Element for off-target gene expression in dorsal root ganglion and / or liver

Abstract

The present disclosure provides sequences that reduce the expression of operably linked transgenes in dorsal root ganglion cells and / or hepatocytes. In some aspects, the sequences may be employed in gene therapy vectors to off-target expression of a therapeutic transgene in the dorsal root ganglion and / or hepatocytes of a subject.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of the following U.S. Provisional Application Serial Numbers: 63 / 410,161 filed on September 26, 2022, 63 / 464,772 filed on May 8, 2022, 63 / 414,873 filed on October 10, 2022, and 63 / 412,119 filed on September 30, 2022, which are hereby incorporated by reference.

[0003] Incorporation by Reference of Sequence Listing Provided as an XML File

[0004] The Sequence Listing is provided as the sequence listing XML file ENCO - 006WO_SEQ_LIST, which was created on September 22, 2023, and is 141,924 bytes in size. The contents of the sequence listing XML are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION

[0005] Gene therapy has great potential in treating 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 an essential protein, or have a reduced amount of the essential protein or a dysfunctional form of the essential protein. Expression of the therapeutic payload in these cells rescues the cells, thereby treating the disease. In one example, Tay - Sachs disease (autosomal recessive, caused by a mutation in the HEXA gene located on chromosome 15) can be successfully treated by expressing a functional form of hexA in the brain using adeno - associated virus (AAV) gene therapy.

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

[0007] In view of the above, there is a general need for tools to improve the tissue specificity of gene therapy. SUMMARY OF THE INVENTION

[0008] The present disclosure particularly provides a nucleic acid cassette that contains a transgene encoding an RNA transcript, such as, for example, an mRNA, wherein the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs: 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii). These sequences reduce the expression of the transgene in dorsal root ganglion (DRG) cells relative to other cells or tissues, such as other cells in the brain, and can thus be employed in a variety of gene therapy strategies targeting cells that are not in the DRG.

[0009] Also provided is a nucleic acid cassette that contains a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs: 65, 110, and 112; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) sequences that are 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 sequence reduces the expression of the RNA transcript in hepatocytes.

[0010] Also provided is a nucleic acid cassette that contains a therapeutic transgene encoding an RNA transcript, the RNA transcript comprising a first sequence that is off-target in dorsal root ganglion (DRG) cells and a second sequence that is off-target in hepatocytes. The addition of the first and second sequences results in a reduction in the expression of the RNA transcript or the polypeptide encoded thereby in DRG cells and hepatocytes relative to a target tissue, such as GABAergic cells.

[0011] In certain aspects, the incorporation of one or more of these sequences results in an improved safety profile of gene therapy by reducing or eliminating toxicity in non-target cells, such as DRG and / or hepatocytes, caused by the expression of the transgene in these cells.

[0012] In some embodiments, the nucleic acid cassette is an expression cassette, wherein the expression cassette can operably contain a promoter, a coding sequence, one or more of the above off-target sequences, and a terminator. In some embodiments, at least one sequence present in the expression cassette is heterologous to another sequence in the expression cassette. For example, in some embodiments, the expression cassette of the present disclosure contains a promoter heterologous to the coding sequence operably linked thereto. In some embodiments, the expression cassette may further contain enhancers and / or introns.

[0013] Obviously, in embodiments where the RNA transcript encoded by the transgene is an mRNA encoding a therapeutic protein and containing an off-target sequence, the mRNA encoded by the transgene will contain both the coding sequence for the therapeutic protein and the off-target sequence, such that the expressed mRNA contains both the coding sequence and the DRG off-target sequence in the same transcript. Although the mechanism may not be clear yet, it should be understood that the off-target element can target the RNA transcript (e.g., mRNA molecule) containing the element for preferential degradation in non-target cells.

[0014] Note that synthetic RNA molecules containing the off-target sequences disclosed herein can also be inactivated in the designated tissues (e.g., DRG, liver, or both) when introduced into such cells (e.g., the cells of a subject). For example, a synthetic antisense RNA (containing one or more DRG off-target elements of the present disclosure, one or more liver off-target elements of the present disclosure, or a combination of both) will have reduced activity in off-target tissues when administered to a subject. There is no limitation in this regard.

[0015] In some embodiments, the promoter of the expression cassette may be selective for cells in a specific tissue (e.g., the target tissue), but also drive the expression of the transgene in DRG and / or hepatocytes. In some embodiments, the promoter can be a CNS-selective promoter, for example, a promoter selected from the group consisting of: Ca 2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0016] In any embodiment, the sequence can be located in the 3'UTR, 5'UTR, or intron of the mRNA.

[0017] In any embodiment, the expression cassette may encode a therapeutic protein (e.g., SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, KV3.1, KV3.2, KV3.3, STXBP1, UBE3A, or a transcription factor that regulates (e.g., activates or represses) the endogenous expression of any of these proteins). In some embodiments, the therapeutic protein may be: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, 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 regulates the expression of the gene from (i).

[0018] In some embodiments, the RNA transcript may comprise a combination of sequences (i), (ii), and (iii).

[0019] Also provided is a vector comprising the cassette outlined above. The vector may be a plasmid or viral vector, e.g., an adeno-associated virus (AAV) or lentiviral vector.

[0020] Also provided is an AAV, lentiviral particle, or cell (which may be in single-stranded form if packaged) comprising the cassette outlined above.

[0021] Also provided is an RNA having the sequence characteristics of an RNA encoded by any of the nucleic acid cassettes described herein.

