Elements for detargeting gene expression in the liver

Abstract

The present disclosure provides sequences that reduce expression of an operably linked transgene in hepatocytes. In some embodiments, the sequences can be used in gene therapy vectors to detarget expression of a therapeutic transgene in hepatocytes of a subject.

Description

[Technical field]

[0001] (cross reference) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 331,680, filed April 15, 2022, and U.S. Provisional Patent Application No. 63 / 412,119, filed September 30, 2022, which are incorporated herein by reference.

[0002] Incorporation by reference of a sequence listing provided as a sequence listing XML file The Sequence Listing is provided herewith as Sequence Listing XML, ENCO-005WO_SEQ_LIST, created on April 13, 2023, and having a size of 39,274 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0003] Introduction Gene therapy has great potential for the treatment of human diseases, especially those with underlying genetic causes. In some gene therapy strategies, a therapeutic payload can be recombinantly expressed in target cells that lack or have reduced amounts of essential proteins or have dysfunctional versions of essential proteins. Expression of the therapeutic payload in the cells rescues those cells, thereby treating the disease. In one example, Tay-Sachs disease (which is recessively inherited and caused by mutations in the HEXA gene located on chromosome 15) can be successfully treated by expressing a functional version of hexA in the brain using adeno-associated virus (AAV) gene therapy.

[0004] One of the challenges in gene therapy is how to deliver therapeutic payload to certain tissues and not to others.For example, some therapeutic payloads that have positive effects in one tissue may have adverse effects in another tissue.Therefore, administering gene therapy that targets diseased cells in one tissue may cause side effects in another tissue.In some cases, even the clinical use of gene therapy may be limited by its off-site effects rather than on-site effects.

[0005] In view of the above, there is a general need for tools to increase the tissue specificity of gene therapy. Summary of the Invention

[0006] Provided herein, inter alia, are nucleic acid cassettes comprising a transgene encoding an mRNA, wherein the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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). These sequences reduce expression of the transgene in hepatocytes (e.g., compared to target cells such as neurons), and thus can be used in various gene therapy strategies to target cells not present in the liver. In certain embodiments, incorporation of one or more of these sequences results in an improved safety profile of the gene therapy by reducing or eliminating toxicity to hepatocytes caused by expression of the transgene in these cells.

[0007] In some embodiments, the nucleic acid cassette is an expression cassette, and the expression cassette may include, in operable linkage, a promoter, a coding sequence, a sequence encoding (i), (ii) or (iii) (also referred to as a liver detargeting element), and a terminator. In some embodiments, at least one sequence present in the expression cassette is heterologous to another one of the sequences in the expression cassette. For example, in some embodiments, an expression cassette of the present disclosure includes a promoter that is heterologous to the operably linked coding sequence. In some embodiments, the expression cassette may further include an enhancer and / or an intron.

[0008] In some embodiments, the promoter of the expression cassette may be selective for cells in a particular tissue (eg, a target tissue such as the brain), but may also drive transgene expression in the liver. In some embodiments, the promoter may be a CNS-selective promoter, such as a promoter selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0009] In any embodiment, the sequence may be in the 3'UTR, 5'UTR, or an intron of the mRNA.

[0010] In any embodiment, the expression cassette may encode a therapeutic protein, such as 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 is 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, myoclonin 1 / EF (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 expression of a gene from (i).

[0011] In some embodiments, the RNA transcript may comprise a combination of sequences of (i), (ii), and (iii).

[0012] Also provided is a vector comprising a cassette as summarized above. The vector may be a plasmid or a viral vector, such as an adeno-associated virus (AAV) or lentiviral vector.

[0013] Also provided is an AAV or lentiviral particle or cell that contains a cassette as summarized above (which, when packaged, may be in single-stranded form).

[0014] Also provided is an RNA encoded by the cassettes summarized above.

[0015] Various methods are also provided. In some embodiments, the methods can be used to express a protein. In these embodiments, the methods can include introducing an expression cassette as summarized above or an mRNA encoded thereby into an organism, wherein the sequence reduces expression of the protein in liver cells of the organism.

[0016] Provided herein is a nucleic acid cassette comprising a therapeutic transgene encoding an mRNA, wherein the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

[0017] In some embodiments, the mRNA comprises a sequence of at least 15 consecutive nucleotides of any of SEQ ID NOs: 1-17 or 39 that reduces expression in hepatocytes.

[0018] In some embodiments, the mRNA further comprises a second sequence of (i), (ii), or (iii).

[0019] In some embodiments, the mRNA further comprises a third sequence of (i), (ii), or (iii).

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

[0021] In some embodiments, the mRNA comprises five or more sequences of (i), (ii), or (iii).

[0022] In some embodiments, the mRNA comprises two or more copies of the sequence of (i), (ii) or (iii).

[0023] In some embodiments, the mRNA comprises three or more copies of the sequence of (i), (ii) or (iii).

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

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

[0026] In some embodiments, the sequence of (i), (ii), or (iii) is located in one or more of the 3'UTR region of an mRNA, the 5'UTR of an mRNA, or an intron of an mRNA.

[0027] In some embodiments, the sequence of (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

[0028] In some embodiments, the sequence of (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

[0029] In some embodiments, the sequence of (i), (ii) or (iii) is located in an intron of the mRNA.

[0030] In some embodiments, the nucleic acid cassette is non-naturally occurring.

[0031] In some embodiments, the nucleic acid cassette comprises a CNS-selective promoter.

[0032] In some embodiments, the CNS selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0033] In some embodiments, the nucleic acid cassette comprises an enhancer.

[0034] In some embodiments, the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

[0035] 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 effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), epilepsy of infancy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy continuous spike and wave during sleep sleep, CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures isolated, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hypermotor epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0036] In some embodiments, the therapeutic protein is selected from the group consisting of: (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, PC (ii) a protein encoded by a gene selected from DH19, 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; (iii) 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 expression of a gene from (i).

[0037] In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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.

[0038] In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii), and the nucleic acid cassette is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 The promoter is selected from the group consisting of α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter, and the mRNA is 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) encoding a transcription factor that activates expression of a gene from (i).

[0039] In some embodiments, the sequence of (i), (ii) or (iii) results in decreased expression of a polypeptide encoded by the mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0040] In some embodiments, the sequence of (i), (ii) or (iii) results in a reduction in expression of a polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0041] In some embodiments, the sequence of (i), (ii) or (iii) results in reduced expression of a polypeptide encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0042] In some embodiments, the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in a target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0043] In some embodiments, the sequence of (i), (ii) or (iii) does not reduce expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0044] In some embodiments, the sequence of (i), (ii) or (iii) results in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0045] In some embodiments, the target cell is a neuronal cell.

[0046] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0047] In some embodiments, the neuronal cell is a GABAergic cell.

[0048] In some embodiments, the GABAergic cell is a parvalbumin-expressing cell.

[0049] In some embodiments, the nucleic acid cassette is a linear construct or a vector.

[0050] In some embodiments, the vector is a plasmid.

[0051] In some embodiments, the vector is a viral vector.

[0052] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0053] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0054] In some embodiments, the AAV is a scAAV.

[0055] In some embodiments, the viral vector is a lentiviral vector.

[0056] Also provided is an mRNA having a sequence encoded by the nucleic acid cassettes summarized above.

[0057] Also provided is a nucleic acid cassette comprising a transgene encoding an mRNA, the mRNA encoding a therapeutic protein and comprising 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 complements thereof.

[0058] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0059] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0060] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0061] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0062] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0063] In some embodiments, the nucleic acid cassette may contain 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 complements thereof.

[0064] In some embodiments, the miRNA is miR-22-3p.

[0065] In some embodiments, the miRNA is miR-1258-5p.

[0066] In some embodiments, the miRNA is miR-5589-3p.

[0067] In some embodiments, the miRNA is miR-17-5p.

[0068] In some embodiments, the miRNA is miR-203a.

[0069] In some embodiments, the miRNA is miR-122-3p.

[0070] In some embodiments, the miRNA is miR-93-5p.

[0071] In some embodiments, the miRNA is miR-122-5p.

[0072] In some embodiments, the mRNA additionally comprises a sequence of at least 10 consecutive nucleotides of any of SEQ ID NOs: 12-17, which reduces expression in hepatocytes.

[0073] In some embodiments, the mRNA additionally comprises at least two sequences of at least 20 consecutive nucleotides of any of SEQ ID NOs: 12-17, which reduces expression in hepatocytes.

[0074] In some embodiments, the miRNA binding site is located in one or more of the 3'UTR region of an mRNA, the 5'UTR of an mRNA, or an intron of an mRNA.

[0075] In some embodiments, the miRNA binding site is located in the 3'UTR region of the mRNA.

[0076] In some embodiments, the miRNA binding site is located in the 5'UTR region of the mRNA.

[0077] In some embodiments, the miRNA binding site is located in an intron of the mRNA.

[0078] In some embodiments, the nucleic acid cassette is non-naturally occurring.

[0079] In some embodiments, the nucleic acid cassette comprises a promoter, optionally a neuroselective promoter.

[0080] In some embodiments, the CNS selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0081] In some embodiments, the nucleic acid cassette comprises an enhancer.

[0082] In some embodiments, the mRNA encodes a therapeutic protein.

[0083] In some embodiments, the therapeutic protein is a therapeutic agent for treating a neurological disease or disorder, optionally including Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, Fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy centrotemporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic lid myoclonia Jeavons syndrome, epilepsy of infancy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy during sleep continuous spike and wave CSW S, infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douz syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0084] In some embodiments, the therapeutic transgene is (a) 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, (b) a protein encoded by a gene selected from 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 expression of a gene from (a).

[0085] In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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. In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii), and the nucleic acid cassette is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 The promoter is selected from the group consisting of α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter, and the mRNA is 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, a therapeutic protein encoded by a gene selected from 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) encoding a transcription factor that activates expression of a gene from (i).

[0086] In some embodiments, the miRNA binding site results in decreased expression of a polypeptide encoded by the mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the miRNA binding site.

[0087] In some embodiments, the miRNA binding site results in a reduction in expression of a polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the polypeptide in the hepatocyte from an otherwise equivalent mRNA that does not contain the miRNA binding site.

[0088] In some embodiments, the miRNA binding site results in reduced expression of a polypeptide encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not contain the miRNA binding site.

[0089] In some embodiments, the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in a target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0090] In some embodiments, the sequence of (i), (ii) or (iii) does not reduce expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0091] In some embodiments, the sequence of (i), (ii) or (iii) results in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0092] In some embodiments, the target cell is a neuronal cell.

[0093] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0094] In some embodiments, the neuronal cell is a GABAergic cell.

[0095] In some embodiments, the GABAergic cell is a parvalbumin-expressing cell.

[0096] In some embodiments, the nucleic acid cassette is a linear construct or a vector.

[0097] In some embodiments, the vector is a plasmid.

[0098] In some embodiments, the vector is a viral vector.

[0099] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0100] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0101] In some embodiments, the AAV is a scAAV.

[0102] In some embodiments, the viral vector is a lentiviral vector.

[0103] Also provided is an mRNA encoded by the nucleic acid cassette of any of the embodiments summarized above.

[0104] In some embodiments, the mRNA encodes a polypeptide.

[0105] In some embodiments, the polypeptide is a therapeutic protein.

[0106] Also provided is an mRNA having a sequence encoded by the nucleic acid cassette of any of the embodiments summarized above.

[0107] Also provided is a method of reducing hepatic expression of a therapeutic protein encoded by an mRNA while maintaining expression of the therapeutic protein in a target tissue, the method comprising including in the mRNA a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

[0108] In some embodiments, the mRNA further comprises a second sequence of (i), (ii), or (iii).

[0109] In some embodiments, the mRNA further comprises a third sequence of (i), (ii), or (iii).

[0110] In some embodiments, the mRNA further comprises a fourth sequence of (i), (ii), or (iii).

[0111] In some embodiments, the mRNA comprises five or more sequences of (i), (ii), or (iii).

[0112] In some embodiments, the mRNA comprises two or more copies of the sequence of (i), (ii) or (iii).

[0113] In some embodiments, the mRNA comprises three or more copies of the sequence of (i), (ii) or (iii).

[0114] In some embodiments, the mRNA comprises four or more copies of the sequence of (i), (ii) or (iii).

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

[0116] In some embodiments, the mRNA comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1-17 or 39, which reduces expression in hepatocytes.

[0117] In some embodiments, the sequence of (i), (ii), or (iii) is located in one or more of the 3'UTR region of an mRNA, the 5'UTR of an mRNA, or an intron of an mRNA.

[0118] In some embodiments, the sequence of (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

[0119] In some embodiments, the sequence of (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

[0120] In some embodiments, the sequence of (i), (ii) or (iii) is located in an intron of the mRNA.

[0121] In some embodiments, the methods include administering to a subject a nucleic acid encoding the mRNA.

[0122] In some embodiments, the administering is systemic administration.

[0123] In some embodiments, the administering is a local administration.

[0124] In some embodiments, the nucleic acid is administered locally to the brain or CNS tissue.

[0125] In some embodiments, administering by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0126] In some embodiments, the therapeutic protein is a protein associated with a neurological disease or disorder.

[0127] In some embodiments, the therapeutic protein is selected from the group consisting of: (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, PC (ii) a protein encoded by a gene selected from DH19, 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; (iii) 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 expression of a gene from (i).

[0128] In some embodiments, the subject has a neurological disease or disorder.

[0129] In some embodiments, the subject is diagnosed with Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), epilepsy of infancy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep continuous spike and wave (CSWS), infantile spasms ( Patients with: West syndrome), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention-deficit hyperactivity disorder.

[0130] In some embodiments, the nucleic acid cassette comprises a CNS-selective promoter.

[0131] In some embodiments, the CNS selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0132] In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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.

[0133] In some embodiments, the mRNA comprises a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii), and the nucleic acid cassette is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 The promoter is selected from the group consisting of α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter, and the mRNA is 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) encoding a transcription factor that activates expression of a gene from (i).

[0134] In some embodiments, the sequence of (i), (ii) or (iii) results in a reduction in expression of the protein encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the protein in a hepatocyte from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0135] In some embodiments, the sequence of (i), (ii) or (iii) results in reduced expression of the protein encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the protein in hepatocytes from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0136] In some embodiments, the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of the protein encoded by the mRNA in a target cell compared to expression of the protein in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0137] In some embodiments, the sequence of (i), (ii) or (iii) does not reduce expression of a protein encoded by the mRNA in a target cell compared to expression of the protein in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0138] In some embodiments, the sequence of (i), (ii) or (iii) results in expression of a protein encoded by the mRNA in a target cell at a level that is at least 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 of the protein in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0139] In some embodiments, the target cell is a neuronal cell.

[0140] In some embodiments, the neural cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0141] In some embodiments, the neuronal cell is a GABAergic cell.

[0142] In some embodiments, the GABAergic cell is a parvalbumin-expressing cell.

[0143] In some embodiments, the mRNA is expressed from a nucleic acid cassette.

[0144] In some embodiments, the nucleic acid cassette is a linear construct.

[0145] In some embodiments, the nucleic acid cassette is a vector.

[0146] In some embodiments, the vector is a plasmid.

[0147] In some embodiments, the vector is a viral vector.

[0148] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0149] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0150] In some embodiments, the AAV is a scAAV.

[0151] In some embodiments, the viral vector is a lentiviral vector.

[0152] In some embodiments, the method further comprises administering the vector to a subject.

[0153] In some embodiments, the method further comprises administering the mRNA to the subject.

[0154] In some embodiments, administering comprises intraparenchymal, intrathecal, intracisternal, or intraventricular administration.

[0155] Other features, advantages, and embodiments will become apparent in view of the following description. [Brief description of the drawings]

[0156] [Figure 1] FIG. 1 is a scatter plot showing the log2 change in brain vs. liver activity for several test constructs. [Diagram 2] Graph showing the results of ELISA assays of selected constructs. [Figure 3A] Representative images showing in vivo expression of various constructs in brain and liver are shown, as well as representative images of brain expression from mice treated with a control vector that does not contain a detargeting element. [Figure 3B] Representative images showing in vivo expression of various constructs in brain and liver are shown, as well as representative images of liver expression from mice treated with a control vector that does not contain a detargeting element. [Figure 3C] Representative images showing in vivo expression of various constructs in brain and liver are shown. Representative images of brain expression from mice treated with vectors encoding RNA containing test sequence 12 (SEQ ID NO: 12) are shown. [Figure 3D] Representative images showing in vivo expression of various constructs in brain and liver are shown. Representative images of liver expression from mice treated with vectors encoding RNA containing test sequence 12 (SEQ ID NO: 12) are shown. [Figure 4] FIG. 1 is a graph showing relative expression (log2 fold change) in liver for several different liver detargeting elements in NHPs.

[0157] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0158] The term "AAV" is an abbreviation for adeno-associated virus and may 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(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8, and their hybrids (i.e., chimeric AAV vectors), as well as avian, bovine, canine, equine, primate, non-primate, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. As used herein, "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeat (ITR) sequences. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV). See, e.g., Raj et al., Expert Rev Hematol. 2011 Oct;4(5):539-549. "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector.When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as a "rAAV viral particle" or simply a "rAAV particle." AAV can contain genome components and capsids from multiple serotypes (e.g., pseudotyped vectors). For example, AAV can contain a serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotyped vectors can demonstrate improved transduction efficiency as well as modified tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred. In some embodiments, the recombinant AAV vector is AAV1, AAV8, AAV9, AAVDJ, or a chimeric AAV that contains features of two or more 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 heterologous nucleic acid flanked by ITRs from an AAV serotype other than AAV 9. In certain embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector and comprises heterologous nucleic acid flanked by ITRs of AAV serotype 2 (i.e., ITR2).

[0159] 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 how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or 2 or more standard deviations, as per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0160] In any of the embodiments described herein, "comprising" can be replaced with "consisting essentially of" or "consisting of." For example, embodiments in which certain elements are included using the open-ended term "comprising" encompass embodiments in which the elements are included using the more restrictive term "consisting essentially of" or "consisting of."

[0161] The terms "determine," "measure," "assess," "estimate," "assay," "analyze," and their grammatical equivalents are used interchangeably herein to refer to any form of measurement, including determining whether an element is present (e.g., detecting). These terms include both quantitative and / or qualitative determinations. Estimating can be relative or absolute.

[0162] The term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from, for example, genomic DNA that contains introns or splice sites, expression may include splicing of the mRNA in a eukaryotic cell.

[0163] "Expression cassette" refers to a nucleic acid molecule containing one or more regulatory elements operably linked to a coding sequence (eg, one or more genes) for expression.

[0164] "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, e.g., an mRNA, or a functional RNA, e.g., an antisense RNA. In some embodiments, the transgene of the present disclosure encodes a therapeutic cargo, e.g., a therapeutic protein or a therapeutic RNA, and also includes one or more liver detargeting sequences / elements to reduce expression of the transgene in hepatocytes.

[0165] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition described herein that is sufficient to affect the intended application, including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending on the intended therapeutic application (intracellular or in vivo), or the subject and disease state to be treated, such as the subject's weight and age, the severity of the disease state, the mode of administration, etc., which can be easily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells. The specific dose will vary depending on the particular 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 through which it is delivered.

[0166] A "fragment" of a nucleotide or peptide sequence is meant to refer to a sequence that is shorter than what is considered to be the "full-length" sequence.

[0167] A "functional fragment" of a DNA, RNA, or protein sequence refers to a biologically active fragment of a sequence that is shorter than a full-length or reference DNA, RNA, or protein sequence, but that retains at least one biological activity (either functional or structural) that is 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 expression of a transgene to which it is operably linked in hepatocytes.

[0168] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0169] The term "human-derived" as used herein refers to a sequence found in the human genome (or human genome build) or a sequence homologous thereto. A homologous sequence can be a sequence having a region with at least 80% sequence identity (e.g., as measured by BLAST) compared to a 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 of human origin. In some cases, a regulatory element contains human-derived and non-human-derived sequences such that a portion of the regulatory element has 100% sequence identity (or local sequence identity) to a sequence in the human genome while the entire regulatory element has low sequence identity to the human genome.

[0170] The term "in vitro" refers to an event that occurs outside of a subject's body. For example, an in vitro assay includes any assay that is performed outside of a subject. An in vitro assay includes cell-based assays in which live or dead cells are used. An in vitro assay also includes cell-free assays in which no intact cells are used.

[0171] The term "in vivo" refers to events that take place inside a subject's body.

[0172] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally, at a chromosomal location that is different from its natural chromosomal location, or contains only the coding sequence.

[0173] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents, refer to the juxtaposition of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, and the like, which are in a relationship permitting them to operate in the expected manner. For example, a regulatory element, which may include a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

[0174] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation or composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0175] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered.

[0176] 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, coding sequence, or 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 repeats (LTRs), stability elements, miRNA target sites, post-translational response elements, or polyA sequences, or combinations thereof. Regulatory elements can function at the DNA and / or RNA level, for example, by regulating gene expression at the transcriptional, post-transcriptional, or translational phases of gene expression; by regulating the level of translation (e.g., stabilizing elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination; by recruiting transcription factors to the coding region 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 of protein synthesis from the RNA transcript; and / or by preventing RNA degradation and / or increasing its stability to promote protein synthesis. In exemplary embodiments, regulatory elements refer to enhancers, repressors, promoters, or combinations thereof, particularly enhancer+promoter combinations or repressor+promoter combinations. In exemplary embodiments, the regulatory elements are derived from human sequences.

[0177] Generally, "sequence identity" or "sequence homology", which may be used interchangeably, refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity", also called "percent homology". Percent identity to a reference sequence (e.g., a nucleic acid or amino acid sequence) can be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence, multiplied by 100. Conservative substitutions are not considered as matches when determining the number of matches for sequence identity. It will be understood that if 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 purposes of estimating percent identity, can be performed by any suitable alignment algorithm or program, including, but not limited to, the Needleman-Wunsch algorithm, the BLAST algorithm, the Smith-Waterman algorithm (see, e.g., EMBOSS Water aligner), and the Clustal Omega alignment program (F. Sievers et al., Mol Sys Biol. 7:539 (2011)). Optimal alignment can be estimated using any suitable parameters of the selected algorithm, including default parameters. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), 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).

[0178] 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 may be useful in human therapy, veterinary applications, and / or preclinical research in animal models of a disease or condition.

[0179] As used herein, the terms "treat", "treatment", "therapy" and the like refer to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, mitigating, delaying or slowing the progression, reducing the impact or symptoms, preventing the onset, preventing recurrence, inhibiting the onset of a disease or disorder, ameliorating, obtaining a beneficial or desired outcome with respect to a disease, disorder or medical condition, e.g., a therapeutic benefit and / or a prophylactic benefit. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to or at risk of having the disease, but who has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., halting its development, and (c) relieving the disease, i.e., causing regression of the disease. Therapeutic benefits include eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by eradicating or improving one or more of the physiological symptoms associated with the underlying disease, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disease. In some cases, for prophylactic benefit, the composition is administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of the disease, even if the disease has not been diagnosed. The methods of the present disclosure can be used in any mammal. In some cases, treatment can result in a reduction or cessation of symptoms. Prophylactic benefit includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0180] A "variant" of a nucleotide sequence refers to a sequence that has genetic modifications or mutations compared to the most common wild-type DNA sequence (e.g., a cDNA or sequence referenced by its GenBank Accession Number) or a designated reference sequence. A variant may be shorter than the reference sequence and / or may have one or more mutations relative to the 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 the reference sequence.

[0181] As used herein, a "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 to a cell where it can be replicated or expressed. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is 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.

[0182] 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. A non-target cell is a cell in which expression of the RNA or protein product of a nucleic acid is undesirable. As used herein, "detargeting" generally refers to reducing expression in a non-target cell.

[0183] Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art, and the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology that are within the knowledge of those of ordinary skill in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0184] Detailed Description Before describing the present invention in more detail, it is to be understood that the invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0185] The upper and lower limits of the ranges may be independently included in the range and are also encompassed in the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0186] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in which the publications are cited.

[0187] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" includes a plurality of such proteins, a reference to a "nucleic acid" includes a reference to one or more nucleic acids and equivalents thereof known to those of skill in the art, and so forth. It is further noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0188] It is recognized that certain features of the invention, which are described for clarity in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the invention are specifically embraced by the invention and are disclosed herein just as if each and every combination were individually or explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and are disclosed herein just as if such subcombinations were individually or explicitly disclosed herein.

[0189] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0190] As summarized above, the present disclosure describes a nucleic acid cassette comprising a transgene encoding an RNA, wherein the RNA comprises (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. The transgene may encode an mRNA, or a non-coding RNA, such as a priMicroRNA, preMicroRNA, or MicroRNA, a short non-coding RNA, a long non-coding RNA, a snoRNA, a snRNA, a tRNA, or a rRNA. In some cases, the nucleic acid cassette comprises a transgene encoding an mRNA, wherein the mRNA comprises (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. These sequences reduce expression of the transgene in liver cells compared to target cells (such as neural cells, e.g., neurons), and thus can be used in various gene therapy strategies to target cells not present in the liver. Reducing the expression of transgene in hepatocytes compared to target cells means that the reduction of transgene expression driven by the liver detargeting sequence disclosed herein is greater in hepatocytes than in target cells.Thus, in certain embodiments, a reduction in transgene expression can be observed in target cells, but it is less than that observed in hepatocytes.This reduction in expression in liver can reduce or eliminate liver toxicity in subjects undergoing gene therapy that targets non-hepatic cells or tissues, such as neural cells, e.g., neurons, thereby improving their safety profile.

[0191] The present disclosure further provides an RNA molecule having a sequence characteristic of the RNA encoded by any of the nucleic acid cassettes described herein. In certain embodiments, the RNA is modified to increase its stability and / or activity when administered to a subject, for example, as a pharmaceutical composition. The RNA composition finds use in a variety of therapeutic modalities delivered using a wide range of viral and non-viral delivery systems, the latter including polymeric materials, ionizable lipids, membrane-permeable lipids and zwitterionic lipids, nanoparticles, and dendrimers (see, for example, Kowalski et al., "Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery," Molecular Therapy 2019 v.27(4) pp.710-728 and Paunovska et al. "Drug delivery systems for RNA therapeutics," Nature Reviews genetics 2022) v23 pp.265-280).

[0192] The RNA (e.g., mRNA) encoded by the transgene of the nucleic acid cassette may contain any combination of two, three, four, or five or more of the sequences. For example, an RNA comprising a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant or functional fragment 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) may further comprise a second sequence of (i), (ii) or (iii), a third sequence of (i), (ii) or (iii), a fourth sequence of (i), (ii) or (iii), and / or five or more sequences of (i), (ii) or (iii). In any embodiment, the nucleic acid cassette may include two or more copies (eg, two, three, four, five, or more than five copies) of the sequence of (i), (ii), or (iii).

[0193] In any embodiment, the sequence may be, for example, in the 3'UTR, 5'UTR, or intron of the mRNA. If the mRNA contains more than one of the sequences, the sequences may be in different parts of the mRNA. However, in many embodiments, the sequence is in the 3'UTR of the mRNA. In these embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant or functional fragment 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) may be located in one or more of the 3'UTR region of the mRNA, the 5'UTR of the mRNA, or an intron of the mRNA.

[0194] Any nucleic acid described herein may be non-naturally occurring, and the term "non-naturally occurring" refers to a composition that does not occur in nature. A non-naturally occurring nucleic acid may contain a sequence of nucleotides that is different from the nucleic acid in its natural state (i.e., has less than 100% sequence identity to a naturally occurring nucleic acid sequence). When two portions of a nucleic acid are "heterologous", they are not portions of the same nucleic acid in its natural state. For example, in some embodiments, a nucleic acid cassette may be composed of a promoter, a coding sequence, and a terminator, and the promoter, coding sequence, and terminator are operably linked. In these embodiments, the promoter may be heterologous to the coding sequence, meaning that the promoter does not drive the expression of the coding sequence in wild-type cells. In any embodiment, the nucleic acid cassette may additionally include an enhancer.

[0195] In any embodiment, the RNA encoded by the transgene of the nucleic acid cassette may comprise a functional fragment of 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 of SEQ ID NOs: 1-17 or 39, which may or may not contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mismatches to SEQ ID NOs: 1-17 or 39. Such fragments may begin anywhere in SEQ ID NO: 1-17 or 39, for example, at position 1, 21, 41, 61, 81, 101, or 121.

[0196] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 1, 5, 7, or 10 that is 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 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 for its complete representation in SEQ ID NO: 1, 5, 7, or 10.Thus, a functional fragment of 10 consecutive nucleotides may start at any one of nucleotides 1 to 13 of SEQ ID NO: 1, 5, 7, or 10, a functional fragment of 11 consecutive nucleotides may start at any one of nucleotides 1 to 12 of SEQ ID NO: 1, 5, 7, or 10, a functional fragment of 12 consecutive nucleotides may start at any one of nucleotides 1 to 11 of SEQ ID NO: 1, 5, 7, or 10, a functional fragment of 13 consecutive nucleotides may start at any one of nucleotides 1 to 10 of SEQ ID NO: 1, 5, 7, or 10, a functional fragment of 14 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO: 1, 5, 7, or 10, and a functional fragment of 15 consecutive nucleotides may start at any one of nucleotides 1 to 8 of SEQ ID NO: 1, 5, 7, or 10. a functional fragment of 16 contiguous nucleotides may start at any one of nucleotides 1-7 of SEQ ID NO: 1, 5, 7, or 10; a functional fragment of 17 contiguous nucleotides may start at any one of nucleotides 1-6 of SEQ ID NO: 1, 5, 7, or 10; a functional fragment of 18 contiguous nucleotides may start at any one of nucleotides 1-5 of SEQ ID NO: 1, 5, 7, or 10; a functional fragment of 19 contiguous nucleotides may start at any one of nucleotides 1-4 of SEQ ID NO: 1, 5, 7, or 10; a functional fragment of 20 contiguous nucleotides may start at any one of nucleotides 1-3 of SEQ ID NO: 1, 5, 7, or 10; and a functional fragment of 21 contiguous nucleotides may start at nucleotide 1 or 2 of SEQ ID NO: 1, 5, 7, or 10.

[0197] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO:2 that is 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 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 for its complete representation in SEQ ID NO:2. Thus, a functional fragment of 10 consecutive nucleotides may start at any one of nucleotides 1 to 10 of SEQ ID NO:2, a functional fragment of 11 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO:2, a functional fragment of 12 consecutive nucleotides may start at any one of nucleotides 1 to 8 of SEQ ID NO:2, a functional fragment of 13 consecutive nucleotides may start at any one of nucleotides 1 to 7 of SEQ ID NO:2, a functional fragment of 14 consecutive nucleotides may start at any one of nucleotides 1 to 6 of SEQ ID NO:2, a functional fragment of 15 consecutive nucleotides may start at any one of nucleotides 1 to 5 of SEQ ID NO:2, a functional fragment of 16 consecutive nucleotides may start at any one of nucleotides 1 to 4 of SEQ ID NO:2, a functional fragment of 17 consecutive nucleotides may start at any one of nucleotides 1 to 3 of SEQ ID NO:2 and a functional fragment of 18 consecutive nucleotides may start at nucleotide 1 or 2 of SEQ ID NO:2.

[0198] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 3 or 8 that is 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 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 with any nucleotide in SEQ ID NO: 3 or 8 that allows for its complete representation in SEQ ID NO: 3 or 8. Thus, a functional fragment of 10 consecutive nucleotides may start at any one of nucleotides 1 to 12 of SEQ ID NO: 3 or 8, a functional fragment of 11 consecutive nucleotides may start at any one of nucleotides 1 to 11 of SEQ ID NO: 3 or 8, a functional fragment of 12 consecutive nucleotides may start at any one of nucleotides 1 to 10 of SEQ ID NO: 3 or 8, a functional fragment of 13 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO: 3 or 8, a functional fragment of 14 consecutive nucleotides may start at any one of nucleotides 1 to 8 of SEQ ID NO: 3 or 8, a functional fragment of 15 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO: 3 or 8, a functional fragment of 16 consecutive nucleotides may start at any one of nucleotides 1 to 8 of SEQ ID NO: 3 or 8, , a functional fragment of 16 contiguous nucleotides may start at any one of nucleotides 1 to 7 of SEQ ID NO: 3 or 8, a functional fragment of 17 contiguous nucleotides may start at any one of nucleotides 1 to 5 of SEQ ID NO: 3 or 8, a functional fragment of 18 contiguous nucleotides may start at any one of nucleotides 1 to 4 of SEQ ID NO: 3 or 8, a functional fragment of 19 contiguous nucleotides may start at any one of nucleotides 1 to 3 of SEQ ID NO: 3 or 8, and a functional fragment of 20 contiguous nucleotides may start at nucleotide 1 or 2 of SEQ ID NO: 3 or 8.

[0199] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 4 or 39 that is 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 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 with any nucleotide in SEQ ID NO: 4 or 39 that allows for its complete representation in SEQ ID NO: 4 or 39.Thus, a functional fragment of 10 consecutive nucleotides may start at any one of nucleotides 1 to 14 of SEQ ID NO: 4 or 39, a functional fragment of 11 consecutive nucleotides may start at any one of nucleotides 1 to 13 of SEQ ID NO: 4 or 39, a functional fragment of 12 consecutive nucleotides may start at any one of nucleotides 1 to 12 of SEQ ID NO: 4 or 39, a functional fragment of 13 consecutive nucleotides may start at any one of nucleotides 1 to 11 of SEQ ID NO: 4 or 39, a functional fragment of 14 consecutive nucleotides may start at any one of nucleotides 1 to 10 of SEQ ID NO: 4 or 39, a functional fragment of 15 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO: 4 or 39, a functional fragment of 16 consecutive nucleotides may start at any one of nucleotides 1 to 12 of SEQ ID NO: 4 or 39, a functional fragment of 17 consecutive nucleotides may start at any one of nucleotides 1 to 13 of SEQ ID NO: 4 or 39, a functional fragment of 18 consecutive nucleotides may start at any one of nucleotides 1 to 14 of SEQ ID NO: 4 or 39, a functional fragment of 19 consecutive nucleotides may start at any one of nucleotides 1 to 15 of SEQ ID NO: 4 or 39, a functional fragment of 20 consecutive nucleotides may start at any one of nucleotides 1 to 16 of SEQ ID NO: 4 or 39, a functional fragment of 21 consecutive nucleotides may start at any one of nucleotides 1 to 17 of SEQ ID NO: 4 or 39, a functional fragment of 22 consecutive nucleotides may start at any one of nucleotides 1 to 18 of SEQ ID NO: 4 or 39, a functional fragment may start at any one of nucleotides 1-8 of SEQ ID NO: 4 or 39, a functional fragment of 17 contiguous nucleotides may start at any one of nucleotides 1-7 of SEQ ID NO: 4 or 39, a functional fragment of 18 contiguous nucleotides may start at any one of nucleotides 1-6 of SEQ ID NO: 4 or 39, a functional fragment of 19 contiguous nucleotides may start at any one of nucleotides 1-5 of SEQ ID NO: 4 or 39, a functional fragment of 20 contiguous nucleotides may start at any one of nucleotides 1-4 of SEQ ID NO: 4 or 39, a functional fragment of 21 contiguous nucleotides may start at any one of nucleotides 1-3 of SEQ ID NO: 4 or 39, and a functional fragment of 22 contiguous nucleotides may start at nucleotide 1 or 2 of SEQ ID NO: 4 or 39.

[0200] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 6, 9, or 11 that is 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 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 with any nucleotide in SEQ ID NO: 6, 9, or 11 that allows for its complete representation in SEQ ID NO: 6, 9, or 11. Thus, a functional fragment of 10 consecutive nucleotides may start at any one of nucleotides 1 to 11 of SEQ ID NO: 6, 9, or 11, a functional fragment of 11 consecutive nucleotides may start at any one of nucleotides 1 to 10 of SEQ ID NO: 6, 9, or 11, a functional fragment of 12 consecutive nucleotides may start at any one of nucleotides 1 to 9 of SEQ ID NO: 6, 9, or 11, a functional fragment of 13 consecutive nucleotides may start at any one of nucleotides 1 to 8 of SEQ ID NO: 6, 9, or 11, and a functional fragment of 14 consecutive nucleotides may start at any one of nucleotides 1 to 7 of SEQ ID NO: 6, 9, or 11. a functional fragment of 15 contiguous nucleotides may start at any one of nucleotides 1 to 6 of SEQ ID NO: 6, 9, or 11; a functional fragment of 16 contiguous nucleotides may start at any one of nucleotides 1 to 5 of SEQ ID NO: 6, 9, or 11; a functional fragment of 17 contiguous nucleotides may start at any one of nucleotides 1 to 4 of SEQ ID NO: 6, 9, or 11; a functional fragment of 18 contiguous nucleotides may start at any one of nucleotides 1 to 3 of SEQ ID NO: 6, 9, or 11; and a functional fragment of 19 contiguous nucleotides may start at nucleotide 1 or 2 of SEQ ID NO: 6, 9, or 11.

[0201] In certain embodiments, the functional fragment is 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 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 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least Also 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, 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 127,12-17 that is 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, at least 142, at least 143, at least 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 length. In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NOs: 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, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NOs: 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 is 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, at least 204, at least 205, at least 206, at least 207, at least 208, at least 209, at least 210, at least 211, at least 212, at least 213, at least 214, at least 215,The functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 14 or 17 that is at least 216, at least 217, at least 218, at least 219, at least 220, at least 221, at least 222, at least 223, at least 224, at least 225, or at least 226 nucleotides in length. In certain embodiments, the functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO: 14 that is 227 nucleotides in length. In certain embodiments, a functional fragment of any one of SEQ ID NOs: 12-17 contains 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 its complete representation in SEQ ID NOs: 12-17.

[0202] In any embodiment, the RNA may contain one or more binding sites for a miRNA. In these embodiments, the RNA may contain 6, 7, 8, 9, or 10 consecutive nucleotides at the 3' end of any of SEQ ID NOs: 1-11, which potentially base-pair with the seed region of a miRNA (at the 5' end of these miRNAs), 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. In some embodiments, the RNA may contain 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 complements thereof. In some embodiments, the RNA may contain a binding site for a miRNA produced from the mir-22, mir-1258, mir-5589, mir-17, mir-203a, mir-93, or mir-122 gene. The one or more binding sites for the miRNA may include any of SEQ ID NOs: 1-11 or 39. In some embodiments, the sequence may be identical to SEQ ID NOs: 1-11 or 39, except for having, for example, one, two, three, or four mismatches to SEQ ID NOs: 1-11 or 39.

[0203] As will be apparent, the nucleic acid cassette itself (which is DNA) may contain (i) any of SEQ ID NOs: 18-34 or 40, (ii) a variant, functional fragment, or 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 expression of the protein or RNA encoded by the cassette in liver cells within the organism compared to a target tissue, e.g., neuronal tissue, e.g., neuronal cells within the brain.

[0204] In some embodiments, the mRNA may encode a polypeptide, e.g., a therapeutic protein, which may be, e.g., intracellular, membrane-bound or secreted.

[0205] A transgene that encodes an mRNA that encodes a therapeutic protein may be referred to herein as a therapeutic transgene. In some embodiments, a therapeutic protein is one that is associated with a neurological disease or disorder, e.g., a protein whose abnormal function (e.g., resulting from a genetic mutation or abnormality) is associated with a neurological disease or disorder.

[0206] Neurological diseases and disorders include those associated with one or more genetic mutations and those with 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 Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy central temporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic eyelid myoclonia Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy during sleep continuous spike and wave CSWS, infantile spasms (West syndrome) group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douz syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0207] Several genetic abnormalities are associated with epilepsy, including many of the neurological diseases and disorders mentioned above. Examples of genes affected by these genetic abnormalities, i.e., genes whose activity and / or expression are altered by 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, HCN2 ... These include CN4, 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.

[0208] Thus, in embodiments in which the mRNA encodes a therapeutic protein for the treatment of a neurological disease or disorder, the therapeutic protein may be selected from the group consisting of: (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, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, A functional protein encoded by a gene selected from the group consisting of 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 variant or functional fragment of (i) or (ii), or (iv) a transcription factor that activates 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.

[0209] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant or functional fragment 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) may result in reduced expression of a polypeptide encoded by an mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii). For example, an mRNA containing a sequence of (i), (ii) or (iii) may result in a reduction in expression of the polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold, compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii). In these embodiments, the reduction in expression of the polypeptide in a hepatocyte is greater than the reduction in expression of the polypeptide in a target cell when compared to an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0210] In some embodiments, (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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), is a polypeptide that is expressed in hepatocytes from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii), or (iii). The mRNA may result in a reduction in expression of the polypeptide encoded by the mRNA in hepatocytes to a level 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% lower than the expression of the mRNA of the target cell. In these embodiments, the reduction in expression of the polypeptide in hepatocytes is greater than the reduction in expression of the polypeptide in the target cell when compared to an otherwise equivalent mRNA that does not contain the sequence of (i), (ii), or (iii).

[0211] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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) does not result in a significant decrease in expression of a polypeptide encoded by an mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii), or (iii). In some embodiments, the sequence of (i), (ii) or (iii) may result in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (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 the target cell when compared to an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0212] In some cases, the target cell may be a neuronal cell, a muscle cell, a cardiac 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 may be a neuronal cell, such as a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in these embodiments, the neuronal cell may be a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may 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 did not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0213] The cassette may be linear, circular, and in some embodiments, the nucleic acid cassette may be a plasmid or a viral vector, such as an adeno-associated viral (AAV) vector or a lentiviral vector. In certain embodiments, the viral vector may 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, and AAV-DJ8, and hybrids thereof.

[0214] Also provided is a nucleic acid cassette comprising a transgene encoding an RNA, the RNA comprising 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 complements thereof. In some embodiments, the RNA may comprise a binding site for a miRNA produced from the mir-22, mir-1258, mir-5589, mir-17, or mir-203a, mir-93, or mir-122 gene. The miRNA binding site should not be in the naturally occurring version of the RNA if the RNA is otherwise naturally occurring. In some embodiments, the cassette may contain two or more, three or more, or four or more binding sites for a miRNA 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 complements thereof. 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 site may be anywhere within the mRNA, particularly within a non-coding sequence such as the 3'UTR region, the 5'UTR, an intron, or any combination thereof.

[0215] In any embodiment, the nucleic acid cassette may not be naturally occurring, meaning, for example, that the miRNA binding site may be heterologous to the mRNA. In any embodiment, the nucleic acid cassette may include a promoter and / or an enhancer. In some embodiments, the nucleic acid cassette may be composed of a promoter, a coding sequence, and a terminator, where the promoter, the coding sequence, and the terminator are operably linked. In these embodiments, the promoter may be heterologous to the coding sequence, meaning that the promoter does not drive the expression of the coding sequence in wild-type cells. In any embodiment, the nucleic acid cassette may additionally include an enhancer.

[0216] In some embodiments, the mRNA may encode a polypeptide, e.g., a therapeutic protein, which may be, e.g., intracellular, membrane-bound or secreted.

[0217] In some embodiments, the protein is a protein associated with a neurological disease or disorder.

[0218] Neurological diseases and disorders include those associated with one or more genetic mutations and those with unknown etiology. Broadly defined, neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy central temporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic eyelid myoclonia Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy during sleep continuous spike and wave CSWS, infantile spasms (West syndrome) group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douz syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0219] Several genetic abnormalities are associated with epilepsy, including many of the neurological diseases and disorders mentioned above. Examples of genes affected by these genetic abnormalities, i.e., genes whose activity and / or expression are altered by 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, HCN2 ... These include CN4, 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.

[0220] Thus, in embodiments in which the mRNA encodes a therapeutic protein for the treatment of a neurological disease or disorder, the therapeutic protein may be selected from the group consisting of: (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, GBA1, GRIN2A, GRIN2B, GRN, 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 expression of a gene from (i). The transcription factor encoded by the mRNA may be an engineered transcription factor or a naturally occurring transcription factor. In some embodiments, (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), may result in reduced expression of a polypeptide encoded by an mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).For example, an mRNA containing a sequence of (i), (ii) or (iii) may result in a reduction in expression of a polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold, compared to the expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0221] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii) may result in reduced expression of a polypeptide encoded by the mRNA in a hepatocyte at a level that is 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% lower than expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii), or (iii).

[0222] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii) does not result in a decrease in expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii), or (iii). In some embodiments, the sequence of (i), (ii), or (iii) does not reduce expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii), or (iii). In these embodiments, the sequence of (i), (ii) or (iii) can result in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii). In any of these embodiments, the target cell can be a neuronal cell, such as a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell. For example, in these embodiments, the neuronal cell can be a GABAergic cell, such as a parvalbumin-expressing cell.

[0223] The cassette may be linear, circular, and in some embodiments, the nucleic acid cassette may be a plasmid or a viral vector, such as an adeno-associated viral (AAV) vector or a lentiviral vector, In certain embodiments, the viral vector may be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, and scAAV.

[0224] Also provided is an mRNA encoded by the above-described nucleic acid cassette.

[0225] Also provided are methods of reducing hepatic expression of a polypeptide encoded by an mRNA compared to expression of the polypeptide in a target tissue. In these embodiments, the methods can include constructing a nucleic acid cassette to include a liver detargeting sequence as described herein in the RNA encoded therein. For example, a nucleic acid cassette can be constructed to include in the mRNA encoded therein a sequence of (i) one of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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). Details of the cassettes produced by the methods are described herein. In some embodiments, the methods may include introducing an expression cassette described herein or an mRNA encoded thereby into an organism, e.g., a human subject, wherein inclusion of any one or more of the liver detargeting sequences reduces expression of the protein in liver cells of the organism relative to the target tissue.

[0226] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), results in a reduction in expression of a polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii). In these embodiments, the sequence of (i), (ii) or (iii) may result in reduced expression of a polypeptide encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

[0227] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii) does not result in a significant decrease in expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii), or (iii). For example, the sequence of (i), (ii), or (iii) may not decrease expression of a polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii), or (iii). For example, in some embodiments, the sequence of (i), (ii) or (iii) may result in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

[0228] In any embodiment herein, the target cell can be a nerve 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 nerve cell, such as a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.For example, in some embodiments, the nerve cell is a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may 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 cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0229] In any embodiment, the method may further include administering to the subject a vector (e.g., an AAV or lentiviral vector) encoding an mRNA, e.g., the mRNA encodes a therapeutic protein. In some embodiments, the method may include administering to the subject an mRNA.

[0230] Expression cassette The nucleic acid cassette may include one or more additional regulatory elements (e.g., promoters, terminators, and / or enhancers) that induce expression of the transgene in a particular cell type, or a particular class of cell types. For example, a cell type selective regulatory element may induce gene expression in a particular cell type compared to one or more other cell types. Alternatively, or in addition, a cell type selective regulatory element may induce gene expression in a particular class of cells compared to one or more other classes of cells. In one embodiment, a cell type selective regulatory element of the invention enhances gene expression in a particular cell type, or a particular class of cells. In another embodiment, a cell type selective regulatory element suppresses gene expression in a particular cell type, or a particular class of cells. Cell type selective modulation of gene expression (e.g., enhancing or suppressing gene expression) does not require that gene expression be affected only in the target cell type or class of cells. Rather, cell type selective modulation of gene expression (e.g., enhancing or suppressing gene expression) requires only an increase or decrease in gene expression in the target cell type compared to one or more other cell types or classes of cells.

[0231] 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 (i) SEQ ID NO: 1, (ii) a variant, functional fragment, multiple copies, or a combination thereof, or (iii) a liver detargeting region comprising 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 to 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.

[0232] 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 (i) SEQ ID NO: 2, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to any one of (i) or (ii), comprising a liver detargeting region. 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.

[0233] 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 (i) SEQ ID NO: 3, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to any one of (i) or (ii), comprising a liver detargeting region. 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.

[0234] 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 detargeting region comprising (i) SEQ ID NO: 4, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0235] 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 detargeting region comprising (i) SEQ ID NO:5, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0236] 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 (i) SEQ ID NO: 6, (ii) a variant, functional fragment, multiple copies, or a combination thereof, or (iii) a liver detargeting region comprising 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 to 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.

[0237] 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 detargeting region comprising (i) SEQ ID NO: 7, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0238] 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 (i) SEQ ID NO: 8, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to any one of (i) or (ii), comprising a liver detargeting region. 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.

[0239] 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 (i) SEQ ID NO: 9, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to any one of (i) or (ii), comprising a liver detargeting region. 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.

[0240] 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 detargeting region comprising (i) SEQ ID NO: 10, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0241] 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 detargeting region comprising (i) SEQ ID NO: 11, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0242] 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 detargeting region comprising (i) SEQ ID NO: 12, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0243] 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 detargeting region comprising (i) SEQ ID NO: 13, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[0244] 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 detargeting region comprising (i) SEQ ID NO: 14, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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.

[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 detargeting region comprising (i) SEQ ID NO: 15, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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 detargeting region comprising (i) SEQ ID NO: 16, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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 detargeting region comprising (i) SEQ ID NO: 17, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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 detargeting region comprising (i) SEQ ID NO: 39, (ii) a variant, functional fragment, multiple copies, or a combination thereof, 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 to 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 (i) at least two different sequences selected from SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, multiple copies, or a combination thereof, or (iii) a liver detargeted region comprising 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 to 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 (i) at least three different sequences selected from SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, multiple copies, or a combination thereof, or (iii) a liver detargeted region comprising 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 to 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 (i) at least four different sequences selected from SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, multiple copies, or a combination thereof, or (iii) a liver detargeted region comprising 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 to 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 some embodiments, the nucleic acid cassette may comprise a CNS-selective promoter operably linked to a polynucleotide encoding a therapeutic protein and one or more liver detargeting 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 may 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, a CNS-selective promoter enhances expression in CNS cells (e.g., neurons, or glial cells such as astrocytes) compared 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).

[0253] Examples of CNS-selective promoters include, but are not limited to, Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, as well as PaqR4 promoter. Suitable promoters are also described, for example, in WO 2018 / 187363, the sequences of which are incorporated herein by reference. Other sequences may also be used.

[0254] In some embodiments, the cassette may include a GABAergic neuron-selective promoter operably linked to a polynucleotide encoding a therapeutic protein. GABAergic cells are inhibitory neurons that produce gamma-aminobutyric acid. GABAergic cells can be identified by markers such as the expression of glutamic acid decarboxylase 2 (GAD2), GAD1, NKX2.1, DLX1, DLX5, SST, PV, and VIP. A GABAergic neuron-selective promoter is a regulatory element that specifically regulates gene expression in GABAergic neurons. For example, a GABAergic neuron-selective promoter enhances expression in GABAergic neurons compared to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, astrocytes, microglia, motor neurons, vascular cells, non-GABAergic neurons, or other CNS cells).

[0255] 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 compared to one or more other CNS cell types.

[0256] In certain embodiments, the neuron-selective promoter may be or include sequences that are human-derived. In some cases, the promoter may be or include sequences that are mouse-derived. In some cases, the promoter is not naturally occurring or includes sequences that are not naturally occurring. In some instances, the promoter's sequence may be 100% human-derived. In other cases, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the promoter sequence is human-derived. For example, the promoter may have 50% of its sequence that is human-derived, and the remaining 50% may be non-human-derived (e.g., mouse-derived or fully synthetic).

[0257] In some embodiments, the therapeutic protein encoded by the mRNA is associated with neurological disease or neurological disorder.As mentioned above, neurological disease and disorder include those associated with one or more gene mutations and those with unknown etiology.Examples of neurological disease and neurological disorder include epilepsy seizures, neurodegenerative disorder, and / or neurodevelopmental disorder. Examples of neurological diseases or disorders include Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy central temporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic eyelid myoclonia Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy during sleep continuous spike and wave CSWS, infantile spasms (West syndrome) group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douz syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0258] In these embodiments, the therapeutic protein is selected from the group consisting of: (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, GBA1, GRIN2A, GRIN2B, GRN, HCN1, HCN4, KCNQ2, KCNQ3, 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; (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 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.

[0259] In certain embodiments, the nucleic acid constructs described herein include, in addition to a promoter, other regulatory elements, such as sequences associated with transcription initiation or termination, enhancer sequences, and efficient RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stability elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, or polyadenylation (polyA) sequences, or combinations thereof. Regulatory elements can function to regulate gene expression at the transcriptional, late-transcriptional, or translational phases of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination. In various embodiments, regulatory elements can recruit transcription factors to the coding region that increase gene expression selectivity in the cell type of interest, increase the rate at which RNA transcripts are produced, increase the stability of the RNA produced, and / or increase the rate of protein synthesis from the RNA transcript.

[0260] In certain embodiments, the cassette may further comprise a polyA sequence. Suitable polyA sequences include, for example, artificial polyA (PA75) 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 globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 EIb polyA, growth hormone polyA, or PBGD polyA. In certain embodiments, the polyA sequence is located downstream of the polynucleotide encoding the functional therapeutic protein in the nucleic acid construct described herein.

[0261] vector Expression vectors can be used to deliver nucleic acid molecules to target cells via transfection or transduction. Vectors can be integrative or non-integrative, which refers to the ability of a vector to integrate an expression cassette or transgene into the genome of a host cell. 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 (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC or PAC); episomal vectors; transposons (e.g., PiggyBac); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0262] Expression vectors may be linear oligonucleotides or circular plasmids and can be delivered to cells via a variety of transfection methods, including physical and chemical methods. Physical methods generally refer to delivery methods that use physical forces to counter cell membrane barriers in facilitating intracellular delivery of genetic material. Examples of physical methods include the use of needles, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, and hydroporation. Chemical methods generally refer to methods in which chemical carriers deliver nucleic acid molecules to cells and can include inorganic particles, lipid-based vectors, polymer-based vectors, and peptide-based vectors.

[0263] In some embodiments, the expression vector is administered to the target cells using inorganic particles. Inorganic particles can refer to nanoparticles, such as nanoparticles engineered for various sizes, shapes, and / or porosity to escape the reticuloendothelial system or to protect trapped molecules from degradation. Inorganic nanoparticles can be prepared from metals (e.g., iron, gold, and silver), inorganic salts, or ceramics (e.g., calcium, magnesium, or silicon phosphates or carbonates). The surfaces of these nanoparticles can be coated to facilitate 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.

[0264] In some embodiments, the expression vector is administered to the target cells using cationic lipids (e.g., cationic liposomes). Various types of lipids have been investigated for gene delivery, such as lipid nanoemulsions (e.g., which are a dispersion of one immiscible liquid in another stabilized by an emulsifier) ​​or solid lipid nanoparticles.

[0265] In some embodiments, the expression vector is administered to the target cell using a peptide-based delivery vehicle. A peptide-based delivery vehicle may have the advantage of protecting the genetic material being delivered, targeting specific cell receptors, disrupting the endosomal membrane, and delivering the genetic material into the nucleus. In some embodiments, the expression vector is administered to the target cell using a polymer-based delivery vehicle. The polymer-based delivery vehicle may include natural proteins, peptides, and / or polysaccharides, or synthetic polymers. In one embodiment, the polymer-based delivery vehicle includes polyethylenimine (PEI). PEI can condense DNA into positively charged particles that bind to anionic cell surface residues and are transported into the cell via endocytosis. In other embodiments, the polymer-based delivery vehicle may include poly-L-lysine (PLL), poly(DL-lactic acid) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, histones, protamine, dendrimers, chitosan, synthetic amino derivatives of dextran, and / or cationic acrylic polymers. In certain embodiments, the polymer-based delivery vehicle may include a mixture of polymers, such as, for example, PEG and PLL.

[0266] In certain embodiments, the expression vector may be a viral vector suitable for gene therapy.The preferred features of viral gene therapy vectors or gene delivery vectors include the ability to be reproducibly and stably propagated and purified to high titers; the ability to mediate targeted delivery (e.g., deliver transgenes specifically to tissues or organs of interest without widespread vector spread to other locations); and the ability to mediate gene delivery and transgene expression without inducing adverse side effects.

[0267] Some types of viruses, such as the non-pathogenic parvoviruses called adeno-associated viruses, have been engineered for gene therapy purposes by utilizing the viral infection pathway but avoiding replication and subsequent expression of viral genes that may lead to toxicity. Such viral vectors can be obtained by deleting all or part of the coding region from the viral genome, but leaving intact sequences (e.g., terminal repeat sequences) 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 host chromatin.

[0268] In various embodiments, suitable viral vectors include negative stranded RNA viruses such as retroviruses (e.g., types A, B, C, and D viruses), adenoviruses, parvoviruses (e.g., adeno-associated viruses or AAV), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive stranded RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Examples of retroviruses include avian leukosis virus, human T-lymphotrophic virus type 1 (HTLV-1), bovine leukemia virus (BLV), lentivirus, and spumaretrovirus. Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Viral vectors can be classified into two groups, integrative and nonintegrative, according to their ability to integrate into the host genome. Oncoretroviruses and lentiviruses can integrate into host cell chromatin, while adenoviruses, adeno-associated viruses, and herpesviruses persist primarily in the cell nucleus as extrachromosomal episomes.

[0269] In certain embodiments, the suitable viral vector is a retroviral vector. Retrovirus refers to viruses of the Retroviridae family. Examples of retroviruses include oncoretroviruses, such as murine leukemia virus (MLV), and lentiviruses, such as human immunodeficiency virus 1 (HIV-1). Retroviral genomes are single-stranded (ss) RNA and contain various genes that can be provided in cis or trans. For example, retroviral genomes can contain cis-acting sequences, such as two long terminal repeats (LTRs), along with elements for gene expression, reverse transcription, and integration into host chromosomes. Other components include a packaging signal (psi or ψ) for specific RNA packaging into newly formed virions, and a polypurine tract (PPT), which is the initiation site for plus-strand DNA synthesis during reverse transcription. In addition, retroviral genomes can contain gag, pol, and env genes. The gag gene encodes structural proteins, the pol gene encodes an enzyme that associates with ssRNA and reverse transcribes viral RNA into DNA, and the env gene encodes the viral envelope. In general, gag, pol and env are provided in trans for viral replication and packaging.

[0270] In certain embodiments, the retroviral vector provided herein may be a lentiviral vector. At least five serogroups or serotypes of lentiviruses have been recognized. Viruses of different serotypes may differentially infect certain cell types and / or hosts. Lentiviruses include, for example, 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 visna virus. Lentiviruses or lentivectors may be capable of transducing quiescent cells. Similar to oncoretroviral vectors, the design of lentivectors may be based on the separation of cis- and trans-acting sequences.

[0271] In an exemplary embodiment, the viral vector provided herein is an adeno-associated virus (AAV). AAV is a small, replication-defective, non-enveloped animal virus that infects humans and some other primate species. AAV is not known to cause human disease and induces a mild immune response. AAV vectors can also infect both dividing and quiescent cells without being integrated into the host cell genome.

[0272] The AAV genome consists of linear, single-stranded DNA approximately 4.7 kb in length. The genome consists of two open reading frames (ORFs) flanked by inverted terminal repeat (ITR) sequences that are approximately 145 bp in length. The ITRs consist of a nucleotide sequence at the 5' end (5'ITR) and a nucleotide sequence located at the 3' end (3'ITR) that contains a palindromic sequence. The ITRs function in cis by folding to form a T-shaped hairpin structure by complementary base pairing that serves as a primer during the initiation of DNA replication for second strand synthesis. The two open reading frames encode the rep and cap genes, which are involved in the replication and packaging of virions. In an exemplary embodiment, the AAV vectors provided herein do not contain the rep or cap genes. Such genes can be provided in trans to produce virions, as further described below.

[0273] In certain embodiments, the AAV vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode 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., relative to the polynucleotide encoding the therapeutic protein and the regulatory sequence located between the 5'ITR sequence and the 3'ITR sequence).

[0274] There are various 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, and AAV-DJ8. These serotypes differ in their tropism, or the type of cells they infect. AAV can contain genomes and capsids from multiple serotypes (e.g., pseudotypes). For example, AAV can contain a genome (e.g., ITRs) of serotype 2 packaged in a capsid from serotype 5 or serotype 9. Pseudotypes can improve transduction efficiency as well as change tropism.

[0275] 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 functional therapeutic proteins to cells, cell types, or tissues, and can be performed either in vivo, ex vivo, or in vitro. In exemplary embodiments, such AAV vectors are replication-deficient. In some embodiments, AAV viruses are engineered or genetically modified so that they can replicate and generate virions only in the presence of helper factors.

[0276] In certain embodiments, viral vectors can be selected to produce virions with high infectivity without selectivity for a particular cell type. 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). Differential expression of the transgene 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 selective expression of the polynucleotide in a target cell, cell type, or tissue, but one or more regulatory elements suppress transgene expression in off-target cells, cell types, or tissues, or confer significantly low, insignificant, or statistically low gene expression in one or more off-target cells, cell types, or tissues.

[0277] In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred.

[0278] In an exemplary embodiment, the present application provides an expression vector designed for delivery by AAV. The AAV can be any serotype, such as 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 a chimeric, hybrid, or mutant AAV. The AAV can also be a self-complementary AAV (scAAV), a "self-complementary" AAV being one in which the coding region is designed to form an intramolecular double-stranded DNA template. Upon infection with such a vector, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. The design of scAAV vectors has been described in various publications, including McCarty et al Gene Therapy 2001 8:1248-54.

[0279] 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, the construct 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, the construct described above, and a 3'ITR.

[0280] host cell In another aspect, the present invention relates to a host cell comprising the nucleic acid cassette described above. The host cell may 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 that is susceptible to infection by the virus of interest and is suitable for in vitro culture.

[0281] In certain embodiments, the host cells provided herein may be used for ex vivo gene therapy purposes. In such embodiments, the cells are transfected with the above-mentioned nucleic acid molecules or expression cassettes and then transplanted into a patient or subject. The transplanted cells may be of autologous, allogeneic, or xenogeneic origin. For clinical use, cell isolation will generally be performed under Good Manufacturing Practices (GMP) conditions. Prior to transplantation, the quality of the cells and the absence of microorganisms or other contaminants are typically checked, and preconditioning, such as by radiation and / or immunosuppressive treatment, may be performed. Furthermore, the host cells may be transplanted together with growth factors to stimulate cell proliferation and / or differentiation.

[0282] In certain embodiments, the host cell can be used for ex vivo gene therapy to CNS.Preferably, the cell is a eukaryotic cell, such as a mammalian cell, including but not limited to human, non-human primate, such as ape, chimpanzee, monkey, and orangutan, domesticated animals including dog and cat, and livestock animals such as horse, cow, pig, sheep, and goat, or other mammalian species, including but not limited to mouse, rat, guinea pig, rabbit, hamster, etc. Those skilled in the art will select the more suitable cell according to the patient or subject to be transplanted.

[0283] In certain embodiments, the host cell provided herein may be a cell with self-renewal and pluripotency properties, such as a stem cell or an induced pluripotent stem cell. The stem cell is preferably a mesenchymal stem cell. Mesenchymal stem cells (MSCs) can differentiate into at least one of osteoblasts, chondrocytes, adipocytes, or muscle cells, and can be isolated from any type of tissue. Generally, MSCs will be isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. Methods for obtaining them are well known to those skilled in the art. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cells that can be generated directly from adult cells. Yamanaka et al. induced iPS cells by introducing Oct3 / 4, Sox2, Klf4, and c-Myc genes into mouse and human fibroblasts and allowing the cells to express the genes (WO 2007 / 069666). Subsequently, Thomson et al. generated human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (WO 2008 / 118820).

[0284] In an exemplary embodiment, the host cell provided herein is a packaging cell. The cell can be an adherent or suspension cell. The packaging cell and the helper vector or virus, or DNA construct, together provide in trans all the missing functions required for complete replication and packaging of the viral vector.

[0285] Preferably, the packaging cells are eukaryotic cells, such as mammalian cells, including monkey, human, dog, and rodent cells. Examples of human cells are PER.C6 cells (WO 01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus 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.

[0286] As an alternative to mammalian sources, cell lines for use in the present invention may be derived from avian sources such as chicken, duck, goose, quail, or pheasant. Examples of avian cell lines include avian embryonic stem cells (WO 01 / 85938 and WO 03 / 076601), immortalized duck retinal cells (WO 2005 / 042728), and avian embryonic stem cell-derived cells, including chicken cells (WO 2006 / 108846) or duck cells, such as the EB66 cell line (WO 2008 / 129058 and WO 2008 / 142124).

[0287] In another embodiment, the host cell is an insect cell, such as an SF9 cell (ATCC CRL-1711), an Sf21 cell (IPLB-Sf21), an MG1 cell (BTI-TN-MG1), or a High Five™ cell (BTI-TN-5B1-4).

[0288] In certain embodiments, the host cells provided herein comprise a nucleic acid construct (e.g., a plasmid) carrying a recombinant AAV vector / genome containing the cassette described above, and may further comprise one or more additional nucleic acid constructs, such as, for example, (i) a nucleic acid construct encoding the rep and cap genes but not carrying ITR sequences (e.g., an AAV helper plasmid), and / or (ii) a nucleic acid construct (e.g., a plasmid) that provides adenoviral functions necessary for AAV replication. In exemplary embodiments, the host cells provided herein comprise i) a nucleic acid construct or expression vector described above, ii) a nucleic acid construct encoding the AAV rep and cap genes that does not carry ITR sequences, and iii) a nucleic acid construct comprising adenoviral helper genes (described further below).

[0289] In certain embodiments, the rep gene, the cap gene, and the adenovirus helper genes 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). Thus, in another exemplary embodiment, the host cell provided herein comprises i) a nucleic acid molecule or expression cassette, and ii) a plasmid encoding the AAV rep and cap genes that does not carry an ITR sequence, and further comprises an adenovirus helper gene. Alternative methods are known. For example, the rep, cap, and adenovirus helper genes do not have to be on the same plasmid, but can be provided on different plasmids, or the rep and cap genes can be provided on a different plasmid than the adenovirus helper genes.

[0290] In certain embodiments, suitable hosts 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 AAV rep, AAV cap, and the AAV vector to be packaged, respectively. The recombinant baculovirus vectors will provide the viral helper gene functions necessary for viral replication and / or packaging.

[0291] Further guidance on the construction and production of virions for gene therapy according to the invention can be found in: Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Vol. 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. ROSnyder 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.); Third. Edition. 2014 Humana Press (Springer).

[0292] Virions and methods for producing virions In certain embodiments, the present application provides a viral particle comprising a viral vector. The terms "viral particle" and "virion" are used interchangeably herein and refer to an infectious and typically replication-deficient viral particle that includes a viral genome (e.g., a viral expression vector) packaged within a capsid, and in some cases, e.g., in the case of retroviruses, a lipid envelope surrounding the capsid. "Capsid" refers to the structure in which the viral genome is packaged. Capsids consist of several oligomeric structural subunits made of proteins. For example, AAV has an icosahedral capsid formed by the interaction of 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.

[0293] In certain embodiments, the recombinant AAV virion provided herein can be prepared by encapsidating the AAV genome from a specific AAV serotype in a viral particle formed by the native Cap protein corresponding to the AAV of the same specific serotype.In other embodiments, the AAV viral particle provided herein comprises a viral vector that comprises the ITR of a given AAV serotype packaged in a protein from a different serotype.See, for example, Bunning H et al.J Gene Med 2008;10:717-733. For example, viral vectors with ITRs from a given AAV serotype can be expressed as a) viral particles composed of capsid proteins 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 variants (e.g., AAV2 ITRs with AAV1 and AAV9 capsid proteins); c) chimeric viral particles composed of capsid proteins truncated by domain swapping between different AAV serotypes or variants (e.g., AAV2 ITRs with AAV8 capsid proteins with AAV9 domains); or d) targeted viral particles engineered to display selective binding domains that enable stringent interaction with target cell-specific receptors (e.g., AAV5 capsid proteins with AAV9 capsid proteins genetically truncated by insertion of peptide ligands). It may be packaged into the ITRs, or into non-engineered AAV9 capsid proteins by coupling of peptide ligands to the capsid surface.

[0294] Those skilled in the art will understand that the AAV virions provided herein can contain capsid proteins of any AAV serotype. In one embodiment, the viral particle contains capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9, which are more suitable for delivery to the CNS (M. Hocquemiller et al., Hum Gene Ther 27(7):478-496(2016)). In a specific embodiment, the viral particle contains a nucleic acid construct of the present invention, wherein the 5'ITR and 3'ITR sequences of the nucleic acid construct are of the AAV2 serotype, and the capsid protein is of the AAV9 serotype.

[0295] Numerous methods are known in the art for the production of rAAV virions, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J. E. et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. All rAAV production cultures for production of rAAV viral particles require 1) suitable host cells including, for example, a human-derived cell line such as HeLa, A549, or 293 cells, or an insect-derived cell line such as SF-9 in the case of baculovirus production systems; 2) suitable helper virus functions provided by wild-type or mutant adenovirus (such as a temperature sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene flanked by AAV ITR sequences; and 5) suitable media and media components to support rAAV production.

[0296] In various embodiments, the host cells described herein contain three components: (1) rep and cap genes, (2) genes providing helper functions, and (3) transgenes flanked by ITRs. The AAV rep gene, AAV cap gene, and genes providing helper functions can be introduced into the cell by incorporating the genes into a vector, e.g., a plasmid, and introducing the vector into the host cell. The rep, cap, and helper function genes can be incorporated into the same plasmid or different plasmids. In a preferred embodiment, the AAV rep and cap genes may be incorporated into one plasmid and the genes providing helper functions are incorporated into another plasmid. The various plasmids (e.g., including the AAV rep and cap genes, helper functions, or transgenes) for the generation of the host cell for virion production can be introduced into the cell by using any suitable method known in the art. Examples of transfection methods include, but are not limited to, co-precipitation with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and biolistic transfection. In certain embodiments, the rep and cap genes, helper functions, and plasmids providing the transgene can be introduced into the cell simultaneously. In another embodiment, the rep and cap genes and plasmids providing the helper functions can be introduced into the cell before or after introduction of the plasmid containing the transgene. In an exemplary embodiment, the cell is transfected with three plasmids simultaneously (e.g., triple transfection method): (1) a plasmid containing the transgene, (2) a plasmid containing the AAV rep and cap genes, and (3) a plasmid containing genes providing the helper functions. Exemplary host cells can be 293, A549, or HeLa cells.

[0297] In other embodiments, one or more of (1) the AAV rep and cap genes, (2) genes providing helper functions, and (3) a transgene (e.g., a PV-selective regulatory element operably linked to a polynucleotide encoding a therapeutic protein disclosed herein) may be carried by the packaging cell either episomally and / or integrated into the genome of the packaging cell. In one embodiment, the host cell may be a packaging cell in which the AAV rep and cap genes and helper functions 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 and cap genes 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 helper functions. In another embodiment, the host cell may be a packaging cell in which the helper functions 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 rep and cap genes. In another embodiment, the host cell can be a producer cell line stably transfected with the rep and cap genes, helper functions, and transgene sequences. Exemplary packaging and producer cells can be derived from 293, A549, or HeLa cells.

[0298] In another embodiment, the producer cell line is an insect cell line (typically Sf9 cells) infected with a baculovirus expression vector providing the Rep and Cap proteins. This system does not require adenovirus helper genes (Ayuso E, et al., Curr. Gene Ther. 2010, 10:423-436).

[0299] 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 a cap protein include the ability to induce capsid formation, promote accumulation of single-stranded DNA, promote AAV DNA packaging into capsids (i.e., encapsidation), bind to a cellular receptor, and promote entry of virions into a host cell. In principle, any Cap protein can be used in the context of the present invention.

[0300] Cap protein has been reported to affect the host tropism, cell, tissue or organ specificity, receptor usage, infection efficiency, and immunogenicity of AAV virus.Therefore, the AAV cap for use in rAAV can be selected, for example, by considering the subject's species (e.g., human or non-human), the subject's immunological status, the subject's suitability for long-term or short-term treatment, or a specific therapeutic application (e.g., treatment of a specific disease or disorder, or delivery to a specific cell, tissue, or organ).In certain embodiments, the cap protein is derived from AAV from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9 serotypes.In an exemplary embodiment, the cap protein is derived from AAV9.

[0301] In some embodiments, an AAV Cap for use 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 its encoding nucleic acid, hi some embodiments, the AAV cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV caps.

[0302] In some embodiments, the AAV cap is chimeric and comprises 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.

[0303] In some embodiments, an AAV cap for use in a 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 evasion can be engineered into the cap protein. Alternatively, or in addition, the cap can be chemically modified so that the surface of the rAAV is pegylated (i.e., PEGylated), which can promote immune evasion. The cap protein can also be mutagenized (e.g., to eliminate its native receptor binding or to mask immunogenic epitopes).

[0304] The term "rep protein" as used herein refers to a polypeptide having at least one functional activity of a native AAV rep protein (e.g., rep40, 52, 68, 78). Examples of functional activities of the rep protein include any activity associated with the physiological function of the protein, including promoting DNA replication by recognition, binding and nicking of the AAV DNA replication origin, and DNA helicase activity. Additional functions include regulating transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into a host chromosome. In certain embodiments, the AAV rep gene may be from an AAV serotype selected from the group consisting of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrhlO, more preferably, AAV1, AAV2, AAV5, AAV8, and AAV9.

[0305] In some embodiments, the AAV rep protein for use in the methods of the invention may 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% or more similar to one or more of the aforementioned AAV rep.

[0306] The phrase "helper function" or "helper gene" as used herein refers to viral proteins that AAV depends on for replication. Helper functions include proteins required for AAV replication, including but not limited to proteins involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression product, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Helper functions include but are not limited to adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, and UL29, and herpesvirus polymerase. In a preferred embodiment, the proteins that AAV depends on for replication are derived from adenovirus.

[0307] In some embodiments, viral proteins on which AAV depends for replication for use in the methods of the invention may be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned viral proteins or its encoding nucleic acid, hi some embodiments, the viral protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned viral proteins.

[0308] Methods for assaying the function of the cap protein, the rep protein, and the viral proteins on which AAV depends for replication are well known in the art.

[0309] The host cells for expressing the transgene of interest can be grown under suitable conditions for the assembly of AAV virions. In certain embodiments, the host cells are grown for a suitable period of time to promote the assembly of AAV virions and the release of virions into the medium. Generally, the cells are grown for 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 up to about 10 days. After about 10 days (or sooner, depending on the culture conditions and the specific host cells used), the level of production generally decreases significantly. Generally, the culture time is measured from the time of virus production. For example, in the case of AAV, virus production generally begins upon providing helper virus functions in a suitable host cell as described herein. Generally, cells are harvested about 48 to about 100 hours after helper virus infection (or after virus production has begun), preferably about 48 to about 96 hours, preferably about 72 to about 96 hours, preferably about 68 to about 72 hours.

[0310] rAAV production cultures can be grown under a variety of conditions (such as over a wide range of temperatures and for various lengths of time) suitable for the particular host cells being utilized. rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include 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 the Wave bag system.

[0311] Suitable media known in the art may be used for the production of rAAV virions. These media include, but are not limited to, media manufactured by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), each of which is incorporated herein by reference in its entirety. In certain embodiments, rAAV production culture media may be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, rAAV vectors may be produced in serum-free conditions, which may also be referred to as animal-derived product-free media.

[0312] After the host cells are cultured to produce AAV virions, the resulting virions can then be harvested and purified. In certain embodiments, AAV virions can be obtained from (1) the host cells of the production culture by lysis of the host cells, and / or (2) the culture medium of the cells after a period of time, preferably 72 hours, after transfection. rAAV virions can be harvested from spent medium from the production culture, provided that the cells are cultured under conditions that cause the release of rAAV virions from intact cells into the medium (see, for example, U.S. Patent No. 6,566,118). Suitable methods for lysing cells are also known in the art, and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0313] After recovery, the rAAV virions may be purified. As used herein, the term "purified" includes preparations of rAAV virions that lack at least some of the other components that may also be present when the rAAV virions are naturally occurring or initially prepared. Thus, for example, purified rAAV virions may be prepared using an isolation technique to enrich them from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as by the percentage of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or it can be measured with respect to secondary interfering substances that may be present in the source mixture, such as contaminants including production culture contaminants or in-process contaminants, including helper viruses, media components, and the like.

[0314] In certain embodiments, the rAAV production culture harvest may be clarified to remove host cell debris, hi some embodiments, the production culture harvest may be clarified using a variety of standard techniques, such as centrifugation or filtration through filters with a pore size of 0.2 μm or greater (e.g., cellulose acetate filters or a series of depth filters).

[0315] In certain embodiments, the rAAV production culture harvest is further treated with Benzonase™ to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase™ digestion is performed under standard conditions, e.g., a final concentration of 1-2.5 Units / ml Benzonase™, at temperatures ranging from room temperature to 37° C. for 30 minutes to several hours.

[0316] In certain embodiments, rAAV virions can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. Methods for purifying rAAV particles can be found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232, U.S. Patent Nos. 6,989,264 and 8,137,948, and WO 2010 / 148143.

[0317] In certain embodiments, purified AAV virions can be dialyzed against PBS, filtered, and stored at −80° C. Viral genome titers 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).

[0318] Pharmaceutical Compositions In certain embodiments, the present application provides a composition comprising a nucleic acid cassette, e.g., an expression cassette, e.g., an rAAV comprising the expression cassette described above, or an RNA, e.g., an mRNA, encoded thereby, and a pharmaceutically acceptable carrier. In some embodiments, a virion containing the cassette and a pharmaceutically acceptable carrier is provided. In exemplary embodiments, such a composition is suitable for gene therapy applications. The pharmaceutical composition is preferably sterile and stable under the conditions of manufacture and storage. Sterile solutions can be achieved, for example, by filtration through a sterile filtration membrane.

[0319] Acceptable carriers and excipients in pharmaceutical compositions are preferably non-toxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients can include buffers such as phosphate, citric acid, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol. The pharmaceutical compositions of the present disclosure can be administered parenterally in the form of an injection formulation. The injectable pharmaceutical composition can be formulated using a sterile solution or any pharma- ceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water and saline.

[0320] The pharmaceutical compositions of the present disclosure can be prepared in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate microcapsules. The pharmaceutical compositions of the present disclosure can also be prepared in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. The pharmaceutical compositions for gene therapy can be in an acceptable diluent or can include a slow release matrix in which the gene delivery vehicle is embedded.

[0321] The pharmaceutical compositions provided herein may be formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration. The pharmaceutical compositions may also be formulated for or administered via nasal, spray, oral, aerosol, rectal, or vaginal administration. In one embodiment, the pharmaceutical compositions provided herein are administered to the CNS or cerebral spinal fluid (CSF), i.e., by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. The tissue target may be specific, e.g., the CNS, or it may be a combination of several tissues, e.g., muscle and CNS tissue. Exemplary tissues or other targets may include liver, skeletal muscle, cardiac muscle, 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 by CNS or CSF injection, i.e., intraparenchymal, intrathecal, intracisternal, or intraventricular injection, one or more of which methods can be used to administer the pharmaceutical compositions of the present disclosure.

[0322] 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.

[0323] The dosage of the pharmaceutical composition of the present disclosure depends on factors including the route of administration, the disease being treated, and the physical characteristics of the subject (e.g., age, weight, general health). The dosage can be adjusted to provide an optimal therapeutic response. Typically, the dosage can be an amount that effectively treats the disease without inducing significant toxicity. In certain embodiments, the pharmaceutical composition can be formed into a unit dose as needed.

[0324] The pharmaceutical composition of the present disclosure can be administered to a subject in need thereof when medically necessary. In an exemplary embodiment, a single administration is sufficient. In one embodiment, the pharmaceutical composition is suitable for use in a human subject and is administered by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. In one embodiment, the pharmaceutical composition is delivered via a peripheral vein by bolus injection. In another embodiment, the pharmaceutical composition is delivered via a peripheral vein by infusion.

[0325] In another aspect, the present application further provides a kit comprising the nucleic acid molecule, vector, host cell, virion or pharmaceutical composition described herein in one or more containers. The kit may include instructions or packaging material describing how to administer the nucleic acid molecule, vector, host cell or virion contained in the kit to a patient. The containers of the kit may be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kit may include one or more ampoules or syringes containing the nucleic acid molecule, vector, host cell, virion or pharmaceutical composition in a suitable liquid or solution form.

[0326] Treatment methods The nucleic acid cassette, expression cassette, expression vector, viral vector, viral particle or pharmaceutical composition of the present invention can be used to treat various disorders, for example, neurological disorders. In some embodiments, the chemical, protein, or nucleic acid molecule of the present invention can be used to treat or ameliorate one or more symptoms associated with a genetic mutation or an under-expressed or non-expressed gene in a subject. In certain embodiments, the treatment can be treating a subject via gene therapy, which is administered directly to the subject in need thereof (e.g., directly to the CNS) or systemically via injection and / or infusion. The therapy can be formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration, or via nasal, spray, oral, aerosol, rectal, or vaginal administration, for example, by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. The tissue target can be specific, for example, the CNS, or it can be a combination of several tissues.

[0327] In any embodiment herein, the target cell can be a nerve 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 nerve cell, such as a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.For example, in some embodiments, the nerve cell is a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may 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 cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0328] In any embodiment, the therapy may be used to increase the production or expression of a target protein in cells, such as GABA neurons or parvalbumin neurons.

[0329] In certain embodiments, the treatments provided herein do not result in adverse reactions to a subject. Treatment with the nucleic acid molecules, expression vectors, pharmaceutical compositions, or virions described herein may cause fewer or less severe adverse reactions in a subject than treatment with a similar gene therapy comprising the same transgene linked to a non-parvalbumin neuron-selective regulatory element.

[0330] Sequence Listing The following table (Table 1) provides sequences that are referred to in other parts of this disclosure.

[0331] SEQ ID NOs:1-17 and 39 provide exemplary liver detargeting elements / sequences that may be present in the RNA transcripts encoded in the nucleic acid cassettes of the present disclosure.

[0332] SEQ ID NOs:18-34 and 40 provide liver detargeting sequences that may be present in a nucleic acid cassette (DNA cassette) encoding an RNA transcript (e.g., mRNA) containing any of SEQ ID NOs:1-17 or 39.

[0333] Additional sequences are provided in Table 1.

[0334] [Table 1-1]

[0335] [Table 1-2] EXAMPLES

[0336] The following examples are offered so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed by the inventors. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular(ly); ip, intraperitoneal(ly); sc, subcutaneous(ly).

[0337] Example 1: Identification of detargeting elements Element selection for library generation: Elements were first retrieved from annotated 3' untranslated regions (3'UTRs) of genomic sequences curated in the AURA database (Atlas of UTR regulatory activity; 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.) based on their proximity to liver-depleted genes according to expression data from the Genotype-Tissue Expression portal. Genes were screened for high expression in at least one non-liver tissue. 100 genes were selected based on depleted expression in liver, but high expression in at least one other tissue. Genomic sequences of the 3'UTRs associated with each of these 100 genes were segmented into overlapping 127 base pair (bp) candidate elements, called "tiles", using a sliding window distance of 25 bp.

[0338] Further elements were extracted from the Functional Annotation of the 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), the mircoRNA Expression and Sequence Analysis database (mESA, Koray D. Kaya, Gokhan Karakulah, Cengiz M. Yakicier, Aybar C. Acar, Ozlen Konu, mESAdb: microRNA Expression and Sequence Analysis Database, Nucleic Acids Research, Volume 39, Issue suppl_1,1 January 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 The miRNAs were retrieved from the human liver. Hum Genomics. 2018;12(1):16. Each miRNA selected was based on a) maintaining minimal liver expression, b) having maximal expression in at least one other tissue type, and c) having maximal expression in all other tissues. The selection of miRNAs was based on whether their expression in liver was significantly different from all other tissues based on Grubbs statistics. Expression was manually checked to ensure that the differential expression of the miRNA was high compared to other tissues. All miRNAs for consideration from the above databases were compiled and checked for redundancy between the names of the miRNAs.

[0339] Control element pool: The control element pool consisted of either published miRNA response elements or previously identified elements. These controls served as benchmarks and diagnostic reference points due to their predictable expression characteristics. The control elements are broadly classified into three categories including a) miRNAs or 3'UTRs with known expression profiles, b) various promoters with known expression profiles, and c) random sequences at the promoter position or 3'UTR. The miRNA, 3'UTR, and random sequence controls were driven by the same promoters used in the screen. The promoter controls contained no elements or sequences in their 3'UTR regions other than those required for amplicon generation and molecular barcode identifiers. Each element in the control pool was assigned a barcode located in the 3'UTR of the gene of interest.

[0340] Library composition: miRNAs were represented as tetrameric repeats with an 8 bp spacer between the given miRNA sequences and contained 2000 unique elements. miRNAs with supporting evidence in the literature or obtained from the FANTOM5 database were assembled into elements containing 1, 2, 3, or 4 copies of a given single element, containing approximately 350 unique elements.

[0341] Unique barcodes assigned to library elements for oligopool synthesis: To prepare a designed library of elements for oligopool synthesis, the sequence of each candidate element was checked for the presence of restriction enzyme sites required for downstream cloning, and those containing these sequences were discarded. 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 detargeted element and its barcode were ligated with flanking sequences for cloning into the vector backbone. The library was synthesized as single-stranded DNA by Twist Biosciences.

[0342] Plasmid library synthesis: Experimental plasmid libraries were constructed from single-stranded oligo pools and ligated into a common plasmid backbone. The single-stranded oligos were amplified via PCR using a common set of primers and further ligated into the 3'UTR of the transgene in the screening plasmid. The plasmids consist of: (minCMV promoter)-(nanoluciferase)-(MCS)-(liver detargeting element)-(amplicon barcode and primer site)-(hGH polyA). The libraries were transformed into electrocompetent E. coli cells and plated onto agar plates for Sanger sequencing and also plated into 200 ml LB liquid cultures. Agar plate colonies were Sanger sequenced to verify that elements were ligated correctly and that no indels were present. The libraries were then isolated from 200 mL cultures using the Zymogen Maxiprep kit (Zymogen). This product was then pooled with a control spike-in plasmid library (see Control Element Pool section) to generate the final library used for vector production.

[0343] AAV vector preparation: All vectors were produced in adherent HEK293T cells in DMEM + 10% FBS. Cells were transfected with a library of element and helper plasmids, including the cis-ITR-containing plasmid, the trans-plasmid pAAVX encoding AAV2 replication, and the AAVX capsid gene and the pALD-X80 adenovirus helper plasmid, using PEI-MAX. AAV was harvested from cells and purified from the lysate using an ultracentrifugation gradient of iodixanol, further refined on an anion exchange column, then concentrated and formulated in PBS containing 0.001% Pluronic.

[0344] Animals: Screening was performed in adult female C57BL / 6J mice (The Jackson Laboratory). Mice were obtained as 6-8 week-old adults and housed on-site for 3 days to acclimate to the new environment. Animals were fed standard chow ad libitum with a 12-day light / 12-day dark cycle. After injection, mice were individually housed in a vivarium until sacrifice.

[0345] AAV injection: Mice (n=5) were injected by intravenous (IV) tail vein injection and stereotaxic direct hippocampal injection. Animals were treated with Rimadyl the day before surgery. Mice received 3E12 gc / animal via IV in a single dose, with a total of 1.8E11 gc / animal spread over four 1.5 μL injections into the left, right, dorsal, and ventral regions of the hippocampus. Animals were incubated with virus in the vivarium for 3 weeks after injection.

[0346] Sample collection: Tissues including hippocampus and liver were collected directly into RNAlater (Sigma Aldrich). Samples were kept in RNAlater at 4°C for 24 hours and then transferred to -80°C until further processing.

[0347] RNA and cDNA generation: RNA from samples was isolated with the RNeasy Mini column method using a standard protocol for total RNA isolation (Qiagen). Total RNA concentrations were normalized before input to the cDNA reaction. cDNA was generated by reverse transcription using the SuperScript IV VILO kit with an oligo-dT primer (Thermo Fisher Scientific).

[0348] Amplicon generation: Amplicons derived from the reporter gene were amplified by PCR using a set of universal primers for all elements in the library. For each sample, there were four technical replicates starting with the first amplicon PCR step. The cycle number of the amplicon PCR was first optimized by qPCR. Step 1 PCR amplifies a region of the 3'UTR of the reporter mRNA under optimized conditions. Briefly, AAV is digested with DNaseI (New England Biolabs) to remove the capsid and then used directly in step 1 PCR as described above. All technical replicate samples for each biological replicate are pooled together in equimolar amounts as the final sequencing library pool.

[0349] Amplicon sequencing: Each sample was combined in equimolar amounts into a final sequencing pool containing PCR samples from each biological replicate, amplicons from the plasmid pool used to generate AAV, and amplicons from the administered AAV library. Samples were then pooled with PhiX in a 60:40 molar ratio for sample pool diversification. The diversified library pool was further diluted and prepared according to the specifications of the Nextseq 500 High Kit (Illumina).

[0350] Candidate Selection: A graph showing brain activity versus liver activity for the constructs tested is provided in FIG. 1. As can be seen in FIG. 1, many different constructs achieved either reduced liver expression while maintaining brain expression, or reduced liver expression to a greater extent than brain expression. The included negative control did not affect either brain or liver expression, while the two positive controls included reduced liver expression to a greater extent than brain expression. Construct activity was estimated based on 1) the log2 fold change (log2 fold change, log2FC) of the construct's expression activity in the liver compared to its abundance in the baseline AAV pool, 2) the log2FC of the construct's expression activity in the hippocampus compared to its abundance in the baseline AAV pool, and 3) the difference between these two log2FC values. Tissue log2FC metrics were calculated using DESeq2 (see below for further details). The log2 fold change in brain and liver expression of selected constructs is also provided in Table 2. The data in Table 2 show a greater reduction in liver expression than in brain expression, indicating that these elements can detarget liver expression. As shown in Table 2, the elements provided herein detarget liver expression both when used alone and when provided as 2x, 3x, or 4x tandem repeats.

[0351] [Table 2]

[0352] Another detargeting element, test sequence 22, was identified in a similarly performed screen. As shown in Table 3, test sequence 22 also detargeted liver expression both when used alone and when provided as 2x, 3x, or 4x tandem repeats.

[0353] [Table 3]

[0354] Window scoring for endogenous 3'UTR tiles: Approximately 7000 constructs in the detargeting screen were sequence elements derived from the 3'UTR regions of endogenous genes that displayed favorable expression patterns (see section "Selection of endogenous 3'UTR-derived elements" for further details). Each endogenous 3'UTR region is spanned by overlapping 127 bp sequence "tiles," each offset by 25 bp from the previous tile. Since adjacent 3'UTR tiles share approximately 80% of their sequence, such tiles are generally expected to have similar activity profiles, and large variations in activity between adjacent tiles may be indicative of noisy measurements.

[0355] An aggregate "window" activity score was calculated for each group of three adjacent tiles by taking a weighted average of the tile's tissue log2FC scores, with the tiles weighted by the inverse of the tile's log2FC variance. As a result, better measured tiles (i.e., less variance in the activity of the five constituent barcodes for a construct) were weighted more heavily toward the window average. The window activity scores were used to filter sequence regions that consistently exhibited favorable activity profiles.

[0356] Selection of elements for validation: Several sequences were selected based on lower liver expression and conserved expression in the hippocampus to be further validated via IHC and ELISA.

[0357] ELISA: Samples for protein analysis were from adult female C57BL / 6J mice. Each mouse was dosed with 5.0E11vg / mouse of AAV9 by intravenous injection (tail vein) over a 3-week incubation period. Vectors were generated as described in AAV vector preparation. The GOI of the vector was as follows: (AAV2 ITR)-(EF1a(short))-mCherry-KASH-spA-(CTCF insulator)-(CMV promoter)-EGFP-KASH-(liver detargeting element)-sPA-(AAV2 ITR). The liver detargeting element was selected as above and is listed as the test sequence in Table 1 above. Both the left lateral lobe of the liver and one hemisphere of the cortex, excluding the olfactory bulb, cerebellum, and other hindbrain tissues, were collected from PBS-perfused mice. Samples were homogenized in a mechanical homogenizer with 2.8 mm ceramic beads (OMNI Intl.) for 1 min at 4°C in Buffer PRT (Abcam, ab171581 and ab221829) and clarified by centrifugation. Total protein was quantified using Micro BCA (ThermoFisher) and normalized in Buffer PRT. EGFP and mCherry proteins were quantified separately using ELISA (Abcam), each quantified based on a standard curve generated from recombinant protein for the respective assay. As can be seen in Figure 2, the majority of detargeted elements tested showed a large reduction in liver expression compared to the control.

[0358] Tissue preparation and Immunohistochemical (IHC) staining: After saline perfusion, whole brain and liver tissues were collected and fixed in 4% neutral buffered formalin for 24 hours, then switched to 70% ETOH and kept at 4°C until processing. Tissues were processed for formalin fixation and paraffin embedding by an external donor. After parasagittal embedding of the brain, 5um sections were cut onto glass slides. Two transverse sections of the liver lobes were collected on one slide for each animal. For IHC, slides were dewaxed, rehydrated, and then heat-induced epitope retrieval was performed in citrate pH 6 buffer at 95°C for 20 minutes. Slides were mounted in Shandon slide holders, washed with phosphate-buffered saline with Tween-20 (PBST), then incubated for 15 h in antibody dilution buffer (phosphate-buffered saline, 1.0% bovine serum albumin, 0.3% Triton-X-100) followed by overnight incubation at 4°C with rabbit anti-Myc [Abcam ab9106] at a dilution of 1:25000 in antibody dilution buffer. This was followed by goat anti-rabbit-HRP [Thermo A16110] for 1 h and then Opal 520 [Akoya Biosciences FP1487001KT] for 10 min. Washes were performed with PBST between each step. Nuclei were stained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) for 15 min and then coverslipped with a #1.5 coverslip. Figures 3A and 3B show representative images of brain and liver expression from mice treated with a control vector that does not contain a detargeting element, and Figures 3C and 3D show images of brain and liver expression from mice treated with a vector encoding an RNA containing test sequence 12 (sequence number 12).

[0359] Example 2: In vitro validation of selected liver detargeting elements Induced pluripotent stem cells (iPSCs) were used to validate the identified liver detargeting elements in human cells. Adeno-associated virus (AAV) particles were prepared using the AAVDJ serotype. Each virus preparation contained a genome containing the EF1a promoter, a coding sequence for enhanced green fluorescent protein fused to the KASH domain (eGFP-KASH), and either a random sequence or a liver detargeting element.

[0360] IPSC-derived glutamatergic neurons and hepatocytes were plated in 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. Cells were then incubated for an additional 72 hours before being harvested for RNA and DNA extraction. Levels of eGFP-KASH mRNA were assayed and compared to an internal control (GAPDH) using quantitative polymerase chain reaction. The results of the in vitro validation are shown in Table 4.

[0361] [Table 4]

[0362] Example 3: Liver detargeting in non-human primates To estimate conservation between mice and non-human primates (NHPs), several detargeting elements were selected for NHP studies. AAVs with different detargeting elements were prepared as described above and pooled for administration by unilateral intracerebroventricular injection at a dose of 10^14 viral genomes / animal to juvenile cynomolgus macaques (n=2) aged 16-21 months. Animals were necropsied approximately 50 days after treatment and liver and brain tissues were harvested for DNA and RNA extraction. AAV-driven transcript expression was assessed by reverse transcription droplet digital PCR analysis using vector-specific primers / probes. Total RNA expression was normalized to AAV genome copy number in each sample. Figure 4 shows the relative expression (log2 fold change) in liver for several different liver detargeting elements in NHPs in each of the treated animals and averaged. As shown in Figure 4, all elements tested showed reduced expression in liver compared to the control sequence, with test sequences 4, 6, and 7 showing greater than 2-fold reduction in liver expression.

[0363] While the present invention has been described in connection with specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the spirit and scope of the subject matter of the present disclosure. All such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A nucleic acid cassette comprising a therapeutic transgene encoding an mRNA, said mRNA comprising a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence 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 consecutive nucleotides of any of SEQ ID NOs: 1-17 or 39, which reduces expression in hepatocytes.

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

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

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

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

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

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

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

11. 11. The nucleic acid cassette of claim 1, wherein the sequence of (i), (ii) or (iii) is located in one or more of the 3'UTR region of the mRNA, the 5'UTR of the mRNA, or an intron of the mRNA.

12. The nucleic acid cassette of claim 11, wherein the sequence of (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

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

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

15. The nucleic acid cassette according to any one of claims 1 to 14, wherein the nucleic acid cassette does not exist in nature.

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

17. 17. The nucleic acid cassette of claim 16, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

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

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

20. The neurological disease or disorder is selected from the group consisting of Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's 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 rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome), and young adult epilepsy.

20. The nucleic acid cassette of claim 19, wherein the nucleic acid cassette is selected from the group consisting of juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

21. The therapeutic protein is (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG 1, 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, 21. The nucleic acid cassette of claim 19 or 20, selected from: a protein encoded by a gene selected from 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 expression of a gene from (i).

22. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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; (c) the mRNA encodes a therapeutic protein associated with a neurological disease or disorder;

23. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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 Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter; (c) the mRNA is 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, and GAB RA1, 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, 23. The nucleic acid cassette of any one of claims 1 to 22, encoding a therapeutic protein encoded by a gene selected from 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 expression of a gene from (i).

24. 24. The nucleic acid cassette of any one of claims 1 to 23, wherein the sequence of (i), (ii) or (iii) results in decreased expression of the polypeptide encoded by the mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii) or (iii).

25. 25. The nucleic acid cassette of claim 24, wherein the sequence of (i), (ii) or (iii) results in a reduction in expression of the polypeptide encoded by the mRNA in hepatocytes at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

26. 25. The nucleic acid cassette of claim 24, wherein the sequence of (i), (ii) or (iii) results in reduced expression of the polypeptide encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not contain the sequence of (i), (ii) or (iii).

27. 27. The nucleic acid cassette of any one of claims 1 to 26, wherein the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of the polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in a target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii) or (iii).

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

29. 28. The nucleic acid cassette of claim 27, wherein the sequence of (i), (ii) or (iii) results in expression of the polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

30. The nucleic acid cassette according to any one of claims 27 to 29, wherein the target cell is a neuronal 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. 32. The nucleic acid cassette of claim 31 , wherein the neuronal cell is a GABAergic cell.

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

34. The nucleic acid cassette according to 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. 37. The nucleic acid cassette of claim 36, wherein the viral vector is an adeno-associated viral (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. 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. 1. A nucleic acid cassette comprising a transgene encoding an mRNA, said mRNA encoding a therapeutic protein and comprising 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 complements thereof.

43. 43. The nucleic acid cassette 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 complements thereof.

44. 43. The nucleic acid cassette 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 complements thereof.

45. 45. The nucleic acid cassette of any 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 complements thereof.

46. 46. ​​The nucleic acid cassette 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 complements thereof.

47. 47. The nucleic acid cassette 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 complements thereof.

48. 47. The nucleic acid cassette 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 complements thereof.

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

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

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

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

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

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

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

56. The nucleic acid cassette according to 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 additionally comprises a sequence of at least 10 consecutive nucleotides of any one of SEQ ID NOs: 12 to 17, which reduces expression in liver cells.

58. The nucleic acid cassette of any one of claims 42 to 56, wherein the mRNA additionally comprises at least two sequences of at least 20 consecutive nucleotides of any one of SEQ ID NOs: 12 to 17, which reduce expression in liver cells.

59. 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 3'UTR region of the mRNA, the 5'UTR of the mRNA, or an intron of the mRNA.

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

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

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

63. 63. The nucleic acid cassette of any one of claims 42 to 62, wherein the nucleic acid cassette is not naturally occurring.

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

65. 65. The nucleic acid cassette of claim 64, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and 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 therapeutic protein is for use in treating a neurological disease or disorder, optionally including Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's LIDS (side effects of Parkinson's drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, Fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy central temporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic palpebral myoclonia Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy during sleep continuous spike and wave (CSWS), infantile spasms (West syndrome), 68. The nucleic acid cassette of claim 67, which is a protein associated with a gene encoding a nucleotide analogue of ...

69. The therapeutic transgene is (a) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOX G1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1 , KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PN 69. The nucleic acid cassette of claim 68, wherein the nucleic acid cassette is selected from: (a) a protein encoded by a gene selected from KP, 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 expression of a gene from (a).

70. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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; (c) the mRNA encodes a therapeutic protein associated with a neurological disease or disorder;

71. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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 Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter; (c) the mRNA is 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, KV 3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, P 71. The nucleic acid cassette of claim 70, encoding a therapeutic protein encoded by a gene selected from OLG1, 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 expression of a gene from (i).

72. 72. The nucleic acid cassette of any one of claims 42 to 71, wherein the miRNA binding site results in decreased expression of the polypeptide encoded by the mRNA in a hepatocyte compared to expression of the polypeptide in a hepatocyte from an otherwise equivalent mRNA that does not contain the miRNA binding site.

73. The nucleic acid cassette of Claim 72, wherein the miRNA binding site results in a reduction in expression of the polypeptide encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the polypeptide in the hepatocyte from an otherwise equivalent mRNA that does not contain the miRNA binding site.

74. 74. The nucleic acid cassette of claim 73, wherein the miRNA binding site results in reduced expression of the polypeptide encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the polypeptide in hepatocytes from an otherwise equivalent mRNA that does not contain the miRNA binding site.

75. 75. The nucleic acid cassette of any one of claims 42 to 74, wherein the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of the polypeptide encoded by the mRNA in a target cell compared to expression of the polypeptide in a target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

76. 76. The nucleic acid cassette of Claim 75, wherein the sequence of (i), (ii) or (iii) does not reduce expression of the polypeptide encoded by the mRNA in the target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

77. 76. The nucleic acid cassette of claim 75, wherein the sequence of (i), (ii) or (iii) results in expression of the polypeptide encoded by the mRNA in a target cell at a level that is at least 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 of the polypeptide in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

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

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

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

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

83. 83. The nucleic acid cassette 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. 85. The nucleic acid cassette of claim 84, wherein the viral vector is an adeno-associated viral (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. 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. 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. 1. A method of reducing hepatic expression of a therapeutic protein encoded by an mRNA while maintaining expression of said therapeutic protein in a target tissue, said method comprising including in said mRNA a sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

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

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

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

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

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

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

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

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

102. The method of any of claims 93-98, wherein the mRNA comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1-17 or 39, which reduces expression in hepatocytes.

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

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

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

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

107. 107. The method of any one of claims 93 to 106, comprising administering to a subject a nucleic acid encoding said mRNA.

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

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

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

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

112. 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 therapeutic protein is (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, KV 3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, PO LG1, 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 expression of a gene from (i).

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

115. The subject is selected from the group consisting of Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy central temporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep continuous spike and wave (CSWS), infantile spasms (West syndrome), juvenile ...

115. The method of claim 114, wherein the patient has oclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse 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. 116. The method of any one of claims 107 to 115, wherein the nucleic acid cassette comprises a CNS-selective promoter.

117. 117. The method of claim 116, wherein the CNS selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

118. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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; (c) the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

119. (a) the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-17 or 39, (ii) a variant, functional fragment, or 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 Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox Dlx5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter; (c) the mRNA is 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, and FOXG1 , GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNK 119. The method of claim 118, wherein the therapeutic protein is encoded by a gene selected from P, 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 expression of a gene from (i).

120. 120. The method of any one of claims 93-119, wherein the sequence of (i), (ii) or (iii) results in a reduction in expression of the protein encoded by the mRNA in a hepatocyte at a level that is at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the protein in a hepatocyte from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

121. 120. The method of any one of claims 93-119, wherein the sequence of (i), (ii) or (iii) results in a reduction in expression of the protein encoded by the mRNA in hepatocytes at a level that is 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% lower than expression of the protein in hepatocytes from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii) or (iii).

122. 122. The method of any one of claims 93 to 121, wherein the sequence of (i), (ii) or (iii) does not result in a significant decrease in expression of the protein encoded by the mRNA in a target cell compared to expression of the protein in a target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

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

124. 124. The method of claim 123, wherein the sequence of (i), (ii) or (iii) results in expression of the protein encoded by the mRNA in a target cell at a level that is at least 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 of the protein in the target cell from an otherwise equivalent mRNA that does not include the sequence of (i), (ii) or (iii).

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

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

128. 128. The method of claim 127, wherein the GABAergic cell is a parvalbumin expressing cell.

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

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

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

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

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

134. 134. The method of claim 133, wherein the viral vector is an adeno-associated viral (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 a scAAV.

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

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

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

140. 131. The method of claim 129 or 130, wherein the administering comprises intraparenchymal, intrathecal, intracisternal, or intraventricular administration.

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