bacterial or eukaryotic plasmids.Viral vectors directly transfect cells. Plasmid DNA can be delivered naked or with the aid of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody-bound) polylysine conjugates, gramacidin S, artificial viral envelopes, or other such intracellular vehicles, as well as by direct injection of the gene construct or by CaPO4 precipitation performed in vivo.

[0141] The preferred approach for in vivo introduction of nucleic acid into cells is by using viral vectors that contain nucleic acid, for example cDNA.The infection of cells with viral vectors has the advantage that the majority of targeted cells can receive nucleic acid.In addition, the molecules that are coded in the viral vector, for example by the cDNA contained in the viral vector, are efficiently expressed in cells that incorporate viral vector nucleic acid.

[0142] Retroviral and adeno-associated viral vectors can be used as recombinant gene delivery systems to transfer exogenous genes in vivo, particularly in humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acid is stably integrated into the host's chromosomal DNA. The development of specialized cell lines (called "packaging cells") that produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy. Defective retroviruses have been characterized for use in gene transfer for gene therapy purposes (for a review, see Miller, Blood 76:271 (1990)). Replication-defective retroviruses can be packaged into virions, which can be used to infect target cells with helper viruses by standard techniques. Protocols for generating recombinant retroviruses and infecting cells with such viruses in vitro or in vivo are described in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those skilled in the art. Examples of packaging virus lines suitable for preparing both ecotropic and amphotropic retroviral systems include ΨCrip, ΨCre, Ψ2, and ΨAm.Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (e.g., Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 89:1011-1012; 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468;PCT application WO 89 / 05345; and PCT application WO 92 / 07573).

[0143] Another viral gene delivery system useful in the method of the present invention utilizes adenovirus-derived vectors. The genome of adenovirus can be engineered to code and express a gene product of interest, but inactivated in terms of its replicative capacity in normal lytic viral life cycle. For example, see Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenovirus vectors derived from adenovirus strain Ad type 5 dl324 or other adenovirus strains (such as Ad2, Ad3, or Ad7) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain situations in that they cannot infect non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, viral particles are relatively stable, can undergo purification and concentration, and can be modified to affect the range of infectivity, as mentioned above.Furthermore, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the genome of the host cell, but remains episomal, thereby avoiding the potential problem that may arise as a result of in situ insertional mutagenesis, i.e., the introduced DNA is integrated into the host genome (e.g., retroviral DNA).In addition, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) compared to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

[0144] Yet another viral vector system useful for delivery of nucleic acid is adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as adenovirus or herpesvirus, as a helper virus for efficient replication and productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol.158:97-129 (1992)). Adeno-associated virus is also one of the few viruses that can integrate its own DNA into non-dividing cells, and shows a high frequency of stable integration (see, for example, Flotte et al., Am.J. Respir. Cell. Mol. Biol.7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989)). Vectors containing as little as 300 base pairs of AAV can be packaged and integrated. Space for exogenous DNA is limited to about 4.5 kb. AAV vectors such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into a variety of cell types using AAV vectors (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993)).

[0145] In addition to viral transfer methods such as those exemplified above, non-viral methods can also be used to induce expression of the nucleic acid compounds described herein, for example, nucleic acid compounds encoding fusion proteins and / or gRNAs described herein. Typically, non-viral methods of gene transfer rely on the normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In some embodiments, non-viral gene delivery systems can rely on endocytosis pathways for the uptake of genes of interest by targeted cells. Exemplary gene delivery systems of this type include liposome-derived systems, polylysine conjugates, lipid nanoparticles, and artificial viral envelopes. Other embodiments include plasmid injection systems such as those described in Meuli et al., J. Invest. Dermatol. 116(1):131-135 (2001); Cohen et al., Gene Ther. 7(22):1896-905 (2000); or Tam et al., Gene Ther. 7(21):1867-74 (2000).

[0146] In some embodiments, the nucleic acid compounds described herein, e.g., nucleic acid compounds encoding fusion proteins and / or gRNAs, are entrapped in liposomes that carry a positive charge on the surface (e.g., lipofectin) and can be tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., No Shinkei Geka 20:547-551 (1992); PCT Publication WO91 / 06309; Japanese Patent Application No. 1047381; and European Patent Publication EP-A-43075).

[0147] In a clinical setting, a gene delivery system of a therapeutic gene can be introduced into a subject by any of several methods, each of which is well known in the art. For example, a pharmaceutical formulation of the gene delivery system can be introduced systemically, for example, by intravenous injection, and specific transduction of the protein in target cells is believed to occur primarily from the specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences that control the expression of the reporter gene, or a combination thereof. In other embodiments, the initial delivery of the recombinant gene is more restricted and the introduction into the subject is fairly localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)).

[0148] The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0149] Treatment The methods described herein include methods for treating disorders associated with haploinsufficiency, e.g., as described herein. Typically, these methods include administering a therapeutically effective amount of a pharmaceutical composition described herein to a subject in need of, or determined to be in need of, such treatment, e.g., other than a gene therapy method described herein.

[0150] In some cases, the treatment methods provided herein can be used to treat a subject (e.g., a human, monkey, dog, cat, mouse) diagnosed with or suspected of having a haploinsufficiency disorder, such as those described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0151] In some cases, the subject may be a human who exhibits one or more symptoms associated with, for example, a haploinsufficiency disorder described herein. Any of the treatment methods provided herein may be used to treat haploinsufficiency disorders at various stages.

[0152] In some cases, the disorder is Dravet syndrome. In some cases, the subject has a mutation selected from the list in Table 4.

[0153] In some cases, the disorder is Rett Syndrome. In some cases, the subject has a mutation selected from the list in Table 6.

[0154] As used in this context, "treat" refers to improving at least one symptom of a disorder associated with a haploinsufficiency disorder. Often, as a result of a haploinsufficiency disorder, the amount of gene product expressed from the mRNA of an active allele is insufficient for proper gene function; thus, treatment can increase the amount of gene product expressed from the mRNA of an active allele, for example, compared to an age-matched untreated subject.

[0155] Dosage An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount necessary to prevent the occurrence of a disease or disease symptoms. An effective amount can be administered in one or more administrations, doses, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will vary depending on the therapeutic compound selected. The composition may be administered one or more times per day to one or more times per week (including once every other day). Those skilled in the art will appreciate that several factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments.

[0156] The dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissues in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0157] Data obtained from cell culture assays and animal studies can be used when determining a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose can be estimated initially from cell culture assays. Dosages can be determined in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0158] variant In some cases, the fusion proteins described herein or the nucleic acid sequences encoding them have at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identity to the amino acid sequences of the sequences provided herein, with differences, e.g., up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the sequences provided herein, including or in addition to the mutations described herein, e.g., substituted with conservative mutations. In preferred embodiments, the variants retain the desired activity of the parent, e.g., the binding activity of an RNA effector protein and the regulatory activity of a regulon portion.

[0159] To determine the percent identity of two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). The length of the reference sequence aligned for comparison is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. The nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, these molecules are identical at that position (as used herein, the "identity" of nucleic acids is equal to the "homology" of nucleic acids). The percent identity between two sequences is a function of the number of identical positions that are common to these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0160] Percent identity between a subject polypeptide or nucleic acid sequence (i.e., query) and a second polypeptide or nucleic acid sequence (i.e., target) can be determined in a variety of ways that are within the skill of the art, for example, using publicly available computer software, such as, for example, Smith-Waterman alignment (Smith, TF and MS Waterman (1981) J Mol Biol 147:195-7); GeneMatcher Plus™ (Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed, pp 353-358) as incorporated herein by reference, "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)); the BLAST program (Basic Local Alignment Search Tool; (Altschul, SF, W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Furthermore, those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Typically, for a target protein or target nucleic acid, the length of comparison can be any length up to the full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of this disclosure, the percent identity is relative to the full length of the query sequence.

[0161] For purposes of this disclosure, comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0162] Typically, conservative substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. EXAMPLES

[0163] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0164] Example 1: PAB Tethered Targeting Figure 3A is a schematic diagram showing the experimental approach applied in this assay. Briefly, PABPC1 was fused to the dCas13b tethering protein and guided to the 3'UTR of the luciferase gene via a designed guide RNA (gRNA) to promote its translation.

[0165] HEK-293 cells were transfected with pJC1211 plasmid expressing dCas13b (pJC1211; SEQ ID NO: 50; Figure 4; Figures 5A-5AB); a plasmid expressing a luc reporter (pJC889; Addgene plasmid no. 18964); and pJC1212 (expressing gRNA1 (Figure 6; Figures 7A-7G; SEQ ID NO: 52)), pJC1313 (expressing gRNA1 (Figure 8; Figures 9A-9F; SEQ ID NO: 53)), or the empty vector Addgene plasmid 103854 (pC0043-PspCas13b crRNA backbone Addgene plasmid no. 103854; Figure 10; Figures 11A-11H; SEQ ID NO: 54) were cotransfected.

[0166] For luciferase detection, cells were lysed 48 hours after transfection and the Promega luciferase assay system was applied according to the manufacturer's protocol (Promega E1500). The results show that the use of two guide RNAs targeting the 3'UTR of the luciferase construct results in approximately 2-3 fold stimulation compared to the control (Figure 3B).

[0167] Example 2: Post-transcriptional regulation of mRNA in vitro and in vivo by PAB1-dCas13b and NAT10-dCas13b fusion proteins Various mRNA regulon therapies are believed to be used as disease-modifying therapies in the treatment of monogenic neurological disorders with haploinsufficiency, such as Dravet syndrome and Rett syndrome. The proteins PABPC1 and NAT10 are tethered to an RNA effector protein, such as dCas13b, and expressed in vitro and in vivo with gRNAs targeting the mRNA of active alleles of genes associated with Dravet syndrome (e.g., SCN1A) or gRNAs targeting the mRNA of active alleles of genes associated with Rett syndrome (e.g., MECP2), respectively.

[0168] Example 3: Enhancement of mRNA expression in vitro Cas13b in vitro optimization The Cas13b / PABPC1 construct was generated by cloning PCR-amplified human PABPC1 into pC0054-CMV-dPspCas13b-long linker-ADAR2DD (Addgene 103870) (pJC1206) (Table 8). The PC1-MS2V5-PABPC1 plasmid (Addgene #65807) was used as a template for PABPC1 amplification (primers are listed in Table 9). Briefly, pJC1206 was site-mutated at nucleotide 5606 to create a unique BamHI site; this construct is referred to below as pJC1210. The ADAR2DD sequence was then removed using BamHI+NotI and replaced with PCR-amplified PABPC1. The resulting construct was pJC1211, which contains the complete human PABPC1 sequence (Table 8). pJC1246 was generated by digesting pJC1211 with BamH1 / Not1 and inserting the PCR product amplified from oJC5001 / oJC5240. pJC1280 was generated by cutting pJC1211 with BamH1 / Not1, blunting the ends, and religating.

[0169] Multiple sgRNAs targeting the 3'UTR of genes of interest were designed using the "nygenome" online tool (Cas13 design (nygenome.org)) for predicting dCas13b guides (Table 7). These guides were individually cloned into the BbsI site of PC0043-Cas3b-crRNA backbone (Addgene #103854) (pJC1208). The reaction mixture containing forward and reverse oligos mixed in 1x NEB buffer 3.1 was incubated at 95°C and 100°C for 5 and 10 min, and then allowed to cool at room temperature for 2 h. The prepared oligos were ligated into pJC1208 using T4 DNA ligase (NEB) according to the GreenGate protocol.

[0170] The RNA Amplifier technology was tested in HEK293, HepG2, and SH-SY5Y (ATCC CRL 2266) cell lines. The HEK293 and HepG2 cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS, and the SH-SY5Y cell line was grown in Eagle's Minimum Essential Medium (EMEM) with 10% FBS, according to ATCC guidelines.

[0171] Cells were transiently co-transfected with individual sgRNAs together with dCas13b-PABPC1 fusion plasmids using Opti-MEM reduced serum medium (Gibco) and GeneXPlus transfection reagent (ATCC ACS-4004) for 48 hours. RNA was isolated using the direct-zol RNA kit (Zymo Research) according to the manufacturer's protocol. cDNA was synthesized using the SuperScript III First Strand Synthesis System (Invitrogen), and qPCR was performed on an Applied Biosystems real-time PCR instrument using PowerUp™ SYBR Green master mix (Applied Biosystems) and designed primers (Table 9). Data was analyzed using CT values ​​compared to no sgRNA transfection and normalized to ACTB as a housekeeping gene.

[0172] Proteins were isolated using RIPA buffer and Western blotting was performed using Mini-protean TGX4-15% gels (BIO-RAD). The following antibodies were used for immunoblotting according to the manufacturer's recommended concentrations: anti-GAPDH (6C5) (Santa Cruz Biotechnology), anti-MeCP2 (D4F3) (Cell Signaling Technology), anti-CHD 2 (Cat. No. 4170) (Cell Signaling Technology), anti-PTEN (Cat. No. 9552) (Cell Signaling Technology), anti-SynGAP (Cat. No. 3200) (Cell Signaling Technology), and an antibody against Shank protein in general, clone N23B / 49 (Cat. No. MABN24) (Millipore).

[0173] Luciferase Assay HEK293 cells were transfected with different ratios of pJC889 (luciferase-pcDNA3 Addgene #18964), pJc1211, and two different guide RNAs targeting the 3'UTR of the firefly luciferase transcript, individually. The optimal ratio of plasmids (0.2:1:1.5) was selected for this experiment. For luminescence detection, cells were lysed in 100 μL of 1× passive lysis buffer (Promega). The lysates were mixed with ONE-Glo EX reagent (Promega) according to the manufacture's protocol, and luminescence was measured using a Lumat LB9507 luminometer (Berthold Technologies).

[0174] AlphaFold method Structural predictions of the fusion proteins and native Cas13b and PABP were generated using AlphaFold v2.0, taken from the github repository located at URL github.com / deepmind / alphafold, commit "1d43aaff941c84dc56311076b58795797e49107b" (Reference 15). Both native and customized fusion FASTAs were processed according to the AlphaFold instructions using the provided Docker script with the following parameters: "--max_template_date=2020-05-14--preset=reduced_dbs". The relaxed predicted structure with the highest pLDDT score was used for interpretation of the corresponding input FASTA.

[0175] Quantitative and statistical analysis All data are presented in figures as bar graphs and quantified as mean ± SEM. Results were considered significant at p<0.05 as indicated throughout the figure legends. All experiments were performed on at least three biological replicates according to common practice unless otherwise stated.

[0176] Table 8. List of backbone plasmids and constructs made. TIFF2024534496000019.tif68165

[0177] Table 9: List of primers used TIFF2024534496000020.tif133156

[0178] We increased mRNA expression by tethering PABPC1, a known translation stimulator, to the 3'UTR of target mRNAs. Tethering was achieved by fusing PABPC1 to the RNA-binding protein dCas13b and co-expressing a guide RNA (gRNA). The gRNA is crucial in that it has antisense homology to a specific mRNA and a short hairpin required for dCas13b binding (Figure 12A). This gRNA-targeted tethering was demonstrated to enhance both reporter and endogenous mRNAs in a gRNA-dependent manner. First, we used HEK293 cells to co-transfect the tethered mRNA amplifier together with a luciferase reporter construct. In the presence of a gRNA targeting the 3'UTR of the luciferase reporter, we observed an approximately 1.5-2-fold increase in the amount of reporter protein. No stimulation occurred when the dCas13b-PABPC1 fusion was expressed alone (Figure 12B, control). We continued to stimulate translation of the endogenous mRNA, MeCP2, in HEK293 cells. Using two different gRNAs targeting the 3'UTR of the endogenous MeCP2 transcript, we observed approximately 1.5-fold stimulation of translation (Figure 12C). In the presence of the mRNA amplifier, we observed a modest 15% increase in the steady-state level of MeCP2 transcript (Figure 12D). These data suggest that the stimulatory role of the tethered mRNA amplifier is due to both mRNA stability and mRNA translation, a known role of PABPC1 in regulating mRNA metabolism.

[0179] Example 4: Promoting mRNA expression in multiple cell types It was further shown that tethered mRNA amplifiers promote mRNA expression in multiple cell types; a stimulatory effect on MeCP2 protein expression was observed in SH-SY5Y (neuronal cell line) and HepG2 (hepatic cell line, FIG. 12E). Finally, it was shown that the effect of tethered mRNA amplifiers can be tuned by moving the gRNA to different positions within the 3'UTR. In the case of MeCP2, the strongest stimulatory effect was observed when the gRNA was moved closer to the 3' end of the transcript (FIG. 12F).

[0180] Example 5: Enhancement of mRNA expression in various mRNA transcripts Next, the tethered mRNA amplifier was tested in other transcripts associated with haploinsufficiency disorders. Using SH-SY5Y cells, a model of neurodegenerative disorders, gRNA-dependent translation stimulation of SYNGAP115 mRNA, SHANK316 mRNA, CHD217 mRNA, and PTEN18 mRNA was observed (Figures 13A-13D). Loss of function of one allele in each of these genes is associated with autism spectrum disorder. In each case, the stimulatory effect observed was 1.2-2.0-fold protein expression, accompanied by an approximately 15-20% increase in mRNA levels. These data demonstrate that the tethered mRNA amplifier can be used as a gene therapy candidate for haploinsufficiency and can be used across multiple transcripts of clinical relevance.

[0181] Example 6: Size minimization of tethered mRNA amplifier fusion proteins Finally, gene therapy vectors such as AAV are known to have a payload size limit of approximately 4.5 kb. To minimize the size of the tethered mRNA amplifier (5.2 kb), we created specific truncations of PABPC1 and tested their effectiveness on MeCP2 expression. PABPC1 contains four RNA recognition motifs (RRM1-4) at its N-terminus, followed by a linker, and a mademoiselle (MLLE) domain at the C-terminus (Figure 14A). The RRM domain binds to the poly(A) tail, while the MLLE domain is known to regulate its stimulatory role in translation. Since PABPC1 was artificially and specifically tethered to mRNA independent of PABPC1 poly(A) binding ability, it was speculated that the RRM would be unnecessary for the function of the tethered mRNA amplifier. Therefore, only the MLLE domain was fused to dCas13b (3.2 KB). As a first test, the predicted folding pattern of this new fusion was analyzed by an in silico approach using AlphaFold v2.0. As shown in Figures 14B-14E, removal of the RRM domain had minimal effect on the folding of either the MLLE domain or dCas13b itself. In HEK293 cells, this minimal construct was also observed to stimulate MeCP2 mRNA expression in a gRNA-dependent manner to a similar extent as the full-length construct (Figure 14F). Thus, the tethered mRNA amplifier approach was shown to be adaptable for use in current clinically effective gene therapy vectors.

[0182] References TIFF2024534496000021.tif223156TIFF2024534496000022.tif176156

[0183] array SEQ ID NO: 1 >NR_148667.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=16] TIFF2024534496000023.tif213156TIFF2024534496000024.tif235156TIFF2024534496000025.tif179156SEQ ID NO: 2 >XR_001738884.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=X2] TIFF2024534496000026.tif36156TIFF2024534496000027.tif235156TIFF2024534496000028.tif138156SEQ ID NO: 3 >XR_001738883.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=X1] TIFF2024534496000029.tif78156TIFF2024534496000030.tif235156TIFF2024534496000031.tif96156SEQ ID NO: 4 >NM_001353949.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=6] TIFF2024534496000032.tif119156TIFF2024534496000033.tif235156TIFF2024534496000034.tif235156TIFF2024534496000035.tif21156SEQ ID NO: 5 >NM_001353958.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=13] TIFF2024534496000036.tif194156TIFF2024534496000037.tif235156TIFF2024534496000038.tif186156SEQ ID NO: 6>NM_001353950.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=7] TIFF2024534496000039.tif29156TIFF2024534496000040.tif235156TIFF2024534496000041.tif235156TIFF2024534496000042.tif115156SEQ ID NO: 7 >NM_001202435.3 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=4] TIFF2024534496000043.tif100156TIFF2024534496000044.tif235156TIFF2024534496000045.tif235156TIFF2024534496000046.tif47156SEQ ID NO: 8 >NM_001353955.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=11] TIFF2024534496000047.tif175156TIFF2024534496000048.tif235156TIFF2024534496000049.tif209156SEQ ID NO: 9 >NM_001353957.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=12] TIFF2024534496000050.tif6156TIFF2024534496000051.tif235156TIFF2024534496000052.tif235156TIFF2024534496000053.tif138156SEQ ID NO: 10 >NM_001353951.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=8] TIFF2024534496000054.tif78156TIFF2024534496000055.tif235156TIFF2024534496000056.tif235156TIFF2024534496000057.tif70156SEQ ID NO: 11 >NM_001353948.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=5] TIFF2024534496000058.tif145156TIFF2024534496000059.tif235156TIFF2024534496000060.tif232156TIFF2024534496000061.tif6156SEQ ID NO: 12 >NM_001353960.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=14] TIFF2024534496000062.tif209156TIFF2024534496000063.tif235156TIFF2024534496000064.tif175156SEQ ID NO: 13 >NM_001353954.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=10] TIFF2024534496000065.tif40156TIFF2024534496000066.tif235156TIFF2024534496000067.tif235156TIFF2024534496000068.tif111156SEQ ID NO: 14 >NM_001165964.3 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=3] TIFF2024534496000069.tif104156TIFF2024534496000070.tif235156TIFF2024534496000071.tif235156TIFF2024534496000072.tif44156SEQ ID NO: 15 >NM_001353952.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=9] TIFF2024534496000073.tif172156TIFF2024534496000074.tif235156TIFF2024534496000075.tif217156SEQ ID NO: 16 >NM_001353961.2 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=15] TIFF2024534496000076.tif224156TIFF2024534496000077.tif235156TIFF2024534496000078.tif164156SEQ ID NO: 17>NM_006920.6 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=2] TIFF2024534496000079.tif51156TIFF2024534496000080.tif235156TIFF2024534496000081.tif235156TIFF2024534496000082.tif104156SEQ ID NO: 18 >NM_001165963.4 SCN1A [organism=Homo sapiens] [GeneID=6323] [transcript=1] TIFF2024534496000083.tif111156TIFF2024534496000084.tif235156TIFF2024534496000085.tif228156SEQ ID NO: 19 >NP_001159435.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=1] TIFF2024534496000086.tif97156SEQ ID NO: 20 >NP_001159436.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=3] TIFF2024534496000087.tif97156SEQ ID NO: 21 >NP_001340878.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=2] TIFF2024534496000088.tif97156SEQ ID NO: 22 >NP_001340883.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=4] TIFF2024534496000089.tif97156SEQ ID NO: 23 >NP_001340889.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=5] TIFF2024534496000090.tif97156SEQ ID NO: 24 >NP_001340890.1 SCN1A [organism=Homo sapiens] [GeneID=6323] [isoform=6] TIFF2024534496000091.tif59156SEQ ID NO: 25 >NM_001386139.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=10] TIFF2024534496000092.tif33156TIFF2024534496000093.tif235156TIFF2024534496000094.tif224156SEQ ID NO: 26 >NM_001110792.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=2] TIFF2024534496000095.tif224156TIFF2024534496000096.tif235156TIFF2024534496000097.tif36156SEQ ID NO: 27 >NM_001369393.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=6] TIFF2024534496000098.tif179156TIFF2024534496000099.tif235156TIFF2024534496000100.tif89156SEQ ID NO: 28 >NM_001386138.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=9] TIFF2024534496000101.tif126156TIFF2024534496000102.tif235156TIFF2024534496000103.tif134156SEQ ID NO: 29 >NM_004992.4 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=1] TIFF2024534496000104.tif81156TIFF2024534496000105.tif235156TIFF2024534496000106.tif186156SEQ ID NO: 30 >NM_001386137.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=8] TIFF2024534496000107.tif29156TIFF2024534496000108.tif235156TIFF2024534496000109.tif232156TIFF2024534496000110.tif6156SEQ ID NO: 31 >NM_001369392.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=5] TIFF2024534496000111.tif202156TIFF2024534496000112.tif235156TIFF2024534496000113.tif70156SEQ ID NO: 32>NM_001369391.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=4] TIFF2024534496000114.tif145156TIFF2024534496000115.tif235156TIFF2024534496000116.tif145156SEQ ID NO: 33>NM_001316337.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=3] TIFF2024534496000117.tif70156TIFF2024534496000118.tif235156TIFF2024534496000119.tif205156SEQ ID NO: 34 >NM_001369394.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=7] TIFF2024534496000120.tif10156TIFF2024534496000121.tif235156TIFF2024534496000122.tif235156TIFF2024534496000123.tif17156SEQ ID NO: 35 >XM_024452383.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=X1] TIFF2024534496000124.tif198156TIFF2024534496000125.tif235156TIFF2024534496000126.tif78156SEQ ID NO: 36 >XM_011531166.2 MECP2 [organism=Homo sapiens] [GeneID=4204] [transcript=X3] TIFF2024534496000127.tif138156TIFF2024534496000128.tif235156TIFF2024534496000129.tif115156SEQ ID NO: 37 >NP_001104262.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [isoform=2] TIFF2024534496000130.tif25156SEQ ID NO: 38 >NP_001303266.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [isoform=3] TIFF2024534496000131.tif18156SEQ ID NO: 39 >NP_001373066.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [isoform=4] TIFF2024534496000132.tif14156SEQ ID NO: 40 >NP_004983.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [isoform=1] TIFF2024534496000133.tif25156SEQ ID NO: 41 >XP_011529468.1 MECP2 [organism=Homo sapiens] [GeneID=4204] [isoform=X1] TIFF2024534496000134.tif21156SEQ ID NO: 42 >sp|P11940|PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens OX=9606 GN=PABPC1 PE=1 SV=2 TIFF2024534496000135.tif33156SEQ ID NO: 43 >sp|P11940-2|PABP1_HUMAN Polyadenylate-binding protein 1 isoform 2 OS=Homo sapiens OX=9606 GN=PABPC1 TIFF2024534496000136.tif29156SEQ ID NO: 44 >sp|Q9H0A0|NAT10_HUMAN RNA cytidine acetyltransferase OS=Homo sapiens OX=9606 GN=NAT10 PE=1 SV=2 TIFF2024534496000137.tif51156SEQ ID NO: 45 >sp|Q9H0A0-2|NAT10_HUMAN RNA cytidine acetyltransferase isoform 2 OS=Homo sapiens OX=9606 GN=NAT10 TIFF2024534496000138.tif48156SEQ ID NO: 46 spCas9 TIFF2024534496000139.tif67156SEQ ID NO: 47 dCas13 TIFF2024534496000140.tif55156SEQ ID NO: 48 PABC1-dCas13 fusion protein TIFF2024534496000141.tif86156SEQ ID NO: 49 PABC1-dCas13 fusion protein TIFF2024534496000142.tif85156SEQ ID NO: 50 >pJC1211 (dCAS13-PABP) (10,647bp) TIFF2024534496000143.tif18156TIFF2024534496000144.tif235156TIFF2024534496000145.tif235156TIFF2024534496000146.tif21156SEQ ID NO: 51 Nuclear export signal TIFF2024534496000147.tif2128SEQ ID NO: 52 >pJC1212(2963bp) TIFF2024534496000148.tif142156SEQ ID NO: 53 >pJC1213(2962bp) TIFF2024534496000149.tif141156SEQ ID NO: 54>addgene-plasmid-103854-sequence-254736(2962bp) TIFF2024534496000150.tif142155

[0184] Other Aspects Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. an RNA effector protein that targets the mRNA of an active allele of a gene associated with a haploinsufficiency disorder; a regulon portion that stimulates and / or stabilizes the mRNA; A fusion protein comprising:

2. (a) the RNA effector protein is a Cas effector protein selected from the group consisting of Cas13, Cas9, Cas12, and Cas14, and / or (b) the regulon portion is PABPC1 or NAT10; The fusion protein of claim 1.

3. the RNA effector protein is Cas13b, or the Cas effector protein is a catalytically inactive Cas protein; The fusion protein of claim 2.

4. The fusion protein of claim 1, further comprising a linker, a spacer, a nuclear export signal, and / or an epitope tag.

5. The fusion protein of claim 1, wherein the RNA effector protein is N-terminal or C-terminal to the regulon portion.

6. 2. The fusion protein of claim 1, comprising or consisting of SEQ ID NO: 48 or SEQ ID NO: 49 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 48 or SEQ ID NO:

49.

7. A polynucleotide encoding the fusion protein of any one of claims 1 to 6.

8. A vector comprising the polynucleotide of claim 7.

9. A cell comprising the vector of claim 8.

10. The fusion protein of any one of claims 1 to 6; and a gRNA that forms a complex with the RNA effector protein and includes a complementary region that hybridizes with the mRNA of the active allele; Including, the system.

11. The system of claim 10, wherein the gRNA targets an mRNA encoding MeCP2, SCN1A, SYNGAP1, SHANK3, CHD2, or PTEN.

12. gRNA, (a) SEQ ID NOs: 1 to 18, and combinations thereof; (b) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, and combinations thereof; (c) SEQ ID NOs: 19 to 24, and combinations thereof; (d) SEQ ID NOs: 25-36, and combinations thereof; or (e) SEQ ID NOs: 37 to 41, and combinations thereof The system of claim 10, comprising a complementary region designed to hybridize to an mRNA selected from the group consisting of:

13. 11. The system of claim 10, wherein the gRNA is selected from the group consisting of SEQ ID NOs: 55-72.

14. One or more polynucleotides encoding the system of any one of claims 10 to 13.

15. 15. One or more vectors comprising one or more polynucleotides of claim 14.

16. 16. A cell comprising one or more vectors of claim 15.

17. The fusion protein of any one of claims 1 to 6, wherein the fusion protein is bound to a gRNA comprising a complementary region that hybridizes with the mRNA of the active allele. A complex comprising:

18. 18. The complex of claim 17, wherein the RNA effector protein is catalytically inactive Cas13b (dCas13b) and the regulon portion is PABP1 or NAT10.

19. gRNA, (a) SEQ ID NOs: 1 to 18, and combinations thereof; (b) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, and combinations thereof; (c) SEQ ID NOs: 19 to 24, and combinations thereof; (d) SEQ ID NOs: 25-36, and combinations thereof; or (e) SEQ ID NOs: 37 to 41, and combinations thereof 19. The complex of claim 17 or 18, comprising a complementary region designed to hybridize to an mRNA selected from the group consisting of:

20. an RNA effector protein that targets the mRNA of an active allele of a gene associated with a haploinsufficiency disorder; a regulon portion that stimulates and / or stabilizes the mRNA; wherein the fusion protein is bound to a gRNA and the mRNA. A complex comprising:

21. The complex of claim 20, wherein the RNA effector protein is catalytically inactive Cas13b (dCas13b) and the regulon portion is PABP1 or NAT10.

22. (a) mRNA, (i) SEQ ID NOs: 1 to 18, and combinations thereof; (ii) SEQ ID NOs: 19-24, and combinations thereof; or (iii) SEQ ID NOs: 25 to 36, and combinations thereof selected from the group consisting of (b) mRNA is (i) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17, and combinations thereof; or (ii) SEQ ID NOs: 37 to 41, and combinations thereof encoding an amino acid selected from the group consisting of:

22. The conjugate of claim 20 or 21.

23. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 6, the polynucleotide of claim 7, the vector of claim 8, the cell of claim 9, or the system of any one of claims 10 to 13, and a pharmaceutically acceptable carrier.

24. A viral vector comprising one or more nucleic acids encoding the fusion protein of any one of claims 1 to 6 or the system of any one of claims 10 to 13.

25. 25. The viral vector of claim 24, which is an adeno-associated viral (AAV) vector.

26. A nanoparticle or liposome comprising the fusion protein of any one of claims 1 to 6, the polynucleotide of claim 7, or the system of any one of claims 10 to 13.

27. Contacting the mRNA with the fusion protein of any one of claims 1 to 6, the polynucleotide of claim 7, the vector of claim 8, or the system of any one of claims 10 to 13. A method for stimulating or stabilizing mRNA in vitro, comprising:

28. 24. A composition for use in a method of treating or preventing a haploinsufficiency disorder in a subject, the composition comprising the pharmaceutical composition of claim 23, the method comprising: the composition, and a gRNA, or a nucleic acid encoding a gRNA, that is designed to form a complex with the RNA effector protein and that includes a complementary region designed to hybridize with the mRNA of the active allele; administering the compound to a subject A composition comprising:

29. The haploinsufficiency disorder is a CNS haploinsufficiency disorder, and the CNS haploinsufficiency disorder is selected from the group consisting of Dravet syndrome, Rett syndrome, recurrent paroxysmal ataxia, familial hemiplegic migraine, CDKL5 deficiency disorder, CHD2 myoclonic encephalopathy, familial focal epilepsy showing variable foci, FOXG1 syndrome, benign familial neonatal seizures, SCN2A-epileptic encephalopathy, SCN2A-developmental encephalopathy, SCN8A-epileptic encephalopathy, SC8A familial infantile epilepsy, and early infantile epilepsy.

29. The composition of claim 28, wherein the epileptic encephalopathy is selected from the group consisting of epileptic encephalopathy, myoclonic atonic epilepsy, early infantile epileptic encephalopathy, SYNGAP1-associated intellectual disability, tuberous sclerosis complex, Lennox-Gastaut syndrome, FoxG1 syndrome, KCNQ2-associated epileptic encephalopathy, PCDH19-associated epilepsy, SLC6A1-associated myoclonic atonic epilepsy, STXBP1-associated epileptic encephalopathy, SYNGAP1 syndrome, and combinations thereof.

30. The target is, (a) AGGFI, ARHGAP31, BMPR2, CHD7, COL2Al, COL3Al, CTLA4, CTNNBI, DLL4, EHMTI, ELN, ENG, FAS, FBNI, FO XGI, GATA3, GLI3, GRN, IRF6, JAGI, KCNQ4, LMXIB, MBD5, MED13L, MITF, MNXI, MYCN, NFIA, NFIX, NOTCH1, NS DI, PAX3, PHIP, PRKARIA, RAil, RBPJ, RPS14, RUNX2, SALL4, SCNIA, SETBPI, SHANK3, SHH, SHOX, SLC2Al / G LUT1, SOXI0, SYNGAPI, TBXI, TBX3, TBX5, TCF4, TCOFI, TGIFI, TNXB, TRPSI, WTI, ZIC2, and combinations thereof, or (b) SCN1A, SCN2A, SCN8A, SCN12A5, SPTAN1, CDKL5, CHD2, FOXG1, KCNQ2, PCDH19, SLC6A1, STXBP1, SYNGAP1, CACNA1A, DEPDC5, MECP2, TSC1, TSC2, and combinations thereof 28. The composition of claim 27, having haploinsufficiency in a gene selected from the group consisting of:

31. 29. The composition of claim 28, wherein the subject is a human.

32. 29. The composition of claim 28, wherein the fusion protein and gRNA are administered as part of a pharmaceutical composition.

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