[0022] A variety of methods are also provided. In some embodiments, the method can be used to express a protein. In these embodiments, the method can include introducing the expression cassette or the mRNA encoded thereby, as outlined above, into an organism, wherein the sequence reduces the expression of the protein in the DRG and / or hepatocytes of the organism. In additional embodiments, where the RNA itself is an active agent (i.e., it is a non-coding RNA, such as, for example, a microRNA, an antisense RNA, etc., as described elsewhere herein), the sequence reduces or eliminates the activity of the RNA in the DRG and / or hepatocytes of the organism. In some embodiments, the method can include administering the expression cassette or the mRNA or the non-coding RNA to a patient suffering from a neurological disease or disorder. In these embodiments, the administration can be systemic or local (e.g., locally administered into the brain or CNS tissue, such as by means of an intraparenchymal, intrathecal, cisterna magna, intraventricular, or intracranial administration method). In these embodiments, the subject to whom the expression cassette or the mRNA or the non-coding RNA is administered can be suffering from, for example, Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency disorder, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X chromosome syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonia epilepsy (Jeavons syndrome), epilepsy in infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with continuous spike and slow waves during sleep (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Ohtahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital epilepsy, Gastaut syndrome, epilepsy with only generalized tonic-clonic seizures, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Doose syndrome), sleep-related hypermotor epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0023] In view of the following description, other features, advantages, and embodiments may become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. These drawings are not intended to limit the scope of the teachings in any way.

[0025] Figure 1A A flowchart showing the design of the DRG off-target regulatory element (RE) library employed in the examples.

[0026] Figure 1B is a scatter plot showing the expression levels of the test construct library in the brain versus in the dorsal root ganglion (DRG). The relative transcript abundances (Log 2 ) of the brain (y-axis) and DRG tissue (x-axis) are shown, normalized to the AAV library abundance and a constitutive control sequence. The relative expression of each sequence element in the library is indicated compared to the mean of promoters paired with random control sequences. Each data point represents a unique sequence screened. Control constructs with random sequences in the off-target region are indicated by lightly shaded circles (within the solid-line box). The dashed region plotted on the scatter plot indicates the brain expression pattern versus the DRG expression pattern used to select candidate DRG off-target elements. Examples of candidate DRG off-target elements are represented by darkly shaded circles.

[0027] Figure 2 Shows the log 2 fold change in EGFP expression in the cortex, DRG, and hippocampus of mice injected with the following vectors compared to an AAV9 construct without an off-target element: (1) an AAV9 vector in which the EGFP transcript expressed by the vector contains 4 copies of the binding site for hsa-mir-183-3p (4x SEQ ID NO:4, provided herein as SEQ ID NO:44); or (2) an AAV9 vector in which the EGFP transcript expressed by the vector contains 4 copies of the binding site for hsa-mir-196b-5p (4x SEQ ID NO:1, provided herein as SEQ ID NO:43). The values used for the calculation are the mean EGFP-KASH transcript levels per μg of total RNA (normalized to VCN / diploid genome).

[0028] Figure 3Representative IHC images are shown that depict EGFP-KASH expression in cortical / hippocampal (top row) and lumbar DRG (bottom row) tissues harvested from mice injected with AAV9 vectors, where the EGFP transcript expressed by the vector: (1) has no off-target elements in off-target regions (no off-target); (2) has a tetrameric miR-183-3p binding site (4x SEQ ID NO:4) in off-target regions; or (3) has a tetrameric miR-196b-5p binding site (4xSEQ ID NO:1) in off-target regions. (The scale of the images is provided in the leftmost image.)

[0029] Figure 4 Shown are the mean and standard deviation (SD) of the percentage of EGFP-positive nuclei from two cortical regions (upper panel) and four DRG regions (lower panel) of brain tissue for each treatment group (n = 5; points in the figure indicate individual animals). Non-manipulated negative controls (UNM) (no AAV9 vector injection) were included in each experiment. Treatment groups included AAV9 vectors where the EGFP transcript expressed by the vector either had no off-target elements in off-target regions (no off-target) or had the indicated off-target elements in off-target regions (provided as SEQ ID NO).

[0030] Figure 5 Examples of representative IHC images are provided that depict EGFP-KASH expression in the brain and DRG from Figure 4 four mice: control mice injected with an AAV9 vector where the EGFP transcript expressed by the vector has no off-target elements in off-target regions (no off-target element); and three mice each injected with a different AAV9 vector where the EGFP transcript expressed by the vector has the indicated off-target elements in off-target regions (provided as SEQ ID NO).

[0031] Figure 6A and Figure 6B : AAV9 with a pan-neuronal promoter and a control UTR (control) or candidate DRG off-target UTRs (SEQ ID NO:46, which contains 4 copies of the hsa-mir-10b-5p binding site; and SEQ ID NO:48, which contains 2 copies of the hsa-mir-196b-5p binding site and 2 copies of the hsa-mir-10b-5p binding site) after ICV administration to mice at P1. Figure 6A Shows % GFP+ nuclei in the DRG (left panel) and brain (right panel). Figure 6BRepresentative images of mouse samples stained for AAV transgene expression from a control vector and a vector are shown. The control vector has no off-target elements in the off-target region, and the off-target region of the transcript in this vector contains four copies of hsa-mir-10b-5p (SEQ ID NO: 46; Figure 6A the same mice as shown in). An AAV vector incorporating the mCherry gene with a myc tag was added to the control under the control of a pan-neuronal promoter to evaluate biodistribution. These experiments showed that the hsa-mir-10b-5p binding sites in the off-target region of the RNA transcript (in this case the 3'UTR) substantially reduced off-target expression in the DRG while maintaining CNS expression. Separate validation of the lead DRG off-target element in mice showed a 5- to 16-fold reduction in DRG expression, while brain expression was not reduced.

[0032] Figure 7A , Figure 7B , Figure 7C and Figure 7D : The DRG off-target element in the AAV9 transcript rescued protein overexpression in the mouse DRG ( Figure 7A shows IHC, and Figure 7B shows the intensity values), with no effect on mouse brain expression ( Figure 7C shows IHC, and Figure 7D shows the intensity values). The 4x hsa-mir-10b-5p DRG off-target element (SEQ ID NO: 46) was evaluated in an AAV9 vector with a neuronal transgene under the control of a pan-neuronal promoter. IHC analysis after ICV administration to mice at P1 showed that the DRG off-target element achieved rescue of protein overexpression in the DRG (i.e., it reduced protein overexpression in the DRG to the level seen in control vehicle injections) without altering expression in brain sections.

[0033] Figure 8A , Figure 8B , Figure 8C , Figure 8D , Figure 8E and Figure 8F : Rescue of protein overexpression in NHP DRG tissue by the off-target element. Figure 8AShows the experimental design of an NHP study for evaluating a DRG off-target element (SEQ ID NO:46) in NHP. NHPs were injected ICV with the following: vehicle (Group 1); AAV with a pan-neuronal promoter that drives the expression of mRNA transcripts encoding neuronal genes (Group 3); or AAV with a pan-neuronal promoter that drives the expression of mRNA transcripts encoding neuronal genes, where the neuronal gene also contains SEQ ID NO:46 in the 3'UTR (DRG off-target sequence) (Group 5). Figure 8B Shows the molecular analysis of AAV transcript expression between Group 3 (promoter, denoted as "P" in the figure) and Group 5 (P + SEQ ID NO:46). Using vector-specific primers / probes, RT-ddPCR analysis was used to evaluate AAV-driven transcript expression in brain and DRG sections. Total RNA expression was normalized to the number of AAV genome copies in each section and expressed as fold change relative to the promoter-only condition. No differences in AAV-driven transgene expression were detected in the forebrain and midbrain, while a >10-fold decrease in AAV-mediated transcript expression was observed in DRG sections (cervical, thoracic, lumbar, and sacral were analyzed). Figure 8C Shows the vector biodistribution in cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal DRG segments. Expression in the DRG was mainly found in sacral DRG tissue. Shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal DRG segments is the protein expression driven by the transgene ( Figure 8D ) and the protein expression driven endogenously by MSD-EEISA ( Figure 8E ) (using specific reagents against the transgene-encoded protein or the endogenous protein). The figure shows that the presence of the DRG off-target element SEQ ID NO:46 in the mRNA transcript (which contains four copies of the hsa-mir-10b-5p binding site) reduces the expression of the transgene-encoded protein but does not significantly affect the expression of endogenous genes. Figure 8F Shows IHC staining of both the transgene-encoded protein and the endogenous protein in sacral (S) DRG sections. The figure shows that in the absence of the DRG off-target element in the transcript, protein expression in sacral DRG tissue is significantly higher (middle panel) compared to endogenous levels (left panel). The inclusion of the binding site for hsa-mir-10b-5p (a DRG off-target element) reduces the protein expression level to endogenous levels (right panel).

[0034] Figure 9 Is a scatter plot showing the screening for liver off-target elements, the log of brain activity versus liver activity 2Variations. Constructs selected as liver off-target elements are shown as dark squares. Control sequences (non-liver off-target) are shown as light squares.

[0035] Figure 10 is a graph showing the results of an ELISA assay comparing constructs in which the transcripts expressed therefrom do not have off-target elements (no-element control) with constructs in which the transcripts expressed therefrom contain the indicated liver off-target elements. SEQ ID NO:113 is a positive control sequence.

[0036] Figure 11A and Figure 11B show representative images that show in vivo expression of various constructs in the brain and liver using a CNS-specific promoter. Figure 11A show representative images of brain and liver expression of a myc-tagged transgene from: (i) mice treated with a control vector that expresses a transcript without off-target elements (encoding the myc-tagged protein) (upper panel; no off-target element); and (ii) mice treated with a vector that expresses a transcript containing SEQ ID NO:66 in its off-target region (encoding the myc-tagged protein) (lower panel). Figure 11A The AAV vectors in are delivered via tail vein injection. Figure 11B show representative images of brain and liver expression of a myc-tagged transgene from: (i) mice injected with a control vector by ICM that expresses a transcript without off-target elements (encoding the myc-tagged protein) under the control of a pan-neuronal promoter (upper panel; no off-target); (ii) mice treated with a vector that expresses a transcript containing SEQ ID NO:110 in its off-target region (encoding the myc-tagged protein) under the control of a pan-neuronal promoter (middle panel; SEQ ID NO:110 contains 2 copies of the hsa-mir-19a-3p binding site (SEQ ID NO:65), 2 copies of the hsa-mir-1258-5p binding site (SEQ ID NO:58), and 2 copies of the hsa-mir-17-5p binding site (SEQ ID NO:60)); and (iii) mice treated with a vector that expresses a transcript containing SEQ ID NO:112 in its off-target region (encoding the myc-tagged protein) under the control of a pan-neuronal promoter (lower panel; SEQ ID NO:112 contains 3 copies of the hsa-mir-122-3p binding site (SEQ ID NO:62)).

[0037] Figure 12A and Figure 12BShown in brain tissue ( Figure 12A ) and liver tissue ( Figure 12B ) in the brain tissue and the liver tissue from Figure 11B Mice in the ICM and mice injected with an AAV vector expressing myc-tagged proteins under the control of the ubiquitous chicken β-actin promoter ("CBA") and without off-target elements. Liver off-target activity of SEQ ID NO: 110 ("110") and SEQ ID NO: 112 ("112") was compared to expression from the CNS promoter (CNS) and the ubiquitous chicken β-actin promoter ("CBA") (*** and **** indicate significant differences in expression).

[0038] Figure 13 Figure 2 shows the relative expression (log fold change) of several different liver off-target elements in NHPs. 2 ). Data and averages of two different NHP animals (animal 1 and animal 2) are shown. SEQ ID NO: 55 is a randomized control.

[0039] Figure 14 A schematic diagram of the design and construction of a combined AAV library using selected DRG off-target elements and liver off-target elements disclosed herein is shown. The AAV vector structure is shown at the top and includes a promoter operably connected to a transgenic, the transgenic comprising a 3'UTR with an off-target region, the off-target region having three positions (position 1, 2 and 3), into which off-target elements or control / reference elements are inserted. All arrangements of elements are present in the AAV library. The SEQ ID NOs of the selected DRG, liver and control / reference elements are shown in the table in DNA and RNA form (DNA is a sequence in an AAV vector; RNA is a sequence in an mRNA transcript expressed from a vector).

[0040] Figure 15 Scatter plots showing the expression patterns of individual vectors from the AAV library in mouse tissues. 2 Brain expression activity (y axis) and log 2 DRG expression activity (x-axis), and the following figure compares the log 2 Brain expression activity (y axis) and log 2Liver expression activity (x-axis). In the upper figure, the highlighted vector points include one or more top DRG off-target elements. In the lower figure, the highlighted vector points include one or more top liver off-target elements. In both figures, the dark-colored vector points are those with only neutral control sequences. This data indicates that DRG and liver off-target elements are highly effective in reducing transgene expression in their respective tissues while maintaining expression in the brain tissue.

[0041] Figure 16 The effects of individual elements in an AAV combinatorial library on transgene expression in the DRG, liver, and forebrain tissues of mice injected with the combinatorial library are shown. These values were determined by recovering transcriptional libraries from DRG, liver, and brain tissue sections using amplicon sequencing of RNA / AAV DNA and NGS quantification for differential expression. A regression-based model of tissue expression shows the most contributing elements among thousands of data points / instances. The data is represented as log2 fold change in the specified tissue in the presence and absence of off-target elements identified by SEQ ID NO on the x-axis. Except for SEQ ID NO:55 (which is the DNA sequence of a random control element in the AAV vector library), each SEQ ID NO represents an RNA sequence present in the off-target region of the expressed transcript. SEQ ID NO:119 is the DRG off-target benchmark, and SEQ ID NO:113 is the liver off-target benchmark. The modeling results identified the top 3'UTR sequence elements with selective DRG and / or liver off-target while maintaining expression in the brain.

[0042] Figure 17 : The combined off-target sequence elements show a range of off-targets in both the liver and DRG (scatter plot, upper figure). Candidate combined off-target regions that exhibit strong off-targets in both the DRG and liver were identified (dark-colored circles highlighted in the scatter plot). These candidate combined off-target regions include at least one DRG and at least one liver off-target element in the off-target region of the 3'UTR. The selected combined off-target elements have no negative impact on transgene expression in the brain tissue (lower figure). This figure shows the log 2 fold change in the specified tissue in the presence and absence of combined off-target elements identified by three SEQ ID NOs on the x-axis. Except for SEQ ID NO:55 and 56 in the last column (which are the DNA sequences of random control elements in the AAV vector library), these three SEQ ID NOs represent RNA sequences present in the off-target region of the expressed transcript.

[0043] Figure 18 : Combinatorial analysis for multiplex validation of DRG off-target sequences in NHP. NHP (n = 2) were administered via intracerebroventricular (ICV) injection asFigure 14 The combined AAV library of the shown design. The DRG off-target coefficient was determined from highly transduced samples from each of the following tissues as Figure 14 described: DRG (sacral section) and brain (hippocampal section). Figure 18 A regression-based model of differential expression is shown to describe the off-target contribution of each element at all points / instances in the recovered library. The SEQ ID NO on the x-axis represents the RNA sequence present in the off-target region of the expressed transcript, except for SEQ ID NO:55 and 56 in the last two columns (which are the DNA sequences of random control elements in the AAV vector library).

[0044] Figure 19 : The correlation of the effect of elements in the combined AAV library on expression in brain, DRG, and liver tissues between mice and NHP is shown. As Figure 19 shown, for each off-target element, the Pearson correlation between the mouse and NHP expression coefficients is 0.86 for brain expression, 0.91 for liver expression, and 0.79 for DRG expression.

[0045] Definition

[0046] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, with respect to the terms "comprising", "including", "having", "carrying", or variants thereof as used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0047] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. This term encompasses all serotypes, subtypes, and both naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes all serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV 12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8, as well as their hybrids (i.e., chimeric AAV vectors), and avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of the 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 public databases such as GenBank. As used herein, an "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence of non-AAV origin (i.e., a polynucleotide heterologous to AAV), wherein the polynucleotide sequence is typically the sequence of interest for cellular genetic transformation. Generally, the heterologous polynucleotide is flanked by at least one, and usually two, AAV inverted terminal repeats (ITRs). rAAV vectors can be single-stranded vectors (ssAAV) or self-complementary vectors (scAAV). See, e.g., Raj et al., Expert Rev Hematol. October 2011, 4(5):539-549. An "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and a packaged polynucleotide rAAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is typically referred to as an "rAAV viral particle" or simply an "rAAV particle". AAV can contain genetic components and capsids from multiple serotypes (e.g., pseudotyped vectors). For example, AAV can contain a serotype 2 genome (e.g., ITRs) encapsulated in a capsid from serotype 5 or serotype 9. Pseudotyped vectors can exhibit improved transduction efficiency and altered tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect CNS cells are preferred. In some aspects, the recombinant AAV vector is AAV1, AAV8, AAV9, AAVDJ, or a chimeric AAV that incorporates features of two or more of these serotypes. In various embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector.In certain embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector and comprises a heterologous nucleic acid flanked by ITRs from a serotype other than AAV9 among the AAV serotypes. In certain embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector and comprises a heterologous nucleic acid flanked by AAV serotype 2 ITRs (i.e., ITR2).

[0048] The term “about” or “approximately” means within an acceptable error range of a particular value as determined by a person 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 measuring system. For example, in accordance with the practice in the art, “about” can mean within one or more standard deviations. Alternatively, “about” can mean a range that varies up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% from a given value.

[0049] In any of the embodiments described herein, “comprising” can be replaced with “consisting essentially of” or “consisting of”. For example, an embodiment that includes a particular element using the open-ended term “comprising” encompasses embodiments that include that element using the more restrictive terms “consisting essentially of” or “consisting of”.

[0050] The terms “determine”, “measure”, “evaluate”, “assess”, “assay”, “analyze” and their grammatical equivalents can be used interchangeably herein to refer to any form of measurement and include determining whether an element is present (e.g., detected). These terms can include quantitative and / or qualitative determinations.

[0051] An assessment can be relative or absolute.

[0052] The term “expression” refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (such as transcribed into mRNA or an encoded RNA transcript), and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a “gene product”. If the polynucleotide includes introns or splice sites, such as derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0053] An “expression cassette” refers to a nucleic acid molecule that contains one or more regulatory elements operably linked to a sequence to be expressed as an RNA transcript that comprises an mRNA (i.e., an RNA molecule that includes a coding sequence (e.g., one or more genes) for expressing a protein) or a non-coding RNA.

[0054] "Transgene" refers to a part of a nucleic acid cassette designed to be expressed in a cell. In some embodiments, the transgene encodes an RNA transcript, such as mRNA or non-coding RNA (e.g., antisense RNA). In some embodiments, the transgenes of the present disclosure encode a therapeutic cargo (e.g., a therapeutic protein or a therapeutic RNA), and further comprise one or more DRG and / or liver off-target sequences / elements to reduce the expression of the transgene in DRG and / or hepatocytes.

[0055] "RNA transcript" refers to an RNA molecule transcribed from a template, such as an RNA molecule transcribed from an expression cassette as described herein. The RNA transcripts expressed from the expression cassettes described herein can be in any desired form, including mRNA encoding a polypeptide / protein or RNA that performs its desired function without serving as a template for protein expression (also referred to as non-coding RNA (ncRNA)). Examples of ncRNA include, but are not limited to: microRNA (miRNA or miR), primary microRNA (pri-miRNA or pri-miR), precursor microRNA (pre-miRNA or pre-miR), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), antisense RNA (asRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme, CRISPR guide RNA (gRNA), etc.

[0056] The term "effective amount" or "therapeutically effective amount" refers to an amount of the compositions described herein sufficient to affect the intended application, including but not limited to the treatment of diseases as defined below. The therapeutically effective amount can vary depending on the intended therapeutic application (in cells or in vivo), or the subject being treated and the disease condition (e.g., the weight and age of the subject, the severity of the disease condition, the mode of administration, etc.), which can be readily determined by one of ordinary skill in the art. The term also applies to the dose that will induce a specific response in the target cell. The specific dose will vary depending on the specific composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carrying the specific dose.

[0057] A "fragment" of a nucleotide or peptide sequence is intended to refer to a sequence that is less than the sequence considered to be the "full-length" sequence.

[0058] A "functional fragment" of a DNA, RNA or protein sequence refers to the following biologically active fragments of the sequence: those that are shorter than the full-length or reference DNA, RNA or protein sequence, but retain at least one biological activity (functionally or structurally) that is substantially similar to that of the full-length or reference DNA, RNA or protein sequence. For example, a "functional fragment" can be a fragment of the sequences disclosed herein that reduces the expression of a transgene operably linked thereto in DRG and / or hepatocytes.

[0059] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", including primary transformed cells and their progeny derived therefrom (regardless of the number of passages). The progeny may not be completely identical to the parental cell in terms of nucleic acid content and may instead contain mutations. This includes mutant progeny having the same function or biological activity as that selected or screened for in the originally transformed cell.

[0060] As used herein, the term "derived from a human" refers to a sequence found in the human genome (or a human genomic construct), or a sequence homologous thereto. A homologous sequence can be a sequence that includes a region having at least 80% sequence identity (e.g., as measured by BLAST) compared to a region of the human genome. For example, sequences 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 are considered to be derived from a human. In some cases, a regulatory element contains sequences that are derived from a human and sequences that are not derived from a human, such that overall, the regulatory element has low sequence identity to the human genome, while a portion of the regulatory element has 100% sequence identity (or local sequence identity) to a sequence in the human genome.

[0061] The term "in vitro" refers to an event that occurs outside the body of a subject. For example, in vitro assays encompass any assay that is run outside of a subject. In vitro assays encompass cell-based assays in which live or dead cells are employed. In vitro assays also encompass cell-free assays in which intact cells are not employed.

[0062] The term "in vivo" refers to an event that occurs inside the body of a subject.

[0063] "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is extrachromosomal, located at a chromosomal location different from its natural chromosomal location, or contains only the coding sequence.

[0064] As used herein, "operably linked", "operably connect", "operably connected" or grammatical equivalents thereof refer to the juxtaposition of genetic elements (e.g., promoters, enhancers, polyadenylation sequences, etc.), where these elements are in a relationship that permits them to function in the intended manner. For example, a regulatory element that can contain a promoter and / or enhancer sequence is operably linked to a coding region if it facilitates the transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region, as long as this functional relationship is maintained.

[0065] "Pharmaceutically acceptable carrier" refers to a non-active ingredient in a pharmaceutical formulation or composition thereof that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0066] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that permits the bioactivity of the active ingredient(s) contained therein to be effective and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered.

[0067] The term "regulatory element" refers to a nucleic acid sequence or genetic element that can affect (e.g., increase, decrease, or modulate) the expression of an operably linked sequence such as a gene, coding sequence, or RNA (e.g., mRNA or ncRNA). Regulatory elements include, but are not limited to, promoters, enhancers, repressors, silencers, insulator sequences, introns, UTRs, inverted terminal repeat (ITR) sequences, long terminal repeat sequences (LTRs), stabilizing elements, miRNA target sites, post-translational response elements, polyA sequences, or combinations thereof. Regulatory elements can act at the DNA and / or RNA level, e.g., by regulating gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression; by regulating translation levels (e.g., stabilizing elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcriptional termination; by recruiting transcription factors to the coding region that increases gene expression; by increasing the rate of RNA transcript production, 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 facilitate protein synthesis. In one exemplary embodiment, the regulatory element refers to an enhancer, repressor, promoter, or combination thereof, particularly a combination of enhancer plus promoter, or a combination of repressor plus promoter. In an exemplary embodiment, the regulatory element is derived from a human sequence.

[0068] Generally speaking, the terms "sequence identity" or "sequence homology", which can be used interchangeably, refer respectively to the exact nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotide or polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity" (also referred to as "percent homology"). The percent identity with a reference sequence (e.g., a nucleic acid or amino acid sequence) can be calculated as the number of exact matches between the two best aligned sequences divided by the length of the reference sequence, multiplied by 100. In determining the number of matches for calculating sequence identity, conservative substitutions are not considered as matches. It should be understood that when the length of the first sequence (A) is not equal to the length of the second sequence (B), the percent identity of the A:B sequences will be different from the percent identity of the B:A sequences. Sequence alignments, such as for the purpose of assessing 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., the EMBOSS Water aligner), and the Clustal Omega alignment program (F. Sievers et al., Mol Sys Biol. 7:539 (2011)). Any suitable parameters of the selected algorithm (including default parameters) can be used to evaluate the best alignment. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and is discussed in the following references: 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).

[0069] The terms "subject" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The methods described herein can be used for human therapy, veterinary applications, and / or preclinical studies in animal models of diseases or disorders.

[0070] As used herein, the terms “treatment,” “therapeutic,” etc. refer to obtaining a desired pharmacological and / or physiological effect, including but not limited to: alleviating, delaying or slowing progression, attenuating the impact or symptoms, preventing the onset of a disease or disorder, preventing recurrence of a disease or disorder, inhibiting, ameliorating the onset of a disease or disorder, obtaining a beneficial or desired outcome with respect to a disease, disorder or medical condition, such as a therapeutic benefit and / or a prophylactic benefit. As used herein, “treatment” encompasses any treatment of a disease in a mammal, particularly a human, including: (a) preventing the disease from occurring in a subject that may be predisposed to the disease or at risk of developing the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. A therapeutic benefit includes eradicating or ameliorating the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder. In some cases, to obtain a prophylactic benefit, these compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the disease may not yet have been diagnosed. The methods of the present disclosure can be used in any mammal. In some cases, treatment can result in alleviation or cessation of symptoms. Prophylactic effects include delaying or eliminating the appearance of a disease or disorder, delaying or eliminating the onset of one or more symptoms of a disease or disorder, slowing, halting or reversing the progression of a disease or disorder, or any combination of these prophylactic effects.

[0071] A “variant” of a nucleotide sequence refers to a sequence that has a genetic alteration or mutation as compared to the most common wild-type DNA sequence (e.g., a cDNA or the sequence referred to by its GenBank accession number) or a designated reference sequence. The variant can be shorter than the reference sequence and / or can have one or more mutations relative to the reference sequence. In some cases, the variant can have a nucleotide sequence that is at least 80% identical, at least 90% identical or at least 95% identical to the reference sequence.

[0072] As used herein, a “vector” refers to a nucleic acid molecule that can be used to mediate delivery of another nucleic acid molecule linked thereto into a cell, where the other nucleic acid molecule can be replicated or expressed. The term includes vectors as self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as “expression vectors.” Other examples of vectors include plasmids and viral vectors.

[0073] 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 not desired. As used herein, "off-target" generally refers to reduced expression in non-target cells.

[0074] Unless otherwise indicated, all terms used herein have the same meaning to those skilled in the art, and the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology, which are within the knowledge of those skilled in the art. Detailed Description

[0075] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as such specific embodiments may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0076] The upper and lower limits of a range may independently be included in the range and are also covered by the present invention, subject to any explicit excluded limits in the stated range. Where the stated range includes one or both of these limits, ranges excluding either or both of the included limits are also included in the present invention.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention 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 now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited.

[0078] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins, reference to "the nucleic acid" includes reference to one or more nucleic acids, and equivalents thereof known to those skilled in the art, and so forth. Also note that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of exclusive terms, such as "solely", "only", etc., in connection with the recitation of claim elements, or use of "negative" limitations.

[0079] It should be understood that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the invention are specifically covered by the invention and are disclosed herein as if each combination were separately and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are also clearly included in the invention and are disclosed herein as if each such sub-combination were separately and explicitly disclosed herein.

[0080] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing in this application should be construed as an admission that the invention is not entitled to antedate such publications by virtue of prior invention.

[0081] In addition, the dates of publication provided may be different from the actual dates of publication, which may need to be independently confirmed.

[0082] Aspects of the present disclosure provide nucleic acid molecules that include one or more DRG off-target elements, one or more liver off-target elements, or a combination of both. In certain embodiments, the nucleic acid molecule is an RNA molecule that further includes a heterologous RNA sequence, e.g., an RNA sequence encoding a protein or non-coding RNA (ncRNA). In some embodiments of these embodiments, the heterologous RNA is a therapeutic RNA, e.g., encoding a therapeutic protein or an ncRNA having a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule that further includes a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript containing one or more off-target elements and a heterologous RNA sequence. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript that includes one or more off-target elements and a heterologous RNA sequence. As described in detail herein and outlined above, the presence of one or more DRG off-target elements in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in DRG cells (e.g., DRG cells of a subject) compared to an RNA molecule having a heterologous RNA sequence without one or more DRG off-target elements. Similarly, the presence of one or more liver off-target elements in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in hepatocytes (e.g., hepatocytes of a subject) compared to an RNA molecule having a heterologous RNA sequence without one or more liver off-target elements. In addition, the presence of one or more DRG off-target elements and one or more liver off-target elements in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in DRG cells and hepatocytes (e.g., DRG cells and hepatocytes of a subject) compared to an RNA molecule having a heterologous RNA sequence without one or more DRG off-target elements and liver off-target elements.

[0083] Aspects of the present disclosure provide nucleic acid molecules that comprise one or more regions that hybridize, under physiological conditions (e.g., in a subject's cells), to hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, hsa-mir-494-3p, or any combination thereof (each of which is an off-target element of the present disclosure). These hybridization regions can be designated as binding sites for specific miRNAs. In certain embodiments, the nucleic acid molecule is an RNA molecule that further comprises a heterologous RNA sequence, e.g., an RNA sequence that encodes a protein or a non-coding RNA (ncRNA). In some of these embodiments, the heterologous RNA is a therapeutic RNA, e.g., an RNA sequence that encodes a therapeutic protein or an ncRNA having a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule that further comprises a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript that contains one or more binding sites. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript that comprises one or more off-target elements and a heterologous RNA sequence. As detailed herein and outlined above, the presence of one or more regions (which hybridize, under physiological conditions, to hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, hsa-mir-494-3p, or any combination thereof) in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in DRG cells (e.g., a subject's DRG cells) compared to an RNA molecule having a heterologous RNA sequence without one or more hybridization regions. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize, under physiological conditions, to hsa-mir-196b-5p (SEQ ID NO:21). Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize, under physiological conditions, to hsa-mir-10b-5p (SEQ ID NO:22). Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize, under physiological conditions, to hsa-mir-24-2-5p (SEQ ID NO:23).Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize to hsa-mir-183-3p (SEQ ID NO:24) under physiological conditions. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize to hsa-mir-196a-5p (SEQ ID NO:25) under physiological conditions. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more regions that hybridize to hsa-mir-494-3p (SEQ ID NO:26) under physiological conditions.

[0084] In some aspects, the present disclosure provides nucleic acid molecules that comprise one or more binding sites for hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, hsa-mir-494-3p, or any combination thereof, each of which is an off-target element of the present disclosure. In certain embodiments, the nucleic acid molecule is an RNA molecule that further comprises a heterologous RNA sequence, e.g., an RNA sequence that encodes a protein or a non-coding RNA (ncRNA). In some of these embodiments, the heterologous RNA is a therapeutic RNA, e.g., an RNA that encodes a therapeutic protein or an ncRNA having a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule that further comprises a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript that contains one or more binding sites. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript that comprises one or more off-target elements and a heterologous RNA sequence. As described in detail herein and outlined above, the presence of one or more binding sites for hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, hsa-mir-494-3p, or any combination thereof in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in DRG cells (e.g., DRG cells of a subject) compared to an RNA molecule having a heterologous RNA sequence without one or more binding sites. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-196b-5p. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-10b-5p. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-24-2-5p. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-183-3p. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-196a-5p. Accordingly, aspects of the present disclosure include nucleic acid molecules that comprise one or more binding sites for hsa-mir-494-3p.

[0085] Aspects of the present disclosure provide nucleic acid molecules that include one or more regions that hybridize, under physiological conditions, to hsa-mir-19a-3p (SEQ ID NO:95), which is an off-target element of the present disclosure. Aspects of the present disclosure provide nucleic acid molecules that include one or more binding sites for hsa-mir-19a-3p. In certain embodiments, the nucleic acid molecule is an RNA molecule that further includes a heterologous RNA sequence, e.g., an RNA sequence that encodes a protein or non-coding RNA (ncRNA). In some embodiments of these embodiments, the heterologous RNA is a therapeutic RNA, e.g., an RNA that encodes a therapeutic protein or an ncRNA having a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule that further includes a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript that contains one or more hsa-mir-19a-3p binding sites. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript that includes one or more hsa-mir-19a-3p binding sites and a heterologous RNA sequence. As detailed herein and outlined above, the presence of one or more hsa-mir-19a-3p binding sites in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in hepatocytes (e.g., hepatocytes of a subject) compared to an RNA molecule that contains a heterologous RNA sequence without one or more hsa-mir-19a-3p binding sites.

[0086] In some embodiments, the nucleic acid molecules of the present disclosure include one or more of the above hybridization regions / miRNA binding sites (in any combination), and one or more additional hybridization regions / miRNA binding sites (e.g., that are off-target for a cell or tissue of interest, such as a cell or tissue as described herein). In some embodiments, the nucleic acid molecules of the present disclosure include one or more of the above hybridization regions and / or miRNA binding sites (in any combination), and one or more additional off-target elements (e.g., for DRG and / or liver, as described herein). There is no limitation in this regard.

[0087] As outlined above, the present disclosure describes a nucleic acid cassette that contains a transgene encoding RNA, where the RNA contains the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a functional fragment of the foregoing sequence; or (iii) a sequence that is at least 80% identical to (i) or (ii), or any combination thereof. The transgene can encode a protein-coding mRNA, or a non-coding RNA (such as pri-miRNA, pre-miRNA, or miRNA), an antisense RNA, a short non-coding RNA, a long non-coding RNA, a snoRNA, a snRNA, a tRNA, or an rRNA. In some cases, the nucleic acid cassette contains a transgene encoding an mRNA, where the mRNA contains the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a functional fragment of the foregoing sequence; or (iii) a sequence that is at least 80% identical to (i) or (ii), or any combination thereof. These sequences reduce the expression of the transgene in dorsal root ganglion cells (DRG) relative to its expression in target cells (such as nerve cells, e.g., neurons), and thus, can be employed in a variety of gene therapy strategies targeting cells that are not in the DRG. Reducing the expression of the transgene in DRG cells relative to its expression in target cells means that the reduction in transgene expression driven by the DRG off-target sequences disclosed herein is greater in DRG cells than in target cells. Thus, while a reduction in transgene expression in target cells can be observed in certain embodiments, it is less than the reduction in transgene expression observed in DRG cells. This reduction in expression in the DRG can mitigate or eliminate DRG toxicity and / or axonal pathologies in subjects receiving gene therapy targeting non-DRG cells or tissues (such as nerve cells, e.g., neurons), thereby improving their safety profile.

[0088] The present disclosure also provides an RNA molecule having the sequence characteristics of an 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, for example, administered to a subject as a pharmaceutical composition. The RNA compositions can be used in a variety of therapeutic modalities delivered using a wide range of viral and non-viral delivery systems, including polymeric materials, ionizable lipids, cell-penetrating lipids and zwitterionic lipids, nanoparticles, and dendrimers (see, e.g., Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery”, Molecular Therapy, Vol. 27, No. 4, 2019, pp. 710-728; and Paunovska et al., “Drug delivery systems for RNA therapeutics”, Nature Reviews genetics, Vol. 23, 2022, pp. 265-280).

[0089] The RNA (e.g., mRNA or ncRNA) encoded by the transgene of the nucleic acid cassette can contain any combination of two, three, four, five, or more of the foregoing sequences. For example, the RNA comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a variant or functional fragment of the foregoing sequence; or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii). It may further comprise a second sequence (i), (ii), or (iii), a third sequence (i), (ii), or (iii), a fourth sequence (i), (ii), or (iii), and / or five or more sequences (i), (ii), or (iii). In any embodiment, the nucleic acid cassette can comprise two or more copies (e.g., two, three, four, five, or more than five copies) of the sequence (i), (ii), or (iii).

[0090] In certain embodiments, the RNA encoded by the transgene of the nucleic acid cassette can contain: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants or functional fragments of the foregoing sequences; or (iii) sequences that are 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).

[0091] In any embodiment, the sequence can be located, for example, in the 3'UTR, 5'UTR, or intron of the mRNA. If the mRNA contains more than one of the foregoing sequences, these sequences can be located in different parts of the mRNA. However, in many embodiments, these sequences are located in the 3'UTR of the mRNA. In these embodiments, the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) can be located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

[0092] Any nucleic acid described herein can be non-naturally occurring, where the term "non-naturally occurring" refers to a composition that does not exist in nature. In some aspects, the non-naturally occurring nucleic acid contains a continuous, uninterrupted nucleotide sequence that does not exist in nature, i.e., it is different from any nucleic acid in its native state (i.e., has less than 100% sequence identity with a naturally occurring nucleic acid sequence). Two regions of a non-naturally occurring nucleic acid are "heterologous" to each other if they are derived from separate genomic regions that are not found to be continuous, uninterrupted nucleic acid sequences in their native state. For example, in some embodiments, the nucleic acid cassette can consist of a promoter, a coding sequence, a sequence encoding a DRG off-target element (as disclosed herein), and a terminator, where the promoter, the coding sequence, the sequence encoding the DRG off-target element, and the terminator are operably linked. In these embodiments, at least one of these elements is heterologous to another of these elements. For example, the coding sequence can be heterologous to the sequence encoding the DRG off-target element, meaning that the sequence encoding the DRG off-target element is not operably linked to the coding sequence in the same manner in wild-type cells. In any embodiment, the nucleic acid cassette can additionally contain an enhancer.

[0093] In any embodiment, the RNA encoded by the transgene of the nucleic acid cassette may comprise a functional fragment of any one of SEQ ID NOs: 1-10 and 43-48, wherein the functional fragment reduces the expression of the RNA operably linked thereto in the DRG. Additionally, the functional fragment may or may not contain mismatches relative to SEQ ID NOs: 1-8, for example, one, two, three, four or more mismatches.

[0094] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of SEQ ID NO:1 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, the functional fragment of SEQ ID NO:1 contains one, two, three or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:1. The functional fragment may begin at any nucleotide in SEQ ID NO:1 that permits its full representation in SEQ ID NO:1.

[0095] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of 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, at least 18, at least 19, at least 20, at least 21 or at least 22 nucleotides in length. In certain embodiments, the functional fragment of SEQ ID NO:2 contains one, two, three or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:2. The functional fragment may begin at any nucleotide in SEQ ID NO:2 that permits its full representation in SEQ ID NO:2.

[0096] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of SEQ ID NO:3 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, the functional fragment of SEQ ID NO:3 contains one, two, three or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:3. The functional fragment may begin at any nucleotide in SEQ ID NO:3 that permits its full representation in SEQ ID NO:3.

[0097] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of SEQ ID NO:4 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, the functional fragment of SEQ ID NO:4 comprises one, two, three, or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:4. The functional fragment can begin at any nucleotide in SEQ ID NO:4 that permits its full representation in SEQ ID NO:4.

[0098] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of SEQ ID NO:5 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, the functional fragment of SEQ ID NO:5 comprises one, two, three, or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:5. The functional fragment can begin at any nucleotide in SEQ ID NO:5 that permits its full representation in SEQ ID NO:5.

[0099] In certain embodiments, the functional fragment comprises any contiguous nucleotide segment of SEQ ID NO:6 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, the functional fragment of SEQ ID NO:6 comprises one, two, three, or four mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:6. The functional fragment can begin at any nucleotide in SEQ ID NO:6 that permits its full representation in SEQ ID NO:6.

[0100] In certain embodiments, the functional fragment comprises a length of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, 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 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, 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, at least 151, at least 152, at least 153, at least 154, at least 155 of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.Any contiguous nucleotide segment of 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, at least 176, at least 177, at least 178, at least 179, 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, at least 216, at least 217, at least 218, at least 219, at least 220, at least 221, at least 222, at least 223 or at least 224 nucleotides. In certain embodiments, the functional fragment of SEQ ID NO:7 or SEQ ID NO:8 contains one, two, three, four, five, six, seven, eight, nine or ten mismatches compared to the corresponding contiguous nucleotide segment in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. The functional fragment can begin at any nucleotide in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0101] In some embodiments, the RNA may comprise miRNA binding sites for miRNAs selected from hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p or hsa-mir-494-3p or their complementary sequences. In certain embodiments, the RNA may comprise one or more binding sites for miRNAs. In these embodiments, the RNA may comprise 6, 7, 8, 9 or 10 contiguous nucleotides that potentially base pair with the seed region of the miRNA, which is, for example, hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p or hsa-mir-494-3p (which is at the 5' end of those miRNAs). In some embodiments, the RNA may comprise 6, 7, 8, 9 or 10 contiguous nucleotides at the 3' end of any one of SEQ ID NOs: 1-6 that potentially base pair with the seed region of the miRNA, which is, for example, hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p or hsa-mir-494-3p (which is at the 5' end of these miRNAs). One or more binding sites for miRNAs may include any one of SEQ ID NOs: 1-6. In some embodiments, the sequence may be the same as SEQ ID NOs: 1-6, except having, for example, one, two, three or four mismatches relative to SEQ ID NOs: 1-6.

[0102] Specifically, sequence (i), (ii) or (iii) may provide a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p and hsa-mir-494-3p.

[0103] Obviously, the nucleic acid cassette itