Element for off-target gene expression in liver

Abstract

The present disclosure provides sequences that reduce expression of operably linked transgenes in hepatocytes. In some aspects, these sequences may be used in gene therapy vectors to off-target expression of a therapeutic transgene in liver cells of a subject.

Description

[0001] Cross-references

[0002] This application claims the benefit of the following U.S. provisional application serial numbers: 63 / 331,680 filed on April 15, 2022, and 63 / 412,119 filed on September 30, 2022, which are incorporated herein by reference.

[0003] The Sequence Listing provided as a Sequence Listing XML file is incorporated by reference

[0004] The Sequence Listing is provided herewith as a Sequence Listing XML file ENCO-005WO_SEQ_LIST, created on April 13, 2023, and is 39,274 bytes in size. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. Background Art

[0005] Gene therapy has great potential in treating human diseases, especially those with potential genetic causes. In some gene therapy strategies, the therapeutic payload can be recombinantly expressed in target cells that lack essential proteins, or have reduced amounts of essential proteins or functionally impaired forms of essential proteins. The expression of the therapeutic payload in these cells saves these cells, thereby treating the disease. In one example, Tay-Sachs disease (which is recessive and caused by mutations in the HEXA gene on chromosome 15) can be successfully treated by expressing a functional form of hexA in the brain using adeno-associated virus (AAV) gene therapy.

[0006] One of the challenges of gene therapy is how to deliver therapeutic payloads to specific tissues, but not to other tissues. For example, some therapeutic payloads that have a positive effect in one tissue may have an adverse effect in another tissue. Therefore, administering a gene therapy that targets diseased cells in one tissue may cause side effects in another tissue. In some cases, the clinical application of gene therapy may even be limited by its off-site effects rather than on-site effects.

[0007] In view of the above, there is a general need for tools to improve the tissue specificity of gene therapy. Summary of the invention

[0008] In particular, provided herein are nucleic acid cassettes comprising a transgene encoding an mRNA, wherein the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment, or a combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii). These sequences reduce the expression of the transgene in hepatocytes (e.g., relative to target cells such as neurons) and, therefore, can be used in a variety of gene therapy strategies that target cells that are not in the liver. In certain aspects, incorporation of one or more of these sequences results in an improved safety profile for gene therapy due to reduction or elimination of hepatocellular toxicity caused by expression of the transgene in these cells.

[0009] In some embodiments, the nucleic acid cassette is an expression cassette, wherein the expression cassette can comprise a promoter, a coding sequence, a sequence encoding (i), (ii) or (iii) (also referred to as a liver off-target element), and a terminator in an operably linked manner. In some embodiments, at least one sequence present in the expression cassette is heterologous to another sequence in the expression cassette. For example, in some embodiments, the expression cassette of the present disclosure comprises a promoter that is heterologous to the coding sequence that is operably linked. In some embodiments, the expression cassette may also comprise an enhancer and / or an intron.

[0010] In some embodiments, the promoter of the expression cassette can be selective for cells in a particular tissue (e.g., a target tissue such as the brain), but also drives transgene expression in the liver. In some embodiments, the promoter can be a CNS selective promoter, for example, a promoter selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0011] In any embodiment, the sequence may be located in the 3'UTR, 5'UTR or an intron of the mRNA.

[0012] In any embodiment, the expression cassette can encode a therapeutic protein, e.g., SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, UBE3A, or a transcription factor that activates endogenous expression of any of these proteins. In some embodiments, the therapeutic protein can be ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, M yoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity to (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

[0013] In some embodiments, the RNA transcript may comprise a combination of sequences (i), (ii), and (iii).

[0014] Also provided is a vector comprising a cassette as outlined above.The vector may be a plasmid or a viral vector, for example, an adeno-associated virus (AAV) or a lentiviral vector.

[0015] Also provided are AAVs, lentiviral particles or cells comprising a cassette as outlined above (which may be in single-stranded form if packaged).

[0016] Also provided is an RNA encoded by the cassettes outlined above.

[0017] Also provided are various methods. In some embodiments, the method can be used to express a protein. In these embodiments, the method can include introducing an expression cassette as outlined above or an mRNA encoded therein into an organism, wherein the sequence reduces the expression of the protein in the organism's hepatocytes.

[0018] Provided herein is a nucleic acid cassette comprising a therapeutic transgene encoding an mRNA, wherein the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

[0019] In some embodiments, the mRNA comprises a sequence of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 1-17 or 39 that reduces expression in hepatocytes.

[0020] In some embodiments, the mRNA further comprises a second sequence (i), (ii) or (iii).

[0021] In some embodiments, the mRNA further comprises a third sequence (i), (ii) or (iii).

[0022] In some embodiments, the mRNA further comprises a fourth sequence (i), (ii) or (iii).

[0023] In some embodiments, the mRNA comprises five or more of the (i), (ii), or (iii) sequences.

[0024] In some embodiments, the mRNA comprises two or more copies of the following sequence: (i), (ii), or (iii).

[0025] In some embodiments, the mRNA comprises three or more copies of the following sequence: (i), (ii), or (iii).

[0026] In some embodiments, the mRNA comprises four or more copies of the following sequence: (i), (ii), or (iii).

[0027] In some embodiments, the mRNA comprises five or more copies of the following sequence: (i), (ii), or (iii).

[0028] In some embodiments, sequence (i), (ii) or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

[0029] In some embodiments, sequence (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

[0030] In some embodiments, sequence (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

[0031] In some embodiments, sequence (i), (ii) or (iii) is located in an intron of the mRNA.

[0032] In some embodiments, the nucleic acid cassette is non-naturally occurring.

[0033] In some embodiments, the nucleic acid cassette comprises a CNS-selective promoter.

[0034] In some embodiments, the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

[0035] In some embodiments, the nucleic acid cassette comprises an enhancer.

[0036] In some embodiments, the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

[0037] In some embodiments, the neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jeavons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSWS), infantile spasms (West syndrome The patient may have any of the following: schizophrenia, leukocorticoid syndrome, juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Doose syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0038] In some embodiments, the therapeutic protein is selected from (i): selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myo clonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

[0039] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); the nucleic acid cassette comprises a CNS-selective promoter; and the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

[0040] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); the nucleic acid cassette comprises a promoter selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic aminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; and the mRNA encodes a protein selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2 , CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KC NQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SC N1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX genes encoded therapeutic proteins, (ii) proteins having at least 90% sequence identity with (i), (iii) functional fragments of (i) or (ii), or (iv) transcription factors that activate the expression of the gene from (i).

[0041] In some embodiments, sequence (i), (ii) or (iii) causes reduced expression in hepatocytes of a polypeptide encoded by the mRNA as compared to expression in hepatocytes of a polypeptide from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii).

[0042] In some embodiments, sequence (i), (ii) or (iii) causes the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced by at most 1 / 2, at most 1 / 5 or at most 1 / 10 as compared to the expression level of a polypeptide in hepatocytes from an mRNA that is otherwise identical but does not contain sequence (i), (ii) or (iii).

[0043] In some embodiments, sequence (i), (ii) or (iii) causes the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of a polypeptide from an otherwise identical mRNA but without sequence (i), (ii) or (iii) in hepatocytes.

[0044] In some embodiments, sequence (i), (ii) or (iii) does not cause expression of a polypeptide encoded by the mRNA in a target cell to be substantially reduced compared to expression of a polypeptide from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii) in a target cell.

[0045] In some embodiments, sequence (i), (ii) or (iii) does not reduce expression in a target cell of a polypeptide encoded by the mRNA as compared to expression in a target cell of a polypeptide from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii).

[0046] In some embodiments, sequence (i), (ii) or (iii) causes expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of a polypeptide from an otherwise identical mRNA but without sequence (i), (ii) or (iii) in a target cell.

[0047] In some embodiments, the target cell is a neural cell.

[0048] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0049] In some embodiments, the neural cell is a GABAergic cell.

[0050] In some embodiments, the GABAergic cell is a cell that expresses parvalbumin.

[0051] In some embodiments, the nucleic acid cassette is a linear construct or vector.

[0052] In some embodiments, the vector is a plasmid.

[0053] In some embodiments, the vector is a viral vector.

[0054] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0055] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0056] In some embodiments, the AAV is a scAAV.

[0057] In some embodiments, the viral vector is a lentiviral vector.

[0058] Also provided is an mRNA having a sequence encoded by the nucleic acid cassette outlined above.

[0059] Also provided is a nucleic acid cassette comprising a transgene encoding an mRNA, wherein the mRNA encodes a therapeutic protein and comprises a miRNA binding site for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or a complementary sequence thereof.

[0060] In some embodiments, the nucleic acid box can comprise miRNA binding sites for two or more miRNAs selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complementary sequences.

[0061] In some embodiments, the nucleic acid box can comprise miRNA binding sites for three or more miRNAs selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complementary sequences.

[0062] In some embodiments, the nucleic acid box can comprise two miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complementary sequences.

[0063] In some embodiments, the nucleic acid box can comprise three miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complements.

[0064] In some embodiments, the nucleic acid box can comprise four miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complements.

[0065] In some embodiments, the nucleic acid box can comprise more than four miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complements.

[0066] In some embodiments, the miRNA is miR-22-3p.

[0067] In some embodiments, the miRNA is miR-1258-5p.

[0068] In some embodiments, the miRNA is miR-5589-3p.

[0069] In some embodiments, the miRNA is miR-17-5p.

[0070] In some embodiments, the miRNA is miR-203a.

[0071] In some embodiments, the miRNA is miR-122-3p.

[0072] In some embodiments, the miRNA is miR-93-5p.

[0073] In some embodiments, the miRNA is miR-122-5p.

[0074] In some embodiments, the mRNA further comprises a sequence of at least 10 contiguous nucleotides of any one of SEQ ID NOs: 12-17, which sequence reduces expression in hepatocytes.

[0075] In some embodiments, the mRNA further comprises at least two sequences of at least 20 contiguous nucleotides of any one of SEQ ID NOs: 12-17, which sequences reduce expression in hepatocytes.

[0076] In some embodiments, the miRNA binding site is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

[0077] In some embodiments, the miRNA binding site is located in the 3'UTR region of the mRNA.

[0078] In some embodiments, the miRNA binding site is located in the 5'UTR region of the mRNA.

[0079] In some embodiments, the miRNA binding site is located in an intron of the mRNA.

[0080] In some embodiments, the nucleic acid cassette is non-naturally occurring.

[0081] In some embodiments, the nucleic acid cassette comprises a promoter, optionally a neural-selective promoter.

[0082] In some embodiments, the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

[0083] In some embodiments, the nucleic acid cassette comprises an enhancer.

[0084] In some embodiments, the mRNA encodes a therapeutic protein.

[0085] In some embodiments, the therapeutic protein is a protein associated with a neurological disease or disorder, which neurological disease or disorder is optionally Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy disease with sustained spike-wave sleep (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gastau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0086] In some embodiments, the therapeutic transgene is selected from (a): selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, M (b) a protein encoded by the genes for yoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (c) a functional fragment of (a) or (b), or (d) a transcription factor that activates the expression of the gene from (a).

[0087] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); the nucleic acid cassette comprises a CNS-selective promoter; and the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

[0088] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii), and the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a pretachykininogen 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic aminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; and the mRNA encodes a protein selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2 , CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KC NQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SC N1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX genes encoded therapeutic proteins, (ii) proteins having at least 90% sequence identity with (i), (iii) functional fragments of (i) or (ii), or (iv) transcription factors that activate the expression of the gene from (i).

[0089] In some embodiments, the miRNA binding site causes reduced expression in a hepatocyte of a polypeptide encoded by the mRNA as compared to expression in a hepatocyte of a polypeptide from an otherwise identical mRNA but lacking the miRNA binding site.

[0090] In some embodiments, the miRNA binding site causes the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced by at most 2-fold, at most 5-fold, or at most 10-fold as compared to the expression level of a polypeptide from an otherwise identical mRNA but without the miRNA binding site in hepatocytes.

[0091] In some embodiments, the miRNA binding site causes at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% decrease in the expression level of a polypeptide encoded by the mRNA in a hepatocyte compared to the expression level of a polypeptide from an otherwise identical mRNA but without the miRNA binding site.

[0092] In some embodiments, sequence (i), (ii) or (iii) does not cause expression of a polypeptide encoded by the mRNA in a target cell to be substantially reduced compared to expression of a polypeptide from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii) in a target cell.

[0093] In some embodiments, sequence (i), (ii) or (iii) does not reduce expression in a target cell of a polypeptide encoded by the mRNA as compared to expression in a target cell of a polypeptide from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii).

[0094] In some embodiments, sequence (i), (ii) or (iii) causes expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of a polypeptide from an otherwise identical mRNA but without sequence (i), (ii) or (iii) in a target cell.

[0095] In some embodiments, the target cell is a neural cell.

[0096] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0097] In some embodiments, the neural cell is a GABAergic cell.

[0098] In some embodiments, the GABAergic cell is a cell that expresses parvalbumin.

[0099] In some embodiments, the nucleic acid cassette is a linear construct or vector.

[0100] In some embodiments, the vector is a plasmid.

[0101] In some embodiments, the vector is a viral vector.

[0102] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0103] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0104] In some embodiments, the AAV is a scAAV.

[0105] In some embodiments, the viral vector is a lentiviral vector.

[0106] Also provided is an mRNA encoded by the nucleic acid cassette of any of the embodiments outlined above.

[0107] In some embodiments, the mRNA encodes a polypeptide.

[0108] In some embodiments, the polypeptide is a therapeutic protein.

[0109] Also provided is an mRNA having a sequence encoded by the nucleic acid cassette of any of the embodiments outlined above.

[0110] Also provided is a method of reducing liver expression of a therapeutic protein encoded by mRNA while maintaining expression of the therapeutic protein in a target tissue, the method comprising applying the following sequence:

[0111] (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii) in the mRNA.

[0112] In some embodiments, the mRNA further comprises a second sequence (i), (ii) or (iii).

[0113] In some embodiments, the mRNA further comprises a third sequence (i), (ii) or (iii).

[0114] In some embodiments, the mRNA further comprises a fourth sequence (i), (ii) or (iii).

[0115] In some embodiments, the mRNA comprises five or more of the (i), (ii), or (iii) sequences.

[0116] In some embodiments, the mRNA comprises two or more copies of the following sequence:

[0117] (i), (ii) or (iii).

[0118] In some embodiments, the mRNA comprises three or more copies of the following sequence:

[0119] (i), (ii) or (iii).

[0120] In some embodiments, the mRNA comprises four or more copies of the following sequence:

[0121] (i), (ii) or (iii).

[0122] In some embodiments, the mRNA comprises five or more copies of the following sequence:

[0123] (i), (ii) or (iii).

[0124] In some embodiments, the mRNA comprises at least 10 contiguous nucleotides of any one of SEQ ID NOs: 1-17 or 39 that reduce expression in hepatocytes.

[0125] In some embodiments, sequence (i), (ii) or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

[0126] In some embodiments, sequence (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

[0127] In some embodiments, sequence (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

[0128] In some embodiments, sequence (i), (ii) or (iii) is located in an intron of the mRNA.

[0129] In some embodiments, the method comprises administering to the subject a nucleic acid encoding the mRNA.

[0130] In some embodiments, the administration is systemic administration.

[0131] In some embodiments, the administration is topical administration.

[0132] In some embodiments, the nucleic acid is administered locally to the brain or CNS tissue.

[0133] In some embodiments, administration is carried out by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0134] In some embodiments, the therapeutic protein is a protein associated with a neurological disease or disorder.

[0135] In some embodiments, the therapeutic protein is selected from (i): selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myo clonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

[0136] In some embodiments, the subject suffers from a neurological disease or disorder.

[0137] In some embodiments, the subject has Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSWS), infantile spasms tic seizures, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0138] In some embodiments, the nucleic acid cassette comprises a CNS-selective promoter.

[0139] In some embodiments, the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

[0140] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); the nucleic acid cassette comprises a CNS-selective promoter; and the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

[0141] In some embodiments, the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); the nucleic acid cassette comprises a promoter selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic aminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; and the mRNA encodes a protein selected from ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2 , CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KC NQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SC N1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX genes encoded therapeutic proteins, (ii) proteins having at least 90% sequence identity with (i), (iii) functional fragments of (i) or (ii), or (iv) transcription factors that activate the expression of the gene from (i).

[0142] In some embodiments, sequence (i), (ii) or (iii) causes the expression level of the protein encoded by the mRNA in hepatocytes to be reduced by at most 1 / 2, at most 1 / 5 or at most 1 / 10 as compared to the expression level of the protein in hepatocytes from an mRNA that is otherwise identical but does not contain sequence (i), (ii) or (iii).

[0143] In some embodiments, sequence (i), (ii) or (iii) causes the expression level of a protein encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of a protein in hepatocytes from an otherwise identical mRNA but without sequence (i), (ii) or (iii).

[0144] In some embodiments, sequence (i), (ii) or (iii) does not cause expression of a protein encoded by the mRNA in a target cell to be substantially reduced compared to expression of a protein from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii) in a target cell.

[0145] In some embodiments, sequence (i), (ii) or (iii) does not reduce expression of a protein encoded by the mRNA in a target cell as compared to expression of a protein from an otherwise identical mRNA but lacking sequence (i), (ii) or (iii) in a target cell.

[0146] In some embodiments, sequence (i), (ii) or (iii) causes expression of a protein encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the protein in the target cell from an otherwise identical mRNA but without sequence (i), (ii) or (iii).

[0147] In some embodiments, the target cell is a neural cell.

[0148] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0149] In some embodiments, the neural cell is a GABAergic cell.

[0150] In some embodiments, the GABAergic cell is a cell that expresses parvalbumin.

[0151] In some embodiments, the mRNA is expressed from a nucleic acid cassette.

[0152] In some embodiments, the nucleic acid cassette is a linear construct.

[0153] In some embodiments, the nucleic acid cassette is a vector.

[0154] In some embodiments, the vector is a plasmid.

[0155] In some embodiments, the vector is a viral vector.

[0156] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0157] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0158] In some embodiments, the AAV is a scAAV.

[0159] In some embodiments, the viral vector is a lentiviral vector.

[0160] In some embodiments, the method further comprises administering the vector to the subject.

[0161] In some embodiments, the method further comprises administering the mRNA to the subject.

[0162] In some embodiments, administration comprises intraparenchymal administration, intrathecal administration, intracisterna magna administration, or intraventricular administration.

[0163] Other features, advantages and embodiments may become apparent in view of the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 is a scatter plot showing the log2 change in brain activity versus liver activity for a number of constructs tested.

[0165] Figure 2 is a graph showing the results of ELISA assays of selected constructs.

[0166] FIG. 3A to FIG. 3D Representative images showing in vivo expression of various constructs in brain and liver are shown. Figure 3A Representative images of brain expression from mice treated with a control vector without off-target elements are shown. Figure 3BRepresentative images of hepatic expression from mice treated with a control vector without off-target elements are shown. Figure 3C Representative images of brain expression from mice treated with a vector encoding RNA containing test sequence 12 (SEQ ID NO: 12) are shown. Figure 3D Representative images of liver expression from mice treated with a vector encoding RNA containing test sequence 12 (SEQ ID NO: 12) are shown.

[0167] Figure 4 is a graph showing the relative expression (log2 of fold change) in the liver of several different liver off-target elements in NHPs.

[0168] definition

[0169] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "includes," "comprising," "having," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0170] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all serotypes, subtypes, and both naturally occurring and recombinant forms unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes all serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, AAV-DJ8 and their hybrids (i.e., chimeric AAV vectors), as well as avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TR), Rep proteins and capsid subunits are known in the art. Such sequences can be found in literature or public databases (such as GenBank). As used herein, "rAAV vector" refers to an AAV vector comprising a polynucleotide sequence from a non-AAV source (i.e., a polynucleotide heterologous to AAV), wherein the polynucleotide sequence is generally a sequence of interest for genetic transformation of cells. Generally speaking, the heterologous polynucleotide is flanked by at least one, usually two, AAV inverted terminal repeats (ITRs). The rAAV vector can be a single-stranded vector (ssAAV) or a self-complementary vector (scAAV). See, for example, Raj et al., Expert Rev Hematol. October 2011, 4 (5): 539–549. "AAV virus" or "AAV virus particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as a "rAAV viral particle", or simply "rAAV particle". AAV can contain genomic components and capsids (e.g., pseudotype vectors) from multiple serotypes. For example, AAV can contain a serotype 2 genome (e.g., ITRs) encapsulated in a capsid from serotype 5 or serotype 9. Pseudotype vectors can exhibit improved transduction efficiency and altered tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect CNS cells are preferred. In some aspects, the recombinant AAV vector is AAV1, AAV8, AAV9, AAVDJ, or a chimeric AAV comprising two or more features of these serotypes. In various embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector.In certain embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector and comprises a heterologous nucleic acid flanked by an ITR from an AAV serotype other than AAV9. In certain embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector and comprises a heterologous nucleic acid flanked by an AAV serotype 2 ITR (i.e., ITR2).

[0171] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the manner in which the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean a difference of only one or more than one standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% from a given value.

[0172] In any embodiments described herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." For example, embodiments in which a particular element is included using the open-ended term "comprising" encompass embodiments in which that element is included using the more restrictive term "consisting essentially of" or "consisting of."

[0173] The terms "determine," "measure," "evaluate," "assess," "assay," "analyze," and their grammatical equivalents are used interchangeably herein to refer to any form of measurement, and include determining whether an element is present (e.g., detected). These terms may include quantitative and / or qualitative determinations. Assessments may be relative or absolute.

[0174] The term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If a polynucleotide includes introns or splice sites, e.g., derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0175] An "expression cassette" refers to a nucleic acid molecule comprising one or more regulatory elements operably linked to a coding sequence (eg, one or more genes) for expression.

[0176] "Transgene" refers to a portion of a nucleic acid cassette designed to be expressed in a cell. In some embodiments, the transgene encodes an RNA transcript, such as an mRNA or a functional RNA, such as an antisense RNA. In some embodiments, the transgene of the present disclosure encodes a therapeutic cargo, such as a therapeutic protein or a therapeutic RNA, and further includes one or more liver off-target sequences / elements to reduce expression of the transgene in hepatocytes.

[0177] The term "effective amount" or "therapeutically effective amount" refers to an amount of the composition described herein sufficient to affect the intended application (including but not limited to disease treatment as defined below). The therapeutically effective amount can vary depending on the intended therapeutic application (in cells or in vivo), or the subject being treated and the disease condition (e.g., the subject's weight and age, the severity of the disease condition, the mode of administration, etc.), which can be readily determined by one of ordinary skill in the art. The term also applies to doses that will induce a specific response in target cells. The specific dose will vary depending on the specific composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system that carries the specific dose.

[0178] A "fragment" of a nucleotide or peptide sequence is intended to refer to a sequence that is smaller than what is considered the "full-length" sequence.

[0179] A "functional fragment" of a DNA, RNA or protein sequence refers to a biologically active fragment of the sequence that is shorter than the full-length or reference DNA, RNA or protein sequence, but retains at least one biological activity (functionally or structurally) substantially similar to the biological activity of the full-length or reference DNA, RNA or protein sequence. For example, a "functional fragment" can be a fragment of a sequence disclosed herein that reduces the expression of a transgene operably linked thereto in hepatocytes.

[0180] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and progeny derived therefrom (regardless of the number of passages). Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the originally transformed cell are included herein.

[0181] As used herein, the term "derived from the human race" refers to a sequence found in the human genome (or human genome construct), or a sequence homologous thereto. Homologous sequences can be sequences comprising a region having at least 80% sequence identity (e.g., as measured by BLAST) compared to a certain region of the human genome. For example, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a human sequence is considered to be derived from the human race. In some cases, a regulatory element comprises a sequence derived from the human race and a sequence not derived from the human race, so that, in general, the regulatory element has a low sequence identity with the human genome, and a part of the regulatory element has 100% sequence identity (or local sequence identity) with a certain sequence in the human genome.

[0182] The term "in vitro" refers to an event that occurs outside the body of a subject. For example, an in vitro assay encompasses any assay that is performed outside a subject. In vitro assays encompass cell-based assays in which live or dead cells are employed. In vitro assays also encompass cell-free assays in which intact cells are not employed.

[0183] The term "in vivo" refers to events that occur within the body of a subject.

[0184] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally, at a chromosomal location different from its natural chromosomal location, or contains only coding sequences.

[0185] As used herein, "operably linked," "operably connected," "operably linked," or their grammatical equivalents, refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, polyadenylation sequences, etc.) wherein these elements are in a relationship that permits them to function in an intended manner. For example, if a regulatory element, which may include a promoter and / or enhancer sequence, helps initiate transcription of a coding sequence, then the regulatory element is operably linked to a coding region. Intervening residues may exist between the regulatory element and the coding region as long as this functional relationship is maintained.

[0186] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition that is not an active ingredient and is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0187] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in effective form permitting the biological activity of the active ingredient contained therein, and contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0188] The term "regulatory element" refers to a nucleic acid sequence or genetic element that can affect (e.g., increase, decrease, or regulate) the expression of an operably linked sequence, such as a gene, a coding sequence, or an RNA (e.g., mRNA). Regulatory elements include, but are not limited to, promoters, enhancers, repressors, silencers, insulator sequences, introns, UTRs, inverted terminal repeat (ITR) sequences, long terminal repeat sequences (LTRs), stabilizing elements, miRNA target sites, post-translational response elements, polyA sequences, or combinations thereof. Regulatory elements can play a role at the DNA and / or RNA level, for example, by regulating gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression; by regulating translation levels (e.g., stabilizing elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination; by recruiting transcription factors to coding regions that increase gene expression; by increasing the rate at which RNA transcripts are produced, increasing the stability of the RNA produced, and / or increasing the rate at which proteins are synthesized from RNA transcripts; and / or by preventing RNA degradation and / or increasing its stability to promote protein synthesis. In an exemplary embodiment, the regulatory element refers to an enhancer, a repressor protein, a promoter, or a combination thereof, in particular a combination of an enhancer plus a promoter, or a combination of a repressor protein plus a promoter. In an exemplary embodiment, the regulatory element is derived from a human sequence.

[0189] In general, "sequence identity" or "sequence homology", which can be used interchangeably, refers to the exact nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity" (also referred to as "percent homology"). The percent identity with a reference sequence (e.g., a nucleic acid or amino acid sequence) can be calculated as the number of exact matches between the two best aligned sequences divided by the length of the reference sequence, multiplied by 100. When determining the number of matches to calculate sequence identity, conservative substitutions are not considered matches. It should be understood that when the length of the first sequence (A) is not equal to the length of the second sequence (B), the percent identity of the A:B sequence will be different from the percent identity of the B:A sequence. Sequence alignment, such as for the purpose of evaluating percent identity, can be performed by any suitable alignment algorithm or program, including but not limited to: Needleman-Wunsch algorithm, BLAST algorithm, Smith-Waterman algorithm (see, e.g., EMBOSS Water aligner) and Clustal Omega alignment program (F. Sievers et al., Mol Sys Biol. 7:539 (2011)). Any suitable parameters (including default parameters) of the selected algorithm can be used to evaluate optimal alignment. The BLAST programs are based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).

[0190] The terms "subject" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The methods described herein can be used for human therapy, veterinary applications, and / or preclinical studies in animal models of a disease or disorder.

[0191] As used herein, the terms "treat", "therapy" and the like refer to obtaining a desired pharmacological and / or physiological effect, including but not limited to: alleviating, delaying or slowing progression, reducing effects or symptoms, preventing the onset of a disease or condition, preventing the recurrence of a disease or condition, inhibiting, ameliorating the onset of a disease or condition, obtaining a beneficial or desired result with respect to a disease, condition or medical condition, such as a therapeutic benefit and / or a preventive benefit. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, including: (a) preventing the disease from occurring in a subject who may be susceptible to the disease or is at risk of developing the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. Therapeutic benefits include eradicating or ameliorating the underlying condition being treated. In addition, therapeutic benefits are achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying condition, such that an improvement is observed in the subject, although the subject may still be suffering from the underlying condition. In some cases, to obtain a preventive benefit, these compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the disease may not have been diagnosed. The methods of the present disclosure can be used in any mammal. In some cases, treatment can cause symptom relief or cessation. The preventive effect includes delaying or eliminating the occurrence of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping or reversing the progression of a disease or condition, or any combination of these preventive effects.

[0192] A "variant" of a nucleotide sequence refers to a sequence that has a genetic change or mutation compared to the most common wild-type DNA sequence (e.g., a cDNA or a sequence referred to by its GenBank accession number) or a specified reference sequence. A variant may be shorter than a reference sequence and / or may have one or more mutations relative to a reference sequence. In some cases, a variant may have a nucleotide sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to a reference sequence.

[0193] As used herein, "vector" refers to a nucleic acid molecule that can be used to mediate the delivery of another nucleic acid molecule to which it is linked into a cell where it can be replicated or expressed. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Other examples of vectors include plasmids and viral vectors.

[0194] As used herein, a "target cell" is generally a cell in which expression of the RNA or protein product of a nucleic acid cassette is desired. Non-target cells are cells in which expression of the RNA or protein product of a nucleic acid is not desired. As used herein, "off-target" generally refers to reducing expression in non-target cells.

[0195] Unless otherwise indicated, all terms used herein have the same meaning as to one skilled in the art, and the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology, which are within the knowledge of one skilled in the art. DETAILED DESCRIPTION

[0196] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as such specific embodiments may vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0197] The upper and lower limits of the ranges may be independently included in these ranges, and are also encompassed within the present invention, subject to any explicitly excluded limits in the stated ranges. Where the range includes one or both of these limits, the range excluding either or both of the included limits is also included in the present invention.

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials related to the cited publications.

[0199] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins, reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. Further, it should be noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0200] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for simplicity may also be provided individually or in any suitable subcombination. All combinations of embodiments belonging to the invention are specifically contemplated by the invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly included in the invention and disclosed herein, just as each such subcombination is individually and explicitly disclosed herein.

[0201] Publications discussed herein are provided only for disclosures prior to the date of filing of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to precede such publications due to prior invention. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0202] As summarized above, the present disclosure describes a nucleic acid cassette comprising a transgene encoding RNA, wherein the RNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the foregoing sequence, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. The transgene may encode a protein encoding an mRNA or a non-coding RNA (such as a pri microRNA, pre microRNA or microRNA), a short non-coding RNA, a long non-coding RNA, a snoRNA, a snRNA, a tRNA or an rRNA. In some cases, the nucleic acid cassette comprises a transgene encoding an mRNA, wherein the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the foregoing sequence, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. These sequences reduce the expression of the transgene in hepatocytes relative to target cells (such as neural cells, e.g., neurons), and therefore, can be used in a variety of gene therapy strategies targeting cells that are not in the liver. Reducing transgene expression in hepatocytes relative to target cells means that the reduction in transgene expression driven by the liver off-target sequences disclosed herein is greater in hepatocytes than in target cells. Thus, while a reduction in transgene expression in target cells may be observed in certain embodiments, it is less than the reduction in transgene expression observed in hepatocytes. This reduction in expression in the liver can reduce or eliminate hepatotoxicity in subjects receiving gene therapy targeting non-hepatocytes or tissues (e.g., neural cells, such as neurons), thereby improving its safety profile.

[0203] The present disclosure also provides an RNA molecule having a sequence feature of an RNA encoded by any nucleic acid box as described herein. In certain embodiments, the RNA is modified to increase its stability and / or activity when, for example, it is applied to a subject as a pharmaceutical composition. RNA compositions can be used in a variety of treatment modes delivered using a wide range of viral and non-viral delivery systems, including polymeric materials, ionizable lipids, lipids that penetrate cells, and zwitterionic lipids, nanoparticles, and dendrimers (see, for example, Kowalski et al., "Delivering the Messenger:Advances in Technologies for Therapeutic mRNA Delivery", Molecular Therapy, Vol. 27, No. 4, 2019, pp. 710–728; and Paunovska et al., "Drug delivery systems forRNA therapeutics", Nature Reviews genetics, Vol. 23, 2022, pp. 265-280).

[0204] The RNA (e.g., mRNA) encoded by the transgene of the nucleic acid cassette can contain any combination of two, three, four, five or more of the foregoing sequences. For example, the RNA comprises the following sequence:

[0205] (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant or functional fragment of the aforementioned sequence, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii), and may also comprise a second sequence (i), (ii) or (iii), a third sequence (i), (ii) or (iii), a fourth sequence (i), (ii) or (iii), and / or five or more sequences (i), (ii) or (iii). In any embodiment, the nucleic acid box may comprise two or more copies (e.g., two, three, four, five or more copies) of sequence (i), (ii) or (iii).

[0206] In any embodiment, the sequence can be, for example, located in 3'UTR, 5'UTR or intron of mRNA. If the mRNA contains more than one aforementioned sequence, these sequences can be located in different parts of the mRNA. However, in many embodiments, these sequences are located in 3'UTR of the mRNA. In these embodiments, the following sequence: (i) any one of SEQ ID NO:1-17 or 39, (ii) variants or functional fragments of the aforementioned sequences, or (iii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences, can be located in one or more of the following: 3'UTR region of the mRNA, 5'UTR of the mRNA or intron of the mRNA.

[0207] Any nucleic acid described herein may be non-naturally occurring, wherein the term "non-naturally occurring" refers to a composition that does not exist in nature. Non-naturally occurring nucleic acids may include a nucleotide sequence that is different from the nucleic acid in its natural state (i.e., having less than 100% sequence identity with a naturally occurring nucleic acid sequence). If two parts of a nucleic acid are "heterologous", they are not components of the same nucleic acid in a natural state. For example, in some embodiments, a nucleic acid box may consist of a promoter, a coding sequence, and a terminator, wherein the promoter, the coding sequence, and the terminator are operably connected. In these embodiments, the promoter may be heterologous to the coding sequence, meaning that the promoter will not drive the expression of the coding sequence in a wild-type cell. In any embodiment, the nucleic acid box may additionally include an enhancer.

[0208] In any embodiment, the RNA encoded by the transgene of the nucleic acid cassette can include a functional fragment having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, or at least 220 contiguous nucleotides of any one of SEQ ID NOs: 1-17 or 39, for example, the functional fragment may or may not contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mismatches relative to SEQ ID NOs: 1-17 or 39. Such fragments can begin at any position in SEQ ID NO: 1-17 or 39, for example, at position 1, 21, 41, 61, 81, 101 or 121.

[0209] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length in SEQ ID NO: 1, 5, 7, or 10. In certain embodiments, a functional fragment of SEQ ID NO: 1, 5, 7, or 10 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 1, 5, 7, or 10. A functional fragment may start at any nucleotide in SEQ ID NO: 1, 5, 7, or 10 that allows it to be fully represented in SEQ ID NO: 1, 5, 7, or 10. Thus, a functional fragment comprising 10 adjacent nucleotides may start at any one of the 1st to the 13th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 11 adjacent nucleotides may start at any one of the 1st to the 12th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 12 adjacent nucleotides may start at any one of the 1st to the 11th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 13 adjacent nucleotides may start at any one of the 1st to the 10th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 14 adjacent nucleotides may start at any one of the 1st to the 9th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 15 adjacent nucleotides may start at any one of the 1st to the 8th nucleotide of SEQ ID NO: 1, 5, 7 or 10, a functional fragment comprising 16 adjacent nucleotides may start at any one of the 1st to the 8th nucleotide of SEQ ID NO: 1, 5, 7 or 10, NO:1, 5, 7 or 10 starts at any one of the 1st to 7th nucleotides, a functional fragment comprising 17 adjacent nucleotides can start at any one of the 1st to 6th nucleotides of SEQ ID NO:1, 5, 7 or 10, a functional fragment comprising 18 adjacent nucleotides can start at any one of the 1st to 5th nucleotides of SEQ ID NO:1, 5, 7 or 10, a functional fragment comprising 19 adjacent nucleotides can start at any one of the 1st to 4th nucleotides of SEQ ID NO:1, 5, 7 or 10, a functional fragment comprising 20 adjacent nucleotides can start at any one of the 1st to 3rd nucleotides of SEQ ID NO:1, 5, 7 or 10, and a functional fragment comprising 21 adjacent nucleotides can start at the 1st or 2nd nucleotide of SEQ ID NO:1, 5, 7 or 10.

[0210] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 nucleotides in length in SEQ ID NO: 2. In certain embodiments, a functional fragment of SEQ ID NO: 2 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 2. A functional fragment may start at any nucleotide in SEQ ID NO: 2 that allows it to be fully represented in SEQ ID NO: 2. Therefore, a functional fragment comprising 10 adjacent nucleotides can start at any one of the 1st to the 10th nucleotide of SEQ ID NO:2, a functional fragment comprising 11 adjacent nucleotides can start at any one of the 1st to the 9th nucleotide of SEQ ID NO:2, a functional fragment comprising 12 adjacent nucleotides can start at any one of the 1st to the 8th nucleotide of SEQ ID NO:2, a functional fragment comprising 13 adjacent nucleotides can start at any one of the 1st to the 7th nucleotide of SEQ ID NO:2, a functional fragment comprising 14 adjacent nucleotides can start at any one of the 1st to the 6th nucleotide of SEQ ID NO:2, a functional fragment comprising 15 adjacent nucleotides can start at any one of the 1st to the 5th nucleotide of SEQ ID NO:2, a functional fragment comprising 16 adjacent nucleotides can start at any one of the 1st to the 4th nucleotide of SEQ ID NO:2, a functional fragment comprising 17 adjacent nucleotides can start at any one of the 1st to the 3rd nucleotide of SEQ ID NO:2, and a functional fragment comprising 18 adjacent nucleotides can start at any one of the 1st to the 3rd nucleotide of SEQ ID NO:2. Start at the first or second nucleotide of ID NO:2.

[0211] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides in length in SEQ ID NO: 3 or 8. In certain embodiments, a functional fragment of SEQ ID NO: 3 or 8 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 3 or 8. A functional fragment may start at any nucleotide in SEQ ID NO: 3 or 8 that allows it to be fully represented in SEQ ID NO: 3 or 8. Thus, a functional fragment comprising 10 adjacent nucleotides may start at any one of the 1st to the 12th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 11 adjacent nucleotides may start at any one of the 1st to the 11th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 12 adjacent nucleotides may start at any one of the 1st to the 10th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 13 adjacent nucleotides may start at any one of the 1st to the 9th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 14 adjacent nucleotides may start at any one of the 1st to the 8th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 15 adjacent nucleotides may start at any one of the 1st to the 7th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 16 adjacent nucleotides may start at any one of the 1st to the 6th nucleotide of SEQ ID NO: 3 or 8, a functional fragment comprising 17 adjacent nucleotides may start at any one of the 1st to the 6th nucleotide of SEQ ID NO: 3 or 8. NO:3 or 8 starts at any one of the 1st to 5th nucleotides, a functional fragment comprising 18 adjacent nucleotides can start at any one of the 1st to 4th nucleotides of SEQ ID NO:3 or 8, a functional fragment comprising 19 adjacent nucleotides can start at any one of the 1st to 3rd nucleotides of SEQ ID NO:3 or 8, and a functional fragment comprising 20 adjacent nucleotides can start at the 1st or 2nd nucleotide of SEQ ID NO:3 or 8.

[0212] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides in length in SEQ ID NO: 4 or 39. In certain embodiments, a functional fragment of SEQ ID NO: 4 or 39 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 4 or 39. A functional fragment may start at any nucleotide in SEQ ID NO: 4 or 39 that allows it to be fully represented in SEQ ID NO: 4 or 39. Thus, a functional fragment comprising 10 adjacent nucleotides may start at any one of the 1st to 14th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 11 adjacent nucleotides may start at any one of the 1st to 13th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 12 adjacent nucleotides may start at any one of the 1st to 12th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 13 adjacent nucleotides may start at any one of the 1st to 11th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 14 adjacent nucleotides may start at any one of the 1st to 10th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 15 adjacent nucleotides may start at any one of the 1st to 9th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 16 adjacent nucleotides may start at any one of the 1st to 8th nucleotides of SEQ ID NO: 4 or 39, a functional fragment comprising 17 adjacent nucleotides may start at any one of the 1st to 8th nucleotides of SEQ ID NO: 4 or 39, NO:4 or 39 starts at any one of the 1st to 7th nucleotides, a functional fragment comprising 18 adjacent nucleotides can start at any one of the 1st to 6th nucleotides of SEQ ID NO:4 or 39, a functional fragment comprising 19 adjacent nucleotides can start at any one of the 1st to 5th nucleotides of SEQ ID NO:4 or 39, a functional fragment comprising 20 adjacent nucleotides can start at any one of the 1st to 4th nucleotides of SEQ ID NO:4 or 39, a functional fragment comprising 21 adjacent nucleotides can start at any one of the 1st to 3rd nucleotides of SEQ ID NO:4 or 39, and a functional fragment comprising 22 adjacent nucleotides can start at the 1st or 2nd nucleotide of SEQ ID NO:4 or 39.

[0213] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 nucleotides in length in SEQ ID NO: 6, 9, or 11. In certain embodiments, a functional fragment of SEQ ID NO: 6, 9, or 11 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 6, 9, or 11. A functional fragment may start at any nucleotide in SEQ ID NO: 6, 9, or 11 that allows it to be fully represented in SEQ ID NO: 6, 9, or 11. Thus, a functional fragment comprising 10 adjacent nucleotides may start at any one of the 1st to 11th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 11 adjacent nucleotides may start at any one of the 1st to 10th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 12 adjacent nucleotides may start at any one of the 1st to 9th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 13 adjacent nucleotides may start at any one of the 1st to 8th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 14 adjacent nucleotides may start at any one of the 1st to 7th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 15 adjacent nucleotides may start at any one of the 1st to 6th nucleotides of SEQ ID NO: 6, 9 or 11, a functional fragment comprising 16 adjacent nucleotides may start at any one of the 1st to 6th nucleotides of SEQ ID NO: 6, 9 or 11, NO:6, 9 or 11 starts at any one of the 1st to 5th nucleotides, a functional fragment comprising 17 adjacent nucleotides can start at any one of the 1st to 4th nucleotides of SEQ ID NO:6, 9 or 11, a functional fragment comprising 18 adjacent nucleotides can start at any one of the 1st to 3rd nucleotides of SEQ ID NO:6, 9 or 11, and a functional fragment comprising 19 adjacent nucleotides can start at the 1st or 2nd nucleotide of SEQ ID NO:6, 9 or 11.

[0214] In certain embodiments, the functional fragment comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66 , at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141 144, at least 145, at least 146, at least 147, at least 148, at least 149, at least 150, or at least 151 nucleotides.In certain embodiments, the functional fragment comprises any contiguous stretch of nucleotides of SEQ ID NO: 12-15 or 17 that is at least 152, at least 153, at least 154, at least 155, at least 156, at least 157, at least 158, at least 159, at least 160, at least 161, at least 162, at least 163, at least 164, at least 165, at least 166, at least 167, at least 168, at least 169, at least 170, at least 171, at least 172, at least 173, at least 174, at least 175, or at least 176 nucleotides in length. In certain embodiments, the functional fragment comprises any contiguous stretch of nucleotides of SEQ ID NO: 13, 14, or 17 that is at least 177, at least 178, or at least 179 nucleotides in length. In certain embodiments, the functional fragment comprises any contiguous stretch of nucleotides of SEQ ID NO: 13, 14, or 17 that is at least 177, at least 178, or at least 179 nucleotides in length. NO: 14 or 17 has a length of at least 180, at least 181, at least 182, at least 183, at least 184, at least 185, at least 186, at least 187, at least 188, at least 189, at least 190, at least 191, at least 192, at least 193, at least 194, at least 195, at least 196, at least 197, at least 198, at least 199, at least 200, at least 201, at least 202, at least 203, or at least 204. In some embodiments, the functional fragment comprises any contiguous stretch of nucleotides of SEQ ID NO: 14 of at least 227 nucleotides in length. In certain embodiments, a functional fragment of any one of SEQ ID NOs: 12-17 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NOs: 12-17. A functional fragment may start at any nucleotide in SEQ ID NOs: 12-17 that allows it to be fully represented in SEQ ID NOs: 12-17.

[0215] In any embodiment, the RNA may comprise one or more binding sites for a miRNA. In these embodiments, the RNA may comprise 6, 7, 8, 9 or 10 adjacent nucleotides at the 3' end of any one of SEQ ID NOs: 1-11, which may potentially base pair with a seed region of a miRNA such as miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p or miR-122-5p (at the 5' end of these miRNAs). In some embodiments, the RNA may include a miRNA binding site for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or its complementary sequence. In some embodiments, the RNA may include a binding site for a miRNA produced from mir-22, mir-1258, mir-5589, mir-17, mir-203a, mir-93 or mir-122 genes. One or more binding sites for miRNA may include any one of SEQ ID NO:1-11 or 39. In some embodiments, the sequence may be identical to SEQ ID NO:1-11 or 39, except that, for example, one, two, three or four mismatches are present relative to SEQ ID NO:1-11 or 39.

[0216] It will be apparent that the nucleic acid cassette itself (which is DNA) may contain (i) any one of SEQ ID NOs: 18-34 or 40, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii), wherein the inclusion of the sequence reduces the expression of the protein or RNA encoded by the nucleic acid cassette in hepatocytes of an organism relative to the expression in a target tissue (e.g., a neuronal tissue, such as neuronal cells in the brain).

[0217] In some embodiments, the mRNA can encode a polypeptide, such as a therapeutic protein, which can be, for example, an intracellular protein, a membrane-bound protein, or a secreted protein.

[0218] A transgene encoding an mRNA encoding a therapeutic protein is sometimes referred to herein as a therapeutic transgene. In some embodiments, the therapeutic protein is a protein associated with a neurological disease or disorder, e.g., a protein whose abnormal function (e.g., caused by a genetic mutation or abnormality) is associated with a neurological disease or disorder.

[0219] Neurological diseases and disorders include those associated with one or more genetic mutations, and those of unknown etiology. In some embodiments, neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X chromosome syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), infantile epilepsy with migratory focal epileptic seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and slow waves in sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0220] Many genetic abnormalities have been associated with epilepsy, including many of the aforementioned neurological diseases and disorders. Examples of genes affected by these genetic abnormalities, i.e., genes whose activity and / or expression has been altered due to genetic mutations, include: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCN Q2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B , SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX.

[0221] Thus, in embodiments in which the mRNA encodes a therapeutic protein for treating a neurological disease or disorder, the therapeutic protein can be: (i) a protein selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PC DH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC 25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWO (ii) a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with (i), (iii) a variant or functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of the gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0222] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii) can cause the expression of a polypeptide encoded by the mRNA in hepatocytes to be reduced compared to the expression of a polypeptide from an mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in hepatocytes. For example, an mRNA containing sequence (i), (ii) or (iii) can cause the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5 or at most 1 / 10 compared to the expression level of a polypeptide in hepatocytes from an mRNA that is otherwise equivalent but does not have sequence (i), (ii) or (iii). In these embodiments, the reduction in expression of the polypeptide in hepatocytes is greater than the reduction in expression of the polypeptide in target cells when compared to an mRNA that is otherwise equivalent but does not have sequence (i), (ii) or (iii).

[0223] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii), can cause the expression level of the polypeptide encoded by the mRNA in hepatocytes to be comparable to that from The expression level of the polypeptide in hepatocytes is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the polypeptide in the mRNA of otherwise equivalent but without sequence (i), (ii) or (iii) in hepatocytes. In these embodiments, the reduction in expression of the polypeptide in hepatocytes is greater than the reduction in expression of the polypeptide in target cells when compared to the mRNA of otherwise equivalent but without sequence (i), (ii) or (iii).

[0224] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii), does not cause the expression of a polypeptide encoded by the mRNA in a target cell to be significantly reduced compared to the expression of a polypeptide from an mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in a target cell. In some embodiments, sequence (i), (ii) or (iii) can cause the expression level of the polypeptide encoded by the mRNA in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the polypeptide in the target cell from an otherwise equivalent mRNA but without sequence (i), (ii) or (iii). In these embodiments, the reduced expression of the polypeptide in hepatocytes is greater than the reduced expression of the polypeptide in the target cell when compared to an otherwise equivalent mRNA but without sequence (i), (ii) or (iii).

[0225] In some cases, the target cell can be a neural cell, a muscle cell, a heart cell, a skin cell, an immune cell, a hematopoietic cell, a cancer cell, a pancreatic cell, or a kidney cell. In any of these embodiments, the target cell can be a neural cell, e.g., a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in these embodiments, the neural cell can be a GABAergic cell, e.g., a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0226] The box can be linear, circular, and in some embodiments, the nucleic acid box can be a vector, such as a plasmid or a viral vector, for example, an adeno-associated virus (AAV) vector or a lentiviral vector. In a specific embodiment, the viral vector can be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, AAV-DJ8 and their hybrids.

[0227] A kind of nucleic acid box of the transgenic comprising coding RNA is also provided, wherein the RNA comprises the miRNA binding site for the miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or its complementary sequence. In some embodiments, the RNA may comprise the binding site for the miRNA produced from mir-22, mir-1258, mir-5589, mir-17, mir-203a, mir-93 or mir-122 gene. If the RNA is naturally occurring in other aspects, the miRNA binding site should not be present in the naturally occurring form of the RNA. In some embodiments, the box can include two or more, three or more, or four or more binding sites for miRNAs selected from, for example, miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or their complementary sequences. In some embodiments, the RNA is an mRNA, for example, an mRNA encoding a therapeutic protein (as described elsewhere herein). In these embodiments, the binding sites can be at any position in the mRNA, particularly in non-coding sequences such as 3'UTR regions, 5'UTR regions, introns, or any combination thereof.

[0228] In any embodiment, the nucleic acid box can be non-naturally occurring, which means that, for example, the miRNA binding site can be heterologous to the mRNA. In any embodiment, the nucleic acid box can include a promoter and / or an enhancer. In some embodiments, the nucleic acid box can be composed of a promoter, a coding sequence and a terminator, wherein the promoter, the coding sequence and the terminator are operably connected. In these embodiments, the promoter can be heterologous to the coding sequence, which means that the promoter will not drive the expression of the coding sequence in wild-type cells. In any embodiment, the nucleic acid box can additionally include an enhancer.

[0229] In some embodiments, the mRNA can encode a polypeptide, such as a therapeutic protein, which can be, for example, an intracellular protein, a membrane-bound protein, or a secreted protein.

[0230] In some embodiments, the protein is a protein associated with a neurological disease or disorder.

[0231] Neurological diseases and disorders include those associated with one or more genetic mutations, and those of unknown etiology. In a broad sense, neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X chromosome syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), infantile epilepsy with migratory focal epileptic seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves in sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0232] Many genetic abnormalities have been associated with epilepsy, including many of the aforementioned neurological diseases and disorders. Examples of genes affected by these genetic abnormalities, i.e., genes whose activity and / or expression has been altered due to genetic mutations, include: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCN Q2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B , SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX.

[0233] Thus, in embodiments where the mRNA encodes a therapeutic protein for treating a neurological disease or disorder, the therapeutic protein can be (i) selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, L GI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor. In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the aforementioned sequence, or (iii) a sequence at least 80% identical to (i) or (ii), can cause the expression of a polypeptide encoded by the mRNA in hepatocytes to be reduced compared to the expression of a polypeptide from an mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in hepatocytes. For example, an mRNA containing sequence (i), (ii) or (iii) can cause the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced to at most 1 / 2, at most 1 / 5 or at most 1 / 10 compared to the expression level of a polypeptide from an mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in hepatocytes.

[0234] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the aforementioned sequence, or (iii) a sequence at least 80% identical to (i) or (ii), can cause the expression level of the polypeptide encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the polypeptide from the mRNA that is otherwise equivalent but does not have sequence (i), (ii) or (iii) in hepatocytes.

[0235] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the aforementioned sequence, or (iii) a sequence at least 80% identical to (i) or (ii), does not cause the expression of the polypeptide encoded by the mRNA in the target cell to be reduced compared to the expression of the polypeptide from the mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in the target cell. In some embodiments, sequence (i), (ii) or (iii) does not cause the expression of the polypeptide encoded by the mRNA in the target cell to be reduced compared to the expression of the polypeptide from the mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in the target cell. In these embodiments, sequence (i), (ii) or (iii) can cause the expression level of the polypeptide encoded by the mRNA in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the polypeptide from the mRNA that is otherwise equivalent but does not have sequence (i), (ii) or (iii) in the target cell. In any of these embodiments, the target cell can be a neural cell, e.g., a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in these embodiments, the neural cell can be a GABAergic cell, e.g., a cell expressing parvalbumin.

[0236] The cassette can be linear, circular, and in some embodiments, the nucleic acid cassette can be a vector, such as a plasmid or a viral vector, for example, an adeno-associated virus (AAV) vector or a lentiviral vector. In a specific embodiment, the viral vector can be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ and scAAV.

[0237] Also provided is an mRNA encoded by the above nucleic acid box.

[0238] A method for reducing the expression of a polypeptide encoded by an mRNA in the liver relative to the expression of the polypeptide in a target tissue is also provided. In these embodiments, the method may include constructing a nucleic acid box to include a liver off-target sequence as described herein in the RNA encoded therein. For example, the nucleic acid box may be constructed to include the following sequence in the mRNA encoded therein: (i) one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment, or a combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii). The details of the box prepared by this method are described herein. In some embodiments, the method can include introducing an expression cassette as described herein or mRNA encoded thereby into an organism (e.g., a human subject), wherein the inclusion of any one or more liver off-target sequences results in reduced expression of the protein in hepatocytes of the organism relative to expression in the target tissue.

[0239] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the aforementioned sequence, or (iii) a sequence at least 80% identical to (i) or (ii), causes the expression level of the polypeptide encoded by the mRNA in hepatocytes to be reduced to at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the polypeptide from the mRNA that is otherwise identical but does not have sequence (i), (ii) or (iii) in hepatocytes. In these embodiments, sequence (i), (ii) or (iii) can cause the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of a polypeptide from an otherwise identical mRNA but without sequence (i), (ii) or (iii) in hepatocytes.

[0240] In some embodiments, the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a functional fragment of the aforementioned sequence, or (iii) a sequence at least 80% identical to (i) or (ii), does not cause the expression of the polypeptide encoded by the mRNA in the target cell to be greatly reduced compared to the expression of the polypeptide from the mRNA of otherwise identical aspects but without sequence (i), (ii) or (iii) in the target cell. For example, sequence (i), (ii) or (iii) may not cause the expression of the polypeptide encoded by the mRNA in the target cell to be reduced compared to the expression of the polypeptide from the mRNA of otherwise identical aspects but without sequence (i), (ii) or (iii) in the target cell. For example, in some embodiments, sequence (i), (ii) or (iii) can cause the expression level of a polypeptide encoded by the mRNA in a target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of a polypeptide from an otherwise identical mRNA but without sequence (i), (ii) or (iii) in a target cell.

[0241] In any of the embodiments herein, the target cell can be a neural cell, a muscle cell, a heart cell, a skin cell, an immune cell, a hematopoietic cell, a cancer cell, a pancreatic cell, or a kidney cell. In some cases, the target cell can be a neural cell, e.g., a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in some embodiments, the neural cell is a GABAergic cell, e.g., a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or other CNS cells (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0242] In any embodiment, the method may further include administering to the subject a vector (e.g., an AAV or lentiviral vector) encoding the mRNA, e.g., wherein the mRNA encodes a therapeutic protein. In some embodiments, the method may include administering to the subject the mRNA.

[0243] Expression cassette

[0244] Nucleic acid box can comprise one or more additional regulatory elements (for example, promoter, terminator and / or enhancer etc.), and it induces transgenic expression in specific cell type or specific cell type kind.For example, cell type selective regulatory element can induce gene expression relative to one or more other cell types in specific cell type.Alternatively or in addition, cell type selective regulatory element can induce gene expression relative to one or more other cell types in specific cell type.In one embodiment, cell type selective regulatory element of the present invention enhances gene expression in specific cell type or specific cell type.In another embodiment, cell type selective regulatory element suppresses gene expression in specific cell type or specific cell type.The cell type selective regulation of gene expression (for example, enhancing or suppressing gene expression) does not need to affect gene expression only in target cell type or cell type.On the contrary, the cell type selective regulation of gene expression (for example, enhancing or suppressing gene expression) only requires gene expression in target cell type to increase or decrease relative to gene expression in one or more other cell types or cell type.

[0245] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 1; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0246] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 2; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0247] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 3; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0248] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 4; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0249] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 5; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0250] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 6; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0251] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 7; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0252] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 8; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0253] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 9; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0254] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 10; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0255] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 11; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0256] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 12; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0257] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 13; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0258] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 14; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0259] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 15; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0260] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 16; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0261] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 17; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0262] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) SEQ ID NO: 39; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0263] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) at least two different sequences selected from SEQ ID NO: 1-17 or 39; (ii) a variant, functional fragment, multiple copies or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0264] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) at least three different sequences selected from SEQ ID NO: 1-17 or 39; (ii) variants, functional fragments, multiple copies or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0265] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an mRNA, wherein the mRNA comprises a liver off-target region, the off-target region comprising: (i) at least four different sequences selected from SEQ ID NO: 1-17 or 39; (ii) variants, functional fragments, multiple copies or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the mRNA encodes a therapeutic protein for a neurological disease or disorder.

[0266] In some embodiments, the nucleic acid box may include a CNS selective promoter operably connected to a polynucleotide encoding a therapeutic protein and one or more liver off-target elements / sequences as disclosed herein. A CNS promoter is a promoter that specifically regulates gene expression in one or more cells of the central nervous system. For example, a CNS selective promoter can specifically regulate gene expression in one or more neurons or glial cells of the CNS. In one embodiment, a CNS selective promoter specifically regulates gene expression in one or more neurons or astrocytes. In another embodiment, a CNS selective promoter specifically regulates gene expression in one or more astrocytes. In certain embodiments, relative to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, microglia, motor neurons, vascular cells, non-GABAergic neurons or other CNS cells), a CNS selective promoter enhances expression in CNS cells (e.g., neurons or glial cells, such as astrocytes).

[0267] Examples of CNS selective promoters include, but are not limited to, Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripheral protein promoter, GAP-43 promoter and PaqR4 promoter. Suitable promoters are also described in, for example, WO 2018 / 187363, the sequences of which are incorporated herein by reference. Other sequences may be used.

[0268] In some embodiments, the nucleic acid box can include a GABA neuron selective promoter operably connected to a polynucleotide encoding a therapeutic protein. GABA cells are inhibitory neurons that produce gamma-aminobutyric acid. GABA cells can be identified by the expression of markers such as glutamate decarboxylase 2 (GAD2), GAD1, NKX2.1, DLX1, DLX5, SST, PV and VIP. GABA neuron selective promoter is a regulatory element that specifically regulates gene expression in GABA neurons. For example, relative to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, astrocytes, microglia, motor neurons, vascular cells, non-GABA neurons or other CNS cells), GABA neuron selective promoter enhances expression in GABA neurons.

[0269] A PV neuron selective promoter is a promoter that specifically regulates gene expression in PV neurons. For example, a PV neuron selective promoter enhances expression in PV neurons relative to one or more other CNS cell types.

[0270] In certain embodiments, the neuron selective promoter can be derived from humans, or include sequences derived from humans. In some cases, the promoter can be derived from mice, or include sequences derived from mice. In some cases, the promoter is non-naturally occurring, or includes sequences that are non-naturally occurring. In some cases, the sequence of the promoter can be 100% derived from humans. In other cases, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the promoter sequence is derived from humans. For example, 50% of the promoter sequence can be derived from humans, and the remaining 50% is non-derived from humans (e.g., derived from mice or fully synthetic).

[0271] In some embodiments, the therapeutic protein encoded by the mRNA is associated with a neurological disease or disorder. As mentioned above, neurological diseases and disorders include those associated with one or more genetic mutations, and those of unknown etiology. Examples of neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or conditions include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, Fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0272] In these embodiments, the therapeutic protein can be: (i) a protein selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, or (ii) a protein encoded by a gene selected from the group consisting of PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX, (ii) a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0273] In certain embodiments, nucleic acid constructs as described herein also include another regulatory element in addition to promoters, such as sequences associated with transcription initiation or termination, enhancer sequences, and effective RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stabilizing elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, or polyadenylation (polyA) sequences or combinations thereof. Regulatory elements can play a role in regulating gene expression in the transcription phase, post-transcription phase, or translation phase of gene expression. At the RNA level, regulation and control can occur at the level of translation (for example, stabilizing mRNA for stabilizing elements for translation), RNA cutting, RNA splicing, and / or transcription termination. In various embodiments, regulatory elements can recruit transcription factors to coding regions, which increase gene expression selectivity, increase the rate of RNA transcript production, increase the stability of the RNA produced, and / or increase the rate of protein synthesis from RNA transcripts in cell types of interest.

[0274] In certain embodiments, the nucleic acid box may also include a polyA sequence. Suitable polyA sequences include, for example, artificial polyA (PA75) of about 75 bp in length (see, for example, WO 2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit beta globulin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5EIb polyA, growth hormone polyA, or PBGD polyA. In certain embodiments, the polyA sequence is positioned downstream of the polynucleotide encoding the therapeutic functional protein in the nucleic acid construct described herein.

[0275] Carrier

[0276] Expression vectors can be used to deliver nucleic acid molecules to target cells via transfection or transduction. Vectors can be integrating or non-integrating vectors, which refer to the ability of the vector to integrate an expression cassette or transgene into the host cell genome. Examples of expression vectors include, but are not limited to: (a) non-viral vectors, such as nucleic acid vectors, including linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs or PACs); episomal vectors; transposons (e.g., PiggyBac); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0277] Expression vectors can be linear oligonucleotides or circular plasmids and can be delivered to cells via various transfection methods, including physical methods and chemical methods. Physical methods generally refer to delivery methods that use physical forces to offset cell membrane barriers to promote intracellular delivery of genetic material. Examples of physical methods include the use of needles, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, and water poration. Chemical methods generally refer to methods in which chemical carriers deliver nucleic acid molecules to cells, and can include inorganic particles, lipid-based carriers, polymer-based carriers, and peptide-based carriers.

[0278] In some embodiments, the expression vector is applied to the target cell using inorganic particles. Inorganic particles can refer to nanoparticles, such as nanoparticles engineered to have various sizes, shapes and / or porosity to escape from the reticuloendothelial system or to protect the embedded molecules from degradation. Inorganic nanoparticles can be prepared by metals (e.g., iron, gold and silver), inorganic salts or ceramics (e.g., phosphates or carbonates of calcium, magnesium or silicon). The surfaces of these nanoparticles can be coated to promote DNA binding or targeted gene delivery. Magnetic nanoparticles (e.g., supermagnetic iron oxide), fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g., cylindrical fullerenes), quantum dots and supramolecular systems can also be used.

[0279] In some embodiments, the expression vector is administered to the target cell using a cationic lipid (e.g., a cationic liposome). Various types of lipids for gene delivery have been studied, such as lipid nanoemulsions (e.g., a dispersion of an immiscible liquid in another immiscible liquid stabilized by an emulsifier) ​​or solid lipid nanoparticles.

[0280] In some embodiments, the expression vector is applied to the target cell using a delivery vehicle based on a peptide. Delivery vehicles based on peptides can have the advantages of protecting the genetic material to be delivered, targeting specific cell receptors, destroying the endosome membrane and delivering the genetic material to the core. In some embodiments, the expression vector is applied to the target cell using a delivery vehicle based on a polymer. Delivery vehicles based on polymers can include natural proteins, peptides, and / or polysaccharides or synthetic polymers. In one embodiment, delivery vehicles based on polymers include polyethyleneimine (PEI). PEI can condense DNA into positively charged particles, which are combined with anionic cell surface residues and then brought into the cell via endocytosis. In other embodiments, delivery vehicles based on polymers can include synthetic amino derivatives of poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), poly-ornithine, polyarginine, histones, protamine, dendrimers, chitosan, dextran, and / or cationic acrylic polymers. In certain embodiments, polymer-based delivery vehicles may include mixtures of polymers such as PEG and PLL.

[0281] In certain embodiments, the expression vector can be a viral vector suitable for gene therapy. Preferred features of viral gene therapy vectors or gene delivery vectors can include the ability to be repeatedly and stably propagated and purified to high titers; mediate targeted delivery (e.g., to specifically deliver the transgene to a tissue or organ of interest without widely distributing the vector elsewhere); and mediate gene delivery and transgene expression without inducing harmful side effects.

[0282] Several types of viruses (e.g., a nonpathogenic parvovirus known as adeno-associated virus) have been engineered for gene therapy purposes by exploiting viral infection pathways but avoiding subsequent expression of viral genes that could cause replication and toxicity. Such viral vectors can be obtained by deleting all or part of the coding regions from the viral genome, but leaving intact those sequences (e.g., terminal repeats) that may be necessary for functions such as packaging of the vector genome into the viral capsid or integration of the vector nucleic acid (e.g., DNA) into the host chromatin.

[0283] In various embodiments, suitable viral vectors include retroviruses (e.g., type A, type B, type C, and type D viruses), adenoviruses, parvoviruses (e.g., adeno-associated viruses or AAV), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Examples of retroviruses include avian leukosis-sarcoma virus, human T-lymphotropic virus type 1 (HTLV-1), bovine leukemia virus (BLV), lentiviruses, and foamy viruses. Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Viral vectors can be divided into two groups based on their ability to integrate into the host genome – integrating and non-integrating. Oncoretroviruses and lentiviruses can integrate into the host cell chromatin, whereas adenoviruses, adeno-associated viruses, and herpes viruses persist primarily in the nucleus as extrachromosomal episomes.

[0284] In certain embodiments, suitable viral vectors are retroviral vectors. Retrovirus refers to a virus of the Retroviridae family. Examples of retroviruses include tumor retroviruses, such as murine leukemia virus (MLV), and slow viruses, such as human immunodeficiency virus 1 (HIV-1). Retroviral genomes are single-stranded (ss) RNAs, including various genes that may be provided in cis or trans. For example, retroviral genomes may include cis-acting sequences, such as two long terminal repeats (LTRs), and elements for gene expression, reverse transcription, and integration into host chromosomes. Other components include packaging signals (psi or ψ) for specific RNA packaging into newly formed virions and polypurine tracts (PPTs), i.e., the starting site for positive strand DNA synthesis during reverse transcription. In addition, retroviral genomes may include gag, pol, and env genes. The gag gene encodes structural proteins, the pol gene encodes enzymes that accompany ssRNA and reverse transcribe viral RNA into DNA, and the env gene encodes viral envelope. Generally speaking, gag, pol, and env are provided in trans form for viral replication and packaging.

[0285] In certain embodiments, the retroviral vector provided herein can be a lentiviral vector. At least five serogroups or serotypes of lentivirus are identified. Viruses of different serotypes can infect certain cell types and / or hosts differently. For example, lentiviruses include primate retroviruses and non-primate retroviruses. Primate retroviruses include HIV and simian immunodeficiency virus (SIV). Non-primate retroviruses include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and sheep myelin shedding virus. Slow virus or lentiviral vectors can be able to transduce quiescent cells. Like tumor retroviral vectors, the design of lentiviral vectors can be based on separating cis-acting sequences from trans-acting sequences.

[0286] In an exemplary embodiment, the viral vector provided herein is an adeno-associated virus (AAV). AAV is a small replication-deficient non-enveloped animal virus that infects humans and other primate species. It is understood that AAV does not cause human disease and only induces a mild immune response. AAV vectors can also infect both dividing cells and quiescent cells without integrating into the host cell genome.

[0287] The AAV genome is composed of a linear single-stranded DNA of about 4.7kb in length. The genome comprises two open reading frames (ORFs), which are flanked by an inverted terminal repeat (ITR) sequence of about 145bp in length. ITR is composed of a nucleotide sequence (5'ITR) at the 5' end and a nucleotide sequence (3'ITR) at the 3' end, which contains a palindromic sequence. ITR folds to form a T-shaped hairpin structure by complementary base pairing and acts as a primer during the initiation of DNA replication for the second strand synthesis. The two open reading frames encode the rep gene and the cap gene involved in virion replication and packaging. In an exemplary embodiment, the AAV vector provided herein does not contain the rep gene and the cap gene. Such genes can be provided in trans for producing virions as further described below.

[0288] In certain embodiments, the AAV vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein or an antibiotic resistance gene, such as kanamycin or ampicillin. In certain embodiments, the stuffer nucleic acid may be located outside the ITR sequence (e.g., compared to the polynucleotide encoding the therapeutic protein and the regulatory sequence between the 5' ITR sequence and the 3' ITR sequence).

[0289] There are various AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ and AAV-DJ8. These serotypes differ in their tropism or the cell types they infect. AAV can comprise genomes and capsids from a variety of serotypes (e.g., pseudotypes). For example, AAV can comprise a serotype 2 genome (e.g., ITR) encapsulated in a capsid from serotype 5 or serotype 9. Pseudotypes can improve transduction efficiency, as well as change tropism.

[0290] In some embodiments, AAV vectors or AAV viral particles or virions can be used to deliver constructs comprising cell-selective regulatory elements operably linked to polynucleotides encoding therapeutic functional proteins to certain cells, cell types or tissues, and can be performed in vivo, ex vivo or in vitro. In an exemplary embodiment, such AAV vectors are replication-deficient vectors. In some embodiments, the AAV virus is engineered or genetically modified so that it can replicate and generate virions only in the presence of auxiliary factors.

[0291] In certain embodiments, viral vectors can be selected to produce virions with high infectivity but no selectivity for specific cell types. In certain embodiments, viral vectors can be designed to produce virions that infect many different cell types, but transgene expression is enhanced and / or optimized in the cell type of interest (e.g., PV neurons), and transgene expression is reduced and / or minimized in other non-target cell types (e.g., non-PV CNS cells). The differential expression of transgenes in different cell types can be controlled, engineered, or manipulated using different regulatory elements that are selective for one or more cell types. In some cases, one or more regulatory elements operably linked to a polynucleotide encoding a therapeutic protein enhance the selective expression of the polynucleotide in a target cell, target cell type, or target tissue, while the one or more regulatory elements suppress the expression of the transgene in an off-target cell, off-target cell type, or off-target tissue, or confer significantly lower, trace, or statistically lower gene expression in one or more off-target cells, off-target cell types, or off-target tissues.

[0292] In some cases, AAV serotypes that can cross the blood-brain barrier or infect CNS cells are preferred.

[0293] In an exemplary embodiment, the application provides an expression vector designed for delivery by AAV. AAV can be any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ and AAV-DJ8, or chimeric, hybrid or variant AAV. AAV can also be self-complementary AAV (scAAV), wherein "self-complementary" AAV is an AAV in which the coding region has been designed to form an intramolecular double-stranded DNA template. Once infected with such a vector, the two complementary halves of scAAV will associate to form a double-stranded DNA (dsDNA) unit, which is ready to replicate and transcribe immediately, rather than waiting for the synthesis of the second chain under cell mediation. The design of scAAV vectors is described in various publications, including McCarty et al., Gene Therapy 20018:1248–54.

[0294] In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR and a 3'ITR. In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR, a promoter, a construct as described above, and a 3'ITR. In certain embodiments, the expression vector designed for delivery by AAV comprises a 5'ITR, an enhancer, a promoter, a construct as described above, and a 3'ITR.

[0295] Host cells

[0296] In another aspect, the present invention relates to a host cell comprising a nucleic acid box as described above. The host cell can be a bacterial cell, a yeast cell, an insect cell or a mammalian cell. In an exemplary embodiment, the host cell refers to any cell line susceptible to infection by a virus of interest and suitable for in vitro culture.

[0297] In certain embodiments, the host cell provided herein can be used for ex vivo gene therapy purposes. In such embodiments, these cells are transfected with nucleic acid molecules or expression cassettes as described above and then transplanted into a patient or subject. Transplanted cells can have autologous, allogeneic or xenogeneic sources. For clinical applications, cell separation will generally be carried out under good manufacturing practice (GMP) conditions. Before transplantation, cell quality is generally checked and it is determined that there are no microbial contaminants or other contaminants, and then pretreatment can be performed, such as with radiation and / or immunosuppressive treatment. In addition, host cells can be transplanted together with growth factors to stimulate cell proliferation and / or differentiation.

[0298] In certain embodiments, host cells can be used to deliver ex vivo gene therapy into the CNS. Preferably, the cells are eukaryotic cells, such as mammalian cells, including but not limited to humans, non-human primates, such as apes, chimpanzees, monkeys, and orangutans; domesticated animals, including dogs and cats; and livestock, such as horses, cattle, pigs, sheep, and goats; or other mammalian species, including but not limited to mice, rats, guinea pigs, rabbits, hamsters, etc. A person skilled in the art will select a more appropriate cell based on the patient or subject to be transplanted.

[0299] In certain embodiments, the host cell provided herein can be a cell with self-renewal properties and pluripotency properties, such as stem cells or induced pluripotent stem cells. Stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells (MSC) can differentiate into at least one of osteoblasts, chondrocytes, adipocytes or myocytes, and can be separated from any type of tissue. Generally speaking, MSC will be separated from bone marrow, adipose tissue, umbilical cord or peripheral blood. The method of obtaining MSC is well known to those skilled in the art. Induced pluripotent stem cells (also referred to as iPS cells or iPSCs) are a class of pluripotent stem cells that can be directly produced from adult cells. Yamanaka et al. induced iPS cells by transferring Oct3 / 4, Sox2, Klf4 and c-Myc genes into mouse and human fibroblasts and forcing cells to express these genes (WO 2007 / 069666). Thomson et al. subsequently used Nanog and Lin28 instead of Klf4 and c-Myc to produce human iPS cells (WO 2008 / 118820).

[0300] In an exemplary embodiment, the host cell provided herein is a packaging cell. The cell may be an adherent cell or a suspension cell. The packaging cell and the auxiliary vector or virus or DNA construct together provide all the missing functions required for complete replication and packaging of the viral vector in a trans manner.

[0301] Preferably, the packaging cell is a eukaryotic cell, such as a mammalian cell, including cells of apes, humans, dogs and rodents. Examples of human cells are PER.C6 cells (WO01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2) and rhesus fetal lung cells (ATCC CL-160). Examples of non-human primate cells are Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650) or COS-7 cells (ATCC CRL-1651). Examples of dog cells are MDCK cells (ATCC CCL-34). Examples of rodent cells are hamster cells, such as BHK21-F, HKCC cells or CHO cells.

[0302] As an alternative to mammalian sources, the cell line used in the present invention can be derived from avian sources, such as chicken, duck, goose, quail or pheasant. The example of avian cell lines includes avian embryonic stem cells (WO01 / 85938 and WO03 / 076601), immortalized duck retinal cells (WO2005 / 042728) and cells derived from avian embryonic stem cells, including chicken cells (WO2006 / 108846) or duck cells, such as EB66 cell lines (WO2008 / 129058 and WO2008 / 142124).

[0303] In another embodiment, the host cell is an insect cell, such as SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB-Sf21), MG1 cells (BTI-TN-MG1), or High Five TM cells (BTI-TN-5B1-4).

[0304] In certain embodiments, the host cells provided herein comprise a nucleic acid construct (e.g., plasmid) carrying a recombinant AAV vector / genome containing a cassette as described above, and may further comprise one or more additional nucleic acid constructs, such as: (i) a nucleic acid construct encoding a rep gene and a cap gene, but not carrying an ITR sequence (e.g., an AAV helper plasmid); and / or (ii) a nucleic acid construct (e.g., a plasmid) providing adenovirus functions necessary for AAV replication. In an exemplary embodiment, the host cells provided herein comprise: i) a nucleic acid construct or expression vector as described above; ii) a nucleic acid construct encoding an AAV rep gene and a cap gene, which does not carry an ITR sequence; iii) a nucleic acid construct comprising an adenovirus helper gene (as further described below).

[0305] In certain embodiments, the rep gene, the cap gene, and the adenovirus helper gene can be combined on a single plasmid (Blouin V et al., J Gene Med. 2004; 6 (suppl): S223-S228; Grimm D et al., Hum. Gene Ther. 2003; 7: 839-850). Therefore, in another exemplary embodiment, the host cell provided herein comprises: i) a nucleic acid molecule or an expression cassette, and ii) a plasmid encoding the AAV rep gene and the cap gene, which does not carry an ITR sequence and further comprises an adenovirus helper gene. Alternative methods are known. For example, the rep gene, the cap gene, and the adenovirus helper gene do not need to be located on the same plasmid, but can be provided on different plasmids, or the rep gene and the cap gene can be provided on a plasmid different from the adenovirus helper gene.

[0306] In certain embodiments, host cells suitable for large-scale production of AAV vectors are insect cells that can be infected with a combination of recombinant baculoviruses (Urabe et al., Hum. Gene Ther. 2002; 13: 1935-1943). For example, SF9 cells can be co-infected with three baculovirus vectors that express the AAV rep, AAV cap, and AAV vector to be packaged, respectively. The recombinant baculovirus vector will provide the viral helper gene functions required for viral replication and / or packaging.

[0307] Further guidance for constructing and producing virions for gene therapy according to the present invention can be found in the following literature: Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Volume 737, Merten and Al-Rubeai (eds.); 2011 Humana Press (Springer); Gene Therapy. M. Giacca. 2010 Springer-Verlag; Heilbronn R. and Weger S. Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics. In: Drug Delivery, Handbook of Experimental Pharmacology 197; M. Schafer-Korting (ed.), 2010 Springer-Verlag; pp. 143-170; Adeno-Associated Virus: Methods and Protocols. R. O. Snyder and P. Moulllier (eds.), 2011 Humana Press (Springer); Bunning H. et al., Recent developments in adeno-associated virus technology. J. Gene Med. 2008; 10:717-733; and Adenovirus: Methods and Protocols. M. Chillon and A. Bosch (eds.); 3rd edition, 2014 Humana Press (Springer).

[0308] Virus and method of producing virus

[0309] In certain embodiments, the application provides viral particles comprising viral vectors. The terms "virion" and "virion" are used interchangeably herein, and relate to infectious and usually replication-defective viral particles, which contain a viral genome (e.g., a viral expression vector) packaged in a capsid, and depending on the circumstances, for example, for retroviruses, it may be a lipid envelope that wraps the capsid. "Capsid" refers to a structure in which a viral genome is packaged. The capsid is composed of several oligomeric structural subunits, which are composed of proteins. For example, AAV has an icosahedral capsid formed by the interaction of the following three capsid proteins: VP1, VP2, and VP3. In one embodiment, the virion provided herein is a recombinant AAV virion or rAAV virion obtained by packaging an AAV vector in a protein shell.

[0310] In certain embodiments, the recombinant AAV virions provided herein can be prepared by encapsidating an AAV genome derived from a specific AAV serotype in a viral particle formed by a native Cap protein of an AAV corresponding to the same specific serotype. In other embodiments, the AAV viral particles provided herein comprise a viral vector containing the ITRs of a given AAV serotype packaged into proteins from a different serotype. See, e.g., Bunning H et al., J Gene Med 2008; 10: 717-733. For example, a viral vector having ITRs from a given AAV serotype can be packaged into: a) viral particles composed of capsid proteins derived from the same or different AAV serotypes (e.g., AAV2 ITRs and AAV9 capsid proteins; AAV2 ITRs and AAV8 capsid proteins; etc.); b) mosaic viral particles composed of a mixture of capsid proteins from different AAV serotypes or mutants (e.g., AAV2 ITRs with AAV1 and AAV9 capsid proteins); c) chimeric viral particles composed of capsid proteins that have been truncated by domain exchange between different AAV serotypes or variants (e.g., AAV2 ITRs with AAV8 capsid proteins having AAV9 domains); or d) targeted viral particles engineered to display a selective binding domain that is capable of strictly interacting with a target cell-specific receptor (e.g., AAV5 ITRs with AAV9 capsid proteins that have been genetically truncated by insertion of a peptide ligand; or AAV9 capsid proteins that have been genetically modified by coupling a peptide ligand to the capsid surface).

[0311] The technician will appreciate that the AAV virions provided herein may include capsid proteins of any AAV serotype. In one embodiment, the viral particles include capsid proteins of AAV serotypes selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9, which are more suitable for delivery to CNS (M. Hocquemiller et al., Hum Gene Ther 27(7): 478-496(2016)). In a specific embodiment, the viral particles include a nucleic acid construct of the present invention, wherein the 5'ITR sequence and 3'ITR sequence of the nucleic acid construct are AAV2 serotypes, and the capsid protein is AAV9 serotype.

[0312] Many methods are known in the art for producing rAAV virions, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71 (11): 8780-8789) and baculovirus-AAV hybrids. The rAAV production culture for producing rAAV virus particles requires: 1) a suitable host cell, including, for example, a cell line derived from humans, such as HeLa, A549 or 293 cells, or a cell line derived from insects, such as SF-9 (for baculovirus production systems); 2) a suitable helper virus function, which is provided by a wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper function; 3) AAV rep and cap genes, and gene products; 4) a transgene flanking the AAV ITR sequence; and 5) a suitable culture medium and culture medium components for supporting rAAV production.

[0313] In various embodiments, the host cells described herein comprise the following three components: (1) rep and cap genes, (2) genes providing auxiliary functions, and (3) transgenes flanking the ITRs. The AAV rep gene, the AAV cap gene, and the genes providing auxiliary functions can be introduced into the cell by incorporating the genes into a vector (such as a plasmid) and then introducing the vector into the host cell. The rep gene, the cap gene, and the auxiliary function gene can be incorporated into the same plasmid or into different plasmids. In a preferred embodiment, the AAV rep gene and the cap gene are incorporated into one plasmid and the gene providing auxiliary function is incorporated into another plasmid. Various plasmids (e.g., comprising the AAV rep gene and the cap gene, auxiliary functions, or transgenes) that generate host cells for virion production can be introduced into the cell using any suitable method well known in the art. Examples of transfection methods include, but are not limited to, calcium phosphate coprecipitation, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and bioballistic transfection. In certain embodiments, a plasmid providing rep and cap genes, auxiliary functions, and a transgene can be introduced into a cell simultaneously. In another embodiment, a plasmid providing rep and cap genes and auxiliary functions can be introduced into a cell before or after the introduction of a plasmid containing a transgene. In an exemplary embodiment, cells are transfected simultaneously with the following three plasmids (e.g., triple transfection): (1) a plasmid containing a transgene, (2) a plasmid containing an AAV rep gene and a cap gene, and (3) a plasmid containing a gene providing auxiliary functions. Exemplary host cells can be 293, A549, or HeLa cells.

[0314] In other embodiments, one or more of (1) the AAV rep gene and the cap gene, (2) a gene providing an auxiliary function, and (3) a transgene (e.g., a PV selective regulatory element operably linked to a polynucleotide encoding a therapeutic protein disclosed herein) can be carried by the packaging cell in episomal form and / or integrated into the genome of the packaging cell. In one embodiment, the host cell can be a packaging cell in which the AAV rep gene and the cap gene and the auxiliary function are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene. In another embodiment, the host cell is a packaging cell in which the AAV rep gene and the cap gene are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene and a plasmid containing the auxiliary function. In another embodiment, the host cell can be a packaging cell in which the auxiliary function is stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene and a plasmid containing the rep gene and the cap gene. In another embodiment, the host cell can be a production cell line stably transfected with the rep gene and the cap gene, the auxiliary function, and the transgene sequence. Exemplary packaging cells and producer cells can be derived from 293, A549 or HeLa cells.

[0315] In another embodiment, the production cell line is an insect cell line (usually Sf9 cells) infected with a baculovirus expression vector providing Rep protein and Cap protein. This system does not require adenovirus helper genes (Ayuso E et al., Curr. Gene Ther. 2010, 10: 423-436).

[0316] As used herein, the term "cap protein" refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3). Examples of functional activities of cap proteins include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote AAV DNA packaging into capsids (i.e., encapsidation), bind to cell receptors, and promote virion entry into host cells. In principle, any Cap protein can be used in the context of the present invention.

[0317] Cap proteins have been reported to have an effect on the host tropism, cell, tissue or organ specificity, receptor utilization, infection efficiency and immunogenicity of AAV viruses. Therefore, the AAV cap used in rAAV can be selected taking into account, for example, the species of the subject (e.g., human or non-human), the immune status of the subject, the adaptability of the subject to long-term or short-term treatment, or a specific therapeutic application (e.g., treatment of a specific disease or condition, or delivery to a specific cell, tissue or organ). In certain embodiments, the cap protein is derived from an AAV in the group consisting of: AAV1, AAV2, AAV5, AAV8 and AAV9 serotypes. In an exemplary embodiment, the cap protein is derived from AAV9.

[0318] In some embodiments, the AAV Cap used in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion or substitution) of one of the aforementioned AAV caps or their encoding nucleic acids. In some embodiments, the AAV cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% similar to one or more of the aforementioned AAV caps, or has a higher degree of similarity.

[0319] In some embodiments, the AAV cap is chimeric, comprising domains from two, three, four or more of the aforementioned AAV caps. In some embodiments, the AAV cap is a mosaic of VP1, VP2 and VP3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned caps.

[0320] In some embodiments, the AAV cap used in the rAAV virion is engineered to contain a heterologous sequence or other modification. For example, a peptide or protein sequence that confers selective targeting or immune escape can be engineered into a cap protein. Alternatively or in addition, the cap can be chemically modified to allow the surface of the rAAV to be PEGylated (i.e., PEG-modified), which can promote immune escape. The cap protein can also be mutagenized (e.g., to remove its natural receptor binding, or to mask immunogenic epitopes).

[0321] As used herein, the term "rep protein" refers to a polypeptide having at least one functional activity of a native AAV rep protein (e.g., rep 40, 52, 68, 78). Examples of functional activities of rep proteins include any activity associated with the physiological function of the protein, including promoting DNA replication by recognizing, binding and cutting the AAV origin of DNA replication, and DNA helicase activity. Additional functions include regulating transcription from AAV (or other heterologous) promoters, and site-specific integration of AAV DNA into host chromosomes. In a specific embodiment, the AAV rep gene can be from serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAVrh10; more preferably, from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8 and AAV9.

[0322] In some embodiments, the AAV rep protein used in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV rep or its encoding nucleic acid. In some embodiments, the AAV rep is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the aforementioned AAV rep, or has a higher degree of similarity.

[0323] As used herein, the expression "auxiliary function" or "auxiliary gene" refers to the viral protein that AAV relies on for replication. Auxiliary functions include those proteins required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis and AAV capsid assembly. Virus-based auxiliary functions can be derived from any known auxiliary virus, such as adenovirus, herpes virus (except herpes simplex virus type 1) and vaccinia virus. Auxiliary functions include but are not limited to adenovirus E1, E2a, VA and E4, or herpes virus UL5, ULB, UL52 and UL29, and herpes virus polymerase. In a preferred embodiment, the protein that AAV relies on for replication is derived from adenovirus.

[0324] In some embodiments, the viral protein used in the methods of the invention on which AAV relies for replication can be generated by mutagenesis (i.e., by insertion, deletion or substitution) of one of the aforementioned viral proteins or nucleic acids encoding them. In some embodiments, the viral protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% similar to one or more of the aforementioned viral proteins, or has a higher degree of similarity.

[0325] Methods for determining the function of the cap protein, rep protein, and viral proteins upon which AAV depends for replication are well known in the art.

[0326] Host cells for expressing transgenic interest can be grown under conditions sufficient to assemble AAV virions. In certain embodiments, host cells grow a suitable time period to promote AAV virion assembly, and virions are released into culture medium. Generally speaking, cells can grow about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days at most. After about 10 days (or faster, depending on culture conditions and the specific host cells used), production levels are usually significantly reduced. Generally speaking, the incubation time is measured from the point of virus production. For example, with respect to AAV, virus production usually starts when the helper virus function is supplied in a suitable host cell as described herein. Generally speaking, after helper virus infection (or after virus production starts) about 48 hours to about 100 hours, preferably about 48 hours to about 96 hours, preferably about 72 hours to about 96 hours, preferably about 68 hours to about 72 hours harvested cells.

[0327] rAAV production cultures can be grown under a variety of conditions appropriate to the particular host cells utilized (over a wide range of temperatures, for varying lengths of time, etc.). rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent containers such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. rAAV vector production cultures can also comprise suspension-adapted host cells such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems.

[0328] Suitable culture media known in the art can be used to produce rAAV virions. These culture media include, but are not limited to, culture media produced by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), each of which is incorporated herein by reference in its entirety. In certain embodiments, rAAV production culture media can be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, rAAV vectors can be produced under serum-free conditions, which can also be referred to as culture media without animal-derived products.

[0329] After culturing the host cells to allow AAV virions to be produced, the resulting virions can be harvested and purified. In certain embodiments, AAV virions can be obtained from host cells (1) by lysing the host cells in the production culture, and / or (2) from the culture medium of the cells after a certain period of time (preferably 72 hours) of transfection. rAAV virions can be harvested from the production culture in the spent culture medium, provided that the cells are cultured under conditions that cause rAAV virions to be released from intact cells into the culture medium (see, e.g., U.S. Patent No. 6,566,118). Suitable methods for lysing cells are also known in the art, including, for example, multiple freeze-thaw cycles, ultrasonic treatment, microfluidization, and treatment with chemicals (such as detergents and / or proteases).

[0330] After harvest, rAAV virions can be purified. As used herein, the term "purified" includes the following preparations of rAAV virions: it does not have rAAV virions naturally present or at least some other components that may also be present when initially prepared. Therefore, for example, purified rAAV virions can be prepared by enrichment from a source mixture (such as a culture lysate or a production culture supernatant) using separation techniques. Enrichment can be measured in a variety of ways, such as by the ratio of DNA enzyme resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or can be measured relative to a second potential interfering substance (such as a pollutant, including a production culture pollutant or a pollutant in the process, including a helper virus, a culture medium component, etc.) present in the source mixture.

[0331] In certain embodiments, the rAAV production culture harvest can be clarified to remove host cell debris. In some embodiments, the production culture harvest can be clarified using a variety of standard techniques, such as centrifugation or filtration through a filter with a pore size of 0.2 μm or greater (e.g., a cellulose acetate filter or a series of depth filters).

[0332] In certain embodiments, Benzonase TM The rAAV production culture harvest is further processed to digest any high molecular weight DNA present in the production culture. TM Digestion was performed under standard conditions, e.g., with Benzonase at a final concentration of 1 to 2.5 units / ml. TM , the temperature ranges from room temperature to 37°C, and the time period ranges from 30 minutes to several hours.

[0333] In certain embodiments, one or more of the following purification steps may be used to isolate or purify rAAV virions: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating rAAV particles; capture of rAAV by apatite chromatography; heat inactivation of helper virus; capture of rAAV by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and capture of rAAV by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. Methods for purifying rAAV particles are found in, for example, Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143.

[0334] In certain embodiments, purified AAV virions can be dialyzed against PBS, filtered and stored at -80° C. The titer of viral genomes can be determined by quantitative PCR using linearized plasmid DNA as a standard curve (see, e.g., Lock M et al., Hum. Gene Ther. 2010; 21: 1273-1285).

[0335] Pharmaceutical composition

[0336] In certain embodiments, the present application provides a composition comprising the above-mentioned nucleic acid box (e.g., expression cassette, e.g., rAAV comprising an expression cassette) or RNA encoded therefrom (e.g., mRNA) and a pharmaceutically acceptable carrier. In some embodiments, a virion comprising the box and a pharmaceutically acceptable carrier is provided. In an exemplary embodiment, such a composition is suitable for gene therapy applications. The pharmaceutical composition is preferably sterile and stable under manufacturing and storage conditions. The sterile solution can be achieved, for example, by filtering through a sterile filtration membrane.

[0337] The acceptable carriers and excipients in the pharmaceutical composition are preferably non-toxic to the recipient at the dosage and concentration adopted. Acceptable carriers and excipients may include buffers such as phosphates, citrates, HEPES and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran and immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, histidine and lysine; and carbohydrates such as glucose, mannose, sucrose and sorbitol. The pharmaceutical composition of the present disclosure can be administered parenterally in the form of an injectable preparation. A sterile solution or any pharmaceutically acceptable liquid can be used as a vehicle to prepare a pharmaceutical composition for injection. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water and saline.

[0338] The pharmaceutical composition of the present disclosure can be prepared in microcapsules such as hydroxymethylcellulose or gelatin-microcapsules and polymethyl methacrylate microcapsules. The pharmaceutical composition of the present disclosure can also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules. The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a sustained release matrix in which a gene delivery vehicle is embedded.

[0339] The pharmaceutical composition provided herein can be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intracerebral administration, intrathecal administration, intracerebellar medullary cistern administration, intraventricular administration or intraperitoneal administration. The pharmaceutical composition can also be formulated for nose, spray, mouth, aerosol, rectum or vaginal administration, or can also be administered via nose, spray, mouth, aerosol, rectum or vagina. In one embodiment, the pharmaceutical composition provided herein is applied to CNS or cerebrospinal fluid (CSF), that is, by intracerebral parenchymal injection, intrathecal injection, intracerebellar medullary cistern injection or intraventricular injection. Tissue target can be specific, such as CNS specific, or can be a combination of several tissues (for example, muscle tissue and CNS tissue). Exemplary tissues or other targets can include liver, skeletal muscle, myocardium, fat deposits, kidney, lung, vascular endothelium, epithelium, hematopoietic cells, cancer cells, CNS and / or CSF. In a preferred embodiment, the pharmaceutical compositions provided herein are administered to the CNS or CSF by injection, i.e., by intraparenchymal injection, intrathecal injection, intracisternal injection or intraventricular injection. One or more of these methods can be used to administer the pharmaceutical compositions of the present disclosure.

[0340] In certain embodiments, the pharmaceutical compositions provided herein comprise an "effective amount" or a "therapeutically effective amount." As used herein, such an amount refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result.

[0341] The dosage of the pharmaceutical composition of the present disclosure depends on various factors, including route of administration, disease to be treated, and physical characteristics (e.g., age, weight, general health) of the subject. The dosage can be adjusted to provide the best therapeutic response. Generally, the dosage can be an amount that effectively treats the disease without inducing significant toxicity. In certain embodiments, the pharmaceutical composition can be formed in unit doses as needed.

[0342] The pharmaceutical composition of the present disclosure can be applied to a subject in need thereof medically if necessary. In an exemplary embodiment, a single administration is sufficient. In one embodiment, the pharmaceutical composition is suitable for human subjects and is administered by intraparenchymal injection, intrathecal injection, intracerebellomedullary cisterna magna injection, or intraventricular injection. In one embodiment, the pharmaceutical composition is delivered via a peripheral vein by bolus injection. In other embodiments, the pharmaceutical composition is delivered via a peripheral vein by infusion.

[0343] In another aspect, the application also provides a kind of test kit, it comprises nucleic acid molecules, carriers, host cells, virions or pharmaceutical compositions as described herein in one or more containers. Test kit may include instructions or packaging materials, which describe how to apply nucleic acid molecules, carriers, host cells or virions contained in the test kit to patients. The container of the test kit may have any suitable material, such as glass, plastics, metals, etc., and may have any suitable size, shape or configuration. In certain embodiments, the test kit may include one or more ampoules or syringes, wherein accommodating nucleic acid molecules, carriers, host cells, virions or pharmaceutical compositions in the form of suitable liquids or solutions.

[0344] Treatment

[0345] The nucleic acid box of the present invention, expression box, expression vector, viral vector, viral particle or pharmaceutical composition can be used for treating multiple diseases, such as nervous system diseases.In some embodiments, the chemical product, protein or nucleic acid molecule of the present invention can be used for treating or improving one or more symptoms associated with gene mutation or low expression or non-expressed gene in the subject.In certain embodiments, treatment can be via gene therapy treatment subject, wherein gene therapy is directly applied to the subject in need thereof (for example, directly applied to the CNS of the subject), or via injection and / or infusion systemic administration.The therapy can be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intracerebral parenchyma administration, intrathecal administration, intracerebellar medullary cisternae administration, intraventricular administration or intraperitoneal administration, or via nose, spray, mouth, aerosol, rectum or vaginal administration, for example, by intracerebral parenchyma injection, intrathecal injection, intracerebellar medullary cisternae injection or intraventricular injection.Tissue target can be specific, such as CNS specific, or can be a combination of several tissues.

[0346] In any of the embodiments herein, the target cell can be a neural cell, a muscle cell, a heart cell, a skin cell, an immune cell, a hematopoietic cell, a cancer cell, a pancreatic cell, or a kidney cell. In some cases, the target cell can be a neural cell, e.g., a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in some embodiments, the neural cell is a GABAergic cell, e.g., a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or other CNS cells (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0347] In any embodiment, the therapies of the invention can be used to increase the production or expression of a target protein in a cell such as a GABA neuron or a parvalbumin neuron.

[0348] In certain embodiments, the treatments provided herein do not cause adverse reactions in the subject. Treatment with the nucleic acid molecules, expression vectors, pharmaceutical compositions, or virions described herein may cause fewer or less severe adverse reactions in the subject than treatment with a similar gene therapy containing the same transgene linked to a non-parvalbumin neuron-selective regulatory element.

[0349] Sequence Listing

[0350] The following table (Table 1) provides sequences referred to in other parts of this disclosure.

[0351] SEQ ID NOs: 1-17 and 39 provide exemplary liver off-target elements / sequences that may be in the RNA transcript encoded in the nucleic acid cassettes of the present disclosure.

[0352] SEQ ID NOs: 18-34 and 40 provide liver off-target sequences that can be in a nucleic acid cassette (DNA cassette) encoding an RNA transcript (eg, mRNA) containing any of SEQ ID NOs: 1-17 or 39.

[0353] Additional sequences are provided as indicated in Table 1.

[0354]

[0355]

[0356]

[0357]

[0358] Example

[0359] The following examples are given in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are neither intended to limit the scope of what the inventors regard as their invention nor to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to quantities used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example: bp, base pair; kb, kilobase; pl, picoliter; s, second; min, minute; h, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0360] Example 1: Identification of off-target elements

[0361] Selecting components for library generation : Annotated 3' untranslated regions (3'UTRs) of genomic sequences initially curated from the AURA database (UTR Regulatory Activity Atlas; Dassi E, Re A, Leo S, Tebaldi T, Pasini L, Peroni D and Quattrone A. (2014) AURA 2: Empowering discovery of post-transcriptional networks. Translation, 2(1): e27738.) were harvested based on proximity to liver depleted genes according to expression data from the genotype-tissue expression portal. Genes highly expressed in at least one non-liver tissue were screened. 100 genes were selected based on reduced expression in the liver but high expression in at least one other tissue. Using a sliding window distance of 25 bp, the genomic sequence of the 3'UTR associated with each of these 100 genes was split into overlapping 127 base pairs (bp) candidate elements, called "tiles".

[0362] Additional elements were harvested from the functional annotation of the following databases: mammalian genome database (Fantom5, Lizio M et al., Gateways to the FANTOM5 promoter level mammalian expression atlas. Genome Biol 16:22 (2015). 10.1186 / s13059-014-0560-6), microRNA expression and sequence analysis database (mESA, Koray D. Kaya, Karakülah,Cengiz M.Yak1c1er,Aybar C.Acar, Konu, mESAdb: microRNA Expression and Sequence Analysis Database, Nucleic Acids Research, Volume 39, Supplement 1, January 1, 2011, pages D170–D180) and Minatel et al. (Minatel BC, Martinez VD, Ng KW et al., Large-scale discovery of previously undetected microRNAs specific to human liver. Hum Genomics. 2018; 12(1): 16). Each miRNA was selected based on the following criteria: a) maintaining minimal liver expression, b) having maximum expression in at least one other tissue type, and c) having maximum expression in all other tissues. The selection of miRNAs was based on whether their expression in the liver was significantly different from all other tissues based on the Grubbs statistic. Expression was manually checked to ensure that the differential expression of the miRNA was higher relative to other tissues. All miRNAs considered in the above databases were compiled and the redundancy between miRNA names was checked.

[0363] Control Component Pool :The control element pool is made of disclosed miRNA response elements or previously identified elements. These controls are used as benchmarks and diagnostic reference points due to their predictable expression characteristics. The control elements are roughly divided into three categories, including: a) miRNA or 3'UTR with known expression profiles, b) various promoters with known expression profiles, and c) random sequences in promoter positions or 3'UTR. The miRNA, 3'UTR and random sequence controls are driven by the same promoter used in the screening. The promoter controls do not contain other elements and sequences in their 3'UTR regions except those elements and sequences required for amplicon generation and molecular barcode identifiers. Each element in the control pool is assigned a barcode in the 3'UTR of the gene of interest.

[0364] Library composition : miRNAs are represented as tetramer repeats with an 8 bp spacer between given miRNA sequences and comprise 2000 unique elements. Those miRNAs with supporting evidence in the literature or obtained from the FANTOM5 database were constructed into elements containing 1, 2, 3 or 4 copies of a given single element and comprise 350 unique elements.

[0365] Assign unique barcodes to library elements for synthesizing oligo pools: To prepare a designed element library for the synthesis of an oligomer pool, the sequence of each candidate element was checked for the presence of restriction enzyme sites necessary for downstream cloning, and those elements containing these sequences were discarded. Those elements whose sequences contained recognition sites for BsrGI, EcoRI, XbaI, AscI, and KpnI were removed from the library pool. A barcode element was assigned to each of the remaining elements. Each off-target element and its barcode were linked to the flanking sequence for cloning into the vector backbone. The library was synthesized as single-stranded DNA by Twist Biosciences.

[0366] Plasmid library synthesis :The experimental plasmid library is constructed by a pool of single-stranded oligomers, and is connected to a common plasmid backbone. Single-stranded oligomers are amplified using a set of universal primers via PCR, and are then further connected to the 3'UTR of the transgenic in the screening plasmid. Plasmid has the following composition: (minCMV promoter)-(nanoluciferase)-(MCS)-(liver off-target element)-(amplicon barcode and primer site)-(hGH polyA). The library is transformed into electrocompetent Escherichia coli (E.coli) cells, then both plated on agar plates for Sanger sequencing, and also inoculated into 200ml LB liquid cultures. Sanger sequencing is carried out on agar plate colonies, to verify that elements are correctly connected and there is no insertion and deletion. Zymogen Maxiprep test kit (Zymogen) is then used to separate the library from 200mL cultures. Subsequently, the product is incorporated into plasmid library (see " control element pool " part) with control and merged to produce the final library for the production of vectors.

[0367] AAV vector preparation : All vectors were produced in adherent HEK293T cells in DMEM + 10% FBS. Using PEI-MAX, cells were transfected with a library of elements and helper plasmids, including a plasmid containing cis ITRs, a trans plasmid pAAVX encoding AAV2 replication, and the AAVX capsid gene and adenoviral helper plasmid pALD-X80. AAV was harvested from cells, purified from lysates using an iodixanol ultracentrifugation gradient, further polished using an anion exchange column, then concentrated and formulated in PBS with 0.001% pluronic.

[0368] animal : Screening was performed in adult female C57BL / 6J mice (The Jackson Laboratory). Adult mice aged 6 to 8 weeks were obtained and housed on site for 3 days to acclimate to the new environment. Animals were allowed to eat a standard normal chow diet ad libitum and received a 12-hour light / 12-hour dark photoperiod. After injection, mice were housed individually in a feeding room until sacrificed.

[0369] AAV injection : Mice (n=5) were injected by intravenous (IV) tail vein injection and stereotaxic direct hippocampal injection. Animals were treated with Rimodi one day before surgery. Mice received 3E12 gc per animal in a single dose via IV, totaling 1.8E11 gc per animal, spread into the left, right, dorsal, and ventral regions of the hippocampus in four 1.5 μL injections. After injection, animals were incubated with the virus in an animal housing room for 3 weeks.

[0370] Sample collection : Tissues including hippocampus and liver were collected directly into RNAlater (SigmaAldrich). Samples were stored in RNAlater at 4°C for 24 hours and then transferred to -80°C until further processing.

[0371] RNA and cDNA generation RNA was isolated from samples using the RNeasy Mini column method using a standard protocol for isolating total RNA (Qiagen). Total RNA concentration was normalized prior to input into the cDNA reaction. cDNA was generated by reverse transcription using the SuperScript IVVILO kit using an oligo dT primer (Thermo Fisher Scientific).

[0372] Amplicon generation : Using a set of universal primers for all elements in the library, the amplicon derived from the reporter gene is amplified via PCR. For each sample, there are four technical replicates starting from the first amplicon PCR step. The number of cycles of the amplicon PCR is first optimized using qPCR. Step 1 PCR amplifies the 3'UTR region of the reporter gene mRNA under optimized conditions. In brief, AAV is digested with DNaseI (New England Biolabs) to remove the capsid and then used directly in the above step 1 PCR. All technical replicates of each biological replicate are combined in equimolar amounts as the final sequencing library pool.

[0373] Amplicon sequencing : Each sample was combined in equimolar amounts into the final sequencing pool, which included PCR samples of each biological replicate, amplicons from the plasmid pool used to prepare AAV, and amplicons from the quantitative AAV library. The samples were then combined with PhiX at a molar ratio of 60:40 to diversify the sample pool. The diversified library pool was further diluted and prepared according to the instructions of the Nextseq 500High kit (Illumina).

[0374] Candidate selection: Figure 1 A graph showing brain activity versus liver activity for the constructs tested is provided. Figure 1 As shown in Figure 2, many different constructs achieve liver expression reduction while maintaining brain expression, or the reduction degree of liver expression is greater than brain expression. The included negative control does not affect brain expression and liver expression, and the included two positive controls reduce liver expression to a greater extent than brain expression. Construct activity is assessed based on the following: the log2 fold change (log2FC) of the expression activity of the construct in the liver compared with its abundance in the baseline AAV pool, 2) the log2FC of the expression activity of the construct in the hippocampus compared with its abundance in the baseline AAV pool, and 3) the difference between these two log2FC values. DESeq2 is used to calculate tissue log2FC measurements (see below for more details). The brain expression and the log2 fold change of liver expression of the selected construct are also provided in Table 2. The data in Table 2 show that liver expression is more reduced than brain expression, indicating that these elements can make liver expression off-target. As shown in Table 2, the elements provided herein are used alone and when provided as 2x, 3x or 4x tandem repeats, liver expression is off-target.

[0375] Table 2

[0376] describe Log2 fold change in liver Log2 fold change in brain Test sequence 1, 1 copy -1.091138 -0.502499 Test sequence 1, 2 copies -2.034107 -1.074236 Test sequence 1, 3 copies -2.008143 -1.313751 Test sequence 1, 4 copies -1.58071 -1.004799 Test sequence 3, 1 copy -0.149644 -0.115088 Test sequence 3, 2 copies -0.620037 -0.477955 Test sequence 3, 3 copies -0.649259 -0.352649 Test sequence 3, 4 copies -1.204078 -0.907602 Test sequence 4, 4 copies -1.886355 -0.388039 Test sequence 5, 4 copies -1.602891 -0.404599 Test sequence 6, 1 copy -1.191418 -0.151467 Test sequence 6, 2 copies -1.55778 -0.738756 Test sequence 6, 3 copies -0.296954 -0.119777 Test sequence 7, 1 copy -2.151987 -0.492311 Test sequence 7, 2 copies -1.815708 -0.787522 Test sequence 7, 3 copies -0.766551 -0.469931 Test sequence 7, 4 copies -0.986991 -0.333258 Positive control 1 -2.813155 -1.276399 Positive Control 2 -0.791072 -0.443886 Negative control 0.029446 -0.000071

[0377] Another off-target element was identified in a similarly performed screen, test sequence 22 As shown in Table 3, test sequence 22 also off-targeted liver expression when used alone and when provided as 2x, 3x, or 4x tandem repeats.

[0378] Table 3

[0379] describe Log2 fold change in liver Log2 fold change in brain Test sequence 22, 1 copy -1.8481975 -0.4314376 Test sequence 22, 2 copies -2.1350587 -0.3656623 Test sequence 22, 4 copies -2.0649475 0.03460468

[0380] Window scoring of endogenous 3'UTR tiles: Approximately 7,000 constructs in the off-target screen were sequence elements derived from 3'UTR regions of endogenous genes that displayed favorable expression patterns (see the "Selection of endogenous 3'UTR-derived elements" section for more details). Each endogenous 3'UTR region was spanned by overlapping 127 bp sequence "tiles", where each tile was offset by 25 bp from the previous tile. Since adjacent 3'UTR tiles share approximately 80% of their sequence, it is expected that these tiles generally have similar activity distributions; large variations in activity between adjacent tiles may be an indicator of noisy measurements.

[0381] The aggregated "window" activity score for each group of 3 adjacent tiles was calculated by taking the weighted average of the tile's tissue log2FC scores, where tiles were weighted by the inverse of the tile's log2FC variance. Thus, tiles with more adequate measurements (i.e., where the variance of the activity of the 5 constituent barcodes of the construct is smaller) are weighted more heavily to the window average. The window activity score is used to screen for sequence regions that consistently exhibit a favorable activity profile.

[0382] Selection of elements for validation: Several sequences were selected based on low liver expression and maintained expression in the hippocampus for further validation via IHC and ELISA.

[0383] ELISA : Samples for protein analysis were from adult female C57BL / 6J mice. Each mouse was given 5.0E11 vg / mouse of AAV9 via intravenous injection (tail vein), and the incubation period lasted for 3 weeks. The vectors were generated as described in the "AAV vector preparation" section. The GOI of the vector is as follows: (AAV2ITR)-(EF1a (short))-mCherry-KASH-spA-(CTCF insulator)-(CMV promoter)-EGFP-KASH-(liver off-target element)-sPA-(AAV2 ITR). The liver off-target element was selected as described above and is listed as a test sequence in Table 1 above. The left lobe of the liver and one hemisphere of the cortex were collected from PBS-perfused mice, excluding the olfactory bulb, cerebellum and other hindbrain tissues. The samples were placed in buffer PRT (Abcam, ab171581 and ab221829) in a mechanical homogenizer, homogenized with 2.8 mm ceramic beads (OMNI Intl.) at 4°C for 1 minute, and then clarified by centrifugation. Total protein was quantified using Micro BCA (ThermoFisher) and then normalized in buffer PRT. EGFP protein and mCherry protein were quantified separately using ELISA (Abcam) and then quantified based on standard curves generated from each corresponding recombinant protein assayed. Figure 2 As shown, most of the off-target elements tested showed a substantial reduction in liver expression compared to the control.

[0384] Tissue preparation and immunohistochemistry (IHC) staining: After saline perfusion, whole brain and liver tissues were collected, fixed in 4% neutral buffered formalin for 24 hours, then exchanged to 70% ETOH and kept at 4°C until processing. Tissues were processed by an external vendor for formalin fixation and paraffin embedding. After parasagittal embedding of the brain, 5 μm sections were cut from the slides. Two cross-sections of the liver lobe were collected on one slide per animal. For IHC, slides were dewaxed, rehydrated, and then subjected to heat-induced epitope retrieval in citrate pH 6 buffer at 95°C for 20 minutes. Slides were mounted on Shandon slide holders, washed with phosphate-buffered saline containing Tween-20 (PBST), and then incubated in antibody dilution buffer (phosphate-buffered saline, 1.0% bovine serum albumin, 0.3% Triton-X-100) for 15 minutes, followed by incubation with rabbit anti-Myc [Abcam ab9106] at a 1:25,000 dilution in antibody dilution buffer at 4°C overnight. They were then treated with goat anti-rabbit HRP [Thermo A16110] for 1 hour and Opal 520 [Akoya Biosciences FP1487001KT] for 10 minutes. Washes were performed with PBST between each step. Nuclei were stained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) for 15 minutes, followed by coverslips #1.5. Figure 3A and Figure 3B Representative images showing brain and liver expression of mice treated with a control vector without off-target elements, Figure 3C and Figure 3D Images showing brain expression and liver expression of mice treated with a vector encoding RNA containing test sequence 12 (SEQ ID NO: 12).

[0385] Example 2 : In vitro validation of selected liver off-target elements

[0386] Induced pluripotent stem cells (IPSCs) were used to validate liver off-target elements identified in human cells. Adeno-associated virus (AAV) particles were prepared using the AAVDJ serotype. Each viral preparation included a genome that included the EF1a promoter, an enhanced green fluorescent protein coding sequence fused to the KASH domain (eGFP-KASH), and either random sequences or liver off-target elements.

[0387] IPSC-derived glutamatergic neurons and hepatocytes were plated into 24-well plates. After 48 hours, IPSC-derived cells were transduced with different AAV constructs at a multiplicity of infection (MOI) of 5×10^5. The cells were then incubated for another 72 hours and harvested for RNA and DNA extraction. Quantitative polymerase chain reaction was used to determine the level of eGFP-KASH mRNA compared to the internal control (GAPDH). The results of in vitro validation are shown in Table 4.

[0388] Table 4

[0389]

[0390]

[0391] Example 3: Liver off-target in non-human primates

[0392] In order to evaluate the conservation between mice and non-human primates (NHPs), several off-target elements were selected for NHP studies. AAV with different off-target elements was prepared as described above, and was administered at a dose of 10^14 viral genomes / animal by unilateral intracerebroventricular injection, which was pooled into young cynomolgus macaques (n=2) between 16 and 21 months of age. The animals were autopsied about 50 days after treatment, and liver and brain tissues were harvested for DNA and RNA extraction. Using vector-specific primers / probes, AAV-driven transcript expression was evaluated by reverse transcription droplet digital PCR analysis. Total RNA expression was normalized relative to the AAV genome copy number in each sample. Figure 4 The relative expression (log2 of fold change) of several different liver off-target elements in NHP in the liver of each treated animal is shown and averaged. Figure 4 As shown, all tested elements showed reduced expression in the liver compared to the control sequence, and tested sequences 4, 6 and 7 showed liver expression reduced by at most 1 / 2.

[0393] Although the present invention has been described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and may be replaced with equivalents without departing from the true intent and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, methods, method steps to the objectives, essence and scope of the present invention. All such modifications are intended to fall within the scope of the appended claims.

Claims

1. A nucleic acid cassette comprising a therapeutic transgene encoding an mRNA, wherein the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii).

2. The nucleic acid cassette of claim 1, wherein the mRNA comprises a sequence of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 1-17 or 39, which sequence reduces expression in hepatocytes.

3. The nucleic acid cassette according to claim 1 or 2, wherein the mRNA further comprises a second sequence (i), (ii) or (iii).

4. The nucleic acid box according to claim 3, wherein the mRNA further comprises a third sequence (i), (ii) or (iii).

5. The nucleic acid box according to claim 4, wherein the mRNA further comprises a fourth sequence (i), (ii) or (iii).

6. The nucleic acid box of claim 5, wherein the mRNA comprises five or more sequences of (i), (ii) or (iii).

7. The nucleic acid cassette of any one of claims 1 to 6, wherein the mRNA comprises two or more copies of the following sequence: (i), (ii) or (iii).

8. The nucleic acid cassette of claim 7, wherein the mRNA comprises three or more copies of the following sequence: (i), (ii) or (iii).

9. The nucleic acid cassette of claim 8, wherein the mRNA comprises four or more copies of the following sequence: (i), (ii) or (iii).

10. The nucleic acid cassette of claim 9, wherein the mRNA comprises five or more copies of the following sequence: (i), (ii) or (iii).

11. The nucleic acid cassette according to any one of claims 1 to 10, wherein the sequence (i), (ii) or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA. 12 . The nucleic acid cassette of claim 11 , wherein the sequence (i), (ii) or (iii) is located in the 3′UTR region of the mRNA.

13. The nucleic acid cassette of claim 11, wherein the sequence (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

14. The nucleic acid cassette of claim 11, wherein the sequence (i), (ii) or (iii) is located in an intron of the mRNA.

15. The nucleic acid cassette of any one of claims 1 to 14, wherein the nucleic acid cassette is non-naturally occurring.

16. The nucleic acid cassette of any one of claims 1 to 15, wherein the nucleic acid cassette comprises a CNS-selective promoter.

17. The nucleic acid cassette of claim 16, wherein the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

18. The nucleic acid cassette of any one of claims 1 to 17, wherein the nucleic acid cassette comprises an enhancer.

19. The nucleic acid cassette of any one of claims 1 to 18, wherein the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

20. The nucleic acid cassette of claim 19, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (C The patient was diagnosed with SWS, infantile spasms (SWS), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gastau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

21. The nucleic acid cassette of claim 19 or 20, wherein the therapeutic protein is selected from (i) a nucleic acid selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, M EF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

22. The nucleic acid cassette according to any one of claims 1 to 21, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence identical to (i) or (ii) sequences that are at least 80% identical; (b) the nucleic acid cassette comprises a CNS-selective promoter; and (c) The mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

23. The nucleic acid cassette according to any one of claims 1 to 22, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence identical to (i) or (ii) sequences that are at least 80% identical; (b) the nucleic acid cassette comprises a promoter selected from the group consisting of: a Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, a synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; and (c) the mRNA encoding a gene selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

24. The nucleic acid cassette according to any one of claims 1 to 23, wherein the sequence (i), (ii) or (iii) causes reduced expression in hepatocytes of a polypeptide encoded by said mRNA as compared to expression in hepatocytes of said polypeptide from an otherwise identical mRNA but lacking said sequence (i), (ii) or (iii).

25. The nucleic acid box of claim 24, wherein the sequence (i), (ii) or (iii) causes the expression level of the polypeptide encoded by the mRNA in hepatocytes to be reduced to at most 1 / 2, at most 1 / 5 or at most 1 / 10 as compared to the expression level of the polypeptide in hepatocytes from an mRNA that is otherwise identical but does not contain the sequence (i), (ii) or (iii).

26. The nucleic acid box of claim 24, wherein the sequence (i), (ii) or (iii) causes the expression level of the polypeptide encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the polypeptide in hepatocytes from an mRNA that is otherwise identical but does not contain the sequence (i), (ii) or (iii).

27. The nucleic acid cassette according to any one of claims 1 to 26, wherein the sequence (i), (ii) or (iii) does not result in substantially reduced expression in a target cell of a polypeptide encoded by the mRNA as compared to the expression in a target cell of the polypeptide from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

28. The nucleic acid cassette of claim 27, wherein the sequence (i), (ii) or (iii) does not reduce expression of the polypeptide encoded by the mRNA in the target cell as compared to expression of the polypeptide in the target cell from an mRNA that is otherwise identical but lacks the sequence (i), (ii) or (iii).

29. The nucleic acid cassette of claim 27, wherein the sequence (i), (ii) or (iii) causes expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the polypeptide in a target cell from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

30. The nucleic acid cassette of any one of claims 27 to 29, wherein the target cell is a neural cell.

31. The nucleic acid cassette of claim 30, wherein the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

32. The nucleic acid cassette of claim 31, wherein the neural cell is a GABAergic cell.

33. The nucleic acid cassette of claim 32, wherein the GABAergic cell is a cell expressing parvalbumin.

34. The nucleic acid cassette of any one of claims 1 to 33, wherein the nucleic acid cassette is a linear construct or a vector.

35. The nucleic acid cassette of claim 34, wherein the vector is a plasmid.

36. The nucleic acid cassette of claim 34, wherein the vector is a viral vector.

37. The nucleic acid cassette of claim 36, wherein the viral vector is an adeno-associated virus (AAV) vector.

38. The nucleic acid cassette of claim 37, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

39. The nucleic acid cassette of claim 37 or 38, wherein the AAV is a scAAV.

40. The nucleic acid cassette of claim 36, wherein the viral vector is a lentiviral vector.

41. An mRNA having a sequence encoded by the nucleic acid cassette of any one of claims 1 to 40.

42. A nucleic acid cassette comprising a transgene encoding an mRNA, wherein the mRNA encodes a therapeutic protein and comprises a miRNA binding site for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or a complement thereof.

43. The nucleic acid box of claim 42, comprising miRNA binding sites for two or more miRNAs selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or their complementary sequences.

44. The nucleic acid box of claim 42, comprising miRNA binding sites for three or more miRNAs selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or their complementary sequences.

45. The nucleic acid box of any one of claims 42 to 44, comprising two miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or their complementary sequences.

46. ​​The nucleic acid box of claim 45, comprising three miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or their complementary sequences.

47. The nucleic acid box of claim 46, comprising four miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p or a complementary sequence thereof.

48. The nucleic acid box of claim 46, comprising more than four miRNA binding sites for a miRNA selected from miR-22-3p, miR-1258-5p, miR-5589-3p, miR-17-5p, miR-203a, miR-122-3p, miR-93-5p, miR-122-5p, or a complementary sequence thereof.

49. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-22-3p.

50. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-1258-5p.

51. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-5589-3p.

52. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-17-5p.

53. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-203a.

54. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-122-3p.

55. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-93-5p.

56. The nucleic acid cassette of any one of claims 42 to 48, wherein the miRNA is miR-122-5p.

57. The nucleic acid cassette of any one of claims 42 to 56, wherein the mRNA further comprises a sequence of at least 10 contiguous nucleotides of any one of SEQ ID NOs: 12-17 that reduces expression in hepatocytes.

58. The nucleic acid cassette of any one of claims 42 to 56, wherein the mRNA further comprises at least two sequences of at least 20 contiguous nucleotides of any one of SEQ ID NOs: 12-17 that reduce expression in hepatocytes.

59. The nucleic acid cassette of any one of claims 42 to 58, wherein the miRNA binding site is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

60. The nucleic acid cassette of claim 59, wherein the miRNA binding site is located in the 3'UTR region of the mRNA.

61. The nucleic acid cassette of claim 59, wherein the miRNA binding site is located in the 5'UTR region of the mRNA.

62. The nucleic acid cassette of claim 59, wherein the miRNA binding site is located in an intron of the mRNA.

63. The nucleic acid cassette of any one of claims 42 to 62, wherein the nucleic acid cassette is non-naturally occurring.

64. The nucleic acid cassette of any one of claims 42 to 63, wherein the nucleic acid cassette comprises a promoter, optionally comprising a neural selective promoter.

65. The nucleic acid cassette of claim 64, wherein the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

66. The nucleic acid cassette of any one of claims 42 to 65, wherein the nucleic acid cassette comprises an enhancer.

67. The nucleic acid cassette of any one of claims 42 to 66, wherein the mRNA encodes a therapeutic protein.

68. The nucleic acid cassette of claim 67, wherein the therapeutic protein is a protein associated with a neurological disease or disorder, optionally Alpers- Hattenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, Fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSWS), infantile spasms (Weiss syndrome syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

69. The nucleic acid cassette of claim 68, wherein the therapeutic transgene is selected from (a) a nucleic acid selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, (a) a protein encoded by a gene encoding MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (b) a protein having at least 90% sequence identity to (a), (c) a functional fragment of (a) or (b), or (d) a transcription factor that activates the expression of a gene from (a).

70. The nucleic acid cassette of any one of claims 42 to 69, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii); (b) the nucleic acid cassette comprises a CNS-selective promoter; and (c) The mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

71. The nucleic acid cassette of claim 70, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of: a Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, a synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; and (c) the mRNA encoding a gene selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

72. The nucleic acid cassette of any one of claims 42 to 71, wherein the miRNA binding site causes reduced expression in hepatocytes of a polypeptide encoded by the mRNA as compared to expression in hepatocytes of the polypeptide from an otherwise identical mRNA but lacking the miRNA binding site.

73. The nucleic acid box of claim 72, wherein the miRNA binding site causes the expression level of a polypeptide encoded by the mRNA in hepatocytes to be reduced by at most 1 / 2, at most 1 / 5, or at most 1 / 10 as compared to the expression level of the polypeptide in hepatocytes from an otherwise identical mRNA but without the miRNA binding site.

74. The nucleic acid box of claim 73, wherein the miRNA binding site causes at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% decrease in the expression level of a polypeptide encoded by the mRNA in a hepatocyte compared to the expression level of the polypeptide in a hepatocyte from an otherwise identical mRNA but without the miRNA binding site.

75. The nucleic acid cassette of any one of claims 42 to 74, wherein the sequences (i), (ii) or (iii) does not result in substantially reduced expression in a target cell of a polypeptide encoded by the mRNA as compared to the expression in a target cell of the polypeptide from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

76. The nucleic acid cassette of claim 75, wherein the sequence (i), (ii) or (iii) does not reduce expression of the polypeptide encoded by the mRNA in the target cell as compared to expression of the polypeptide in the target cell from an mRNA that is otherwise identical but lacks the sequence (i), (ii) or (iii).

77. The nucleic acid box of claim 75, wherein the sequence (i), (ii) or (iii) causes expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the polypeptide in a target cell from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

78. The nucleic acid cassette of any one of claims 42 to 76, wherein the target cell is a neural cell.

79. The nucleic acid cassette of claim 78, wherein the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

80. The nucleic acid cassette of claim 79, wherein the neural cell is a GABAergic cell.

81. The nucleic acid cassette of claim 80, wherein the GABAergic cell is a cell expressing parvalbumin.

82. The nucleic acid cassette of any one of claims 42 to 81, wherein the nucleic acid cassette is a linear construct or a vector.

83. The nucleic acid box of claim 82, wherein the vector is a plasmid.

84. The nucleic acid cassette of claim 82, wherein the vector is a viral vector.

85. The nucleic acid cassette of claim 84, wherein the viral vector is an adeno-associated virus (AAV) vector.

86. The nucleic acid cassette of claim 85, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

87. The nucleic acid cassette of claim 85 or 86, wherein the AAV is a scAAV.

88. The nucleic acid cassette of claim 85, wherein the viral vector is a lentiviral vector.

89. An mRNA encoded by the nucleic acid cassette of any one of claims 42 to 88.

90. The nucleic acid cassette of any one of claims 42 to 88, wherein the mRNA encodes a polypeptide.

91. The nucleic acid cassette of claim 90, wherein the polypeptide is a therapeutic protein.

92. An mRNA having a sequence encoded by the nucleic acid cassette of any one of claims 42 to 91.

93. A method for reducing liver expression of a therapeutic protein encoded by an mRNA while maintaining expression of the therapeutic protein in a target tissue, the method comprising applying the following sequences: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii) in the mRNA.

94. The method of claim 93, wherein the mRNA further comprises a second sequence (i), (ii) or (iii).

95. The method of claim 93, wherein the mRNA further comprises a third sequence (i), (ii) or (iii).

96. The method of claim 93, wherein the mRNA further comprises a fourth sequence (i), (ii) or (iii).

97. The method of claim 93, wherein the mRNA comprises five or more of the sequences of (i), (ii) or (iii).

98. The method of any one of claims 93 to 97, wherein the mRNA comprises two or more copies of the following sequence: (i), (ii) or (iii).

99. The method of any one of claims 93 to 98, wherein the mRNA comprises three or more copies of the following sequence: (i), (ii) or (iii).

100. The method of any one of claims 93 to 99, wherein the mRNA comprises four or more copies of the following sequence: (i), (ii) or (iii).

101. The method of any one of claims 93 to 100, wherein the mRNA comprises five or more copies of the following sequence: (i), (ii) or (iii).

102. The method of any one of claims 93 to 98, wherein the mRNA comprises at least 10 contiguous nucleotides of any one of SEQ ID NOs: 1-17 or 39 that reduce expression in hepatocytes.

103. The method according to any one of claims 93 to 102, wherein the sequence (i), (ii) or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

104. The method of claim 103, wherein the sequence (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

105. The method of claim 103, wherein the sequence (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

106. The method of claim 103, wherein the sequence (i), (ii) or (iii) is located in an intron of the mRNA.

107. The method of any one of claims 93 to 106, wherein the method comprises administering to a subject a nucleic acid encoding the mRNA.

108. The method of any one of claims 93 to 107, wherein the administration is systemic administration.

109. The method of any one of claims 93 to 107, wherein the administration is topical administration.

110. The method of claim 109, wherein the nucleic acid is administered topically into the brain or CNS tissue.

111. The method of claim 109 or 110, wherein the administration is carried out by intraparenchymal, intrathecal, intracisternal, intraventricular or intracranial administration.

112. The method of any one of claims 93 to 111, wherein the therapeutic protein is a protein associated with a neurological disease or disorder.

113. The method of any one of claims 93 to 112, wherein the therapeutic protein is selected from (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2 , MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity to (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

114. The method of any one of claims 107 to 113, wherein the subject suffers from a neurological disease or disorder.

115. The method of claim 114, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

116. The method of any one of claims 107 to 115, wherein the nucleic acid cassette comprises a CNS-selective promoter.

117. The method of claim 116, wherein the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox Dlx5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

118. The method of any one of claims 107 to 117, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii); (b) the nucleic acid cassette comprises a CNS-selective promoter; and (c) The mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

119. The method of claim 118, wherein: (a) the mRNA comprises the following sequence: (i) any one of SEQ ID NO: 1-17 or 39, (ii) a variant, a functional fragment or a combination thereof, or (iii) a sequence that is at least 80% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of: a Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, a synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; and (c) the mRNA encoding a gene selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX, (ii) a protein having at least 90% sequence identity with (i), (iii) a functional fragment of (i) or (ii), or (iv) a transcription factor that activates the expression of a gene from (i).

120. The method according to any one of claims 93 to 119, wherein the sequence (i), (ii) or (iii) causes the expression level of the protein encoded by the mRNA in hepatocytes to be reduced by at most 1 / 2, at most 1 / 5 or at most 1 / 10 as compared to the expression level of the protein in hepatocytes from an mRNA that is otherwise identical but does not contain the sequence (i), (ii) or (iii).

121. The method according to any one of claims 93 to 119, wherein the sequence (i), (ii) or (iii) causes the expression level of the protein encoded by the mRNA in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the protein in hepatocytes from otherwise identical mRNA but without the sequence (i), (ii) or (iii).

122. The method according to any one of claims 93 to 121, wherein the sequence (i), (ii) or (iii) does not result in the expression of a protein encoded by the mRNA in a target cell that is substantially reduced compared to the expression of the protein in a target cell from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

123. The method of claim 122, wherein the sequence (i), (ii) or (iii) does not reduce expression of the protein encoded by the mRNA in the target cell as compared to expression of the protein in the target cell from an mRNA that is otherwise identical but lacks the sequence (i), (ii) or (iii).

124. The method of claim 123, wherein the sequence (i), (ii) or (iii) causes expression of a protein encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the protein in the target cell from an otherwise identical mRNA but lacking the sequence (i), (ii) or (iii).

125. The method of any one of claims 93 to 124, wherein the target cell is a neural cell.

126. The method of claim 125, wherein the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

127. The method of claim 126, wherein the neural cell is a GABAergic cell.

128. The method of claim 127, wherein the GABAergic cells are cells expressing parvalbumin.

129. The method of any one of claims 93 to 128, wherein the mRNA is expressed from a nucleic acid cassette.

130. The method of claim 129, wherein the nucleic acid cassette is a linear construct.

131. The method of claim 129, wherein the nucleic acid cassette is a vector.

132. The method of claim 131, wherein the vector is a plasmid.

133. The method of claim 131, wherein the vector is a viral vector.

134. The method of claim 133, wherein the viral vector is an adeno-associated virus (AAV) vector.

135. The method of claim 134, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

136. The method of claim 134 or 135, wherein the AAV is scAAV.

137. The method of claim 133, wherein the viral vector is a lentiviral vector.

138. The method of any one of claims 131 to 137, further comprising administering the vector to a subject.

139. The method of any one of claims 93 to 138, further comprising administering the mRNA to a subject.

140. The method of any one of claims 129 or 130, wherein the administration comprises intraparenchymal administration, intrathecal administration, intracisterna magna administration, or intraventricular administration.

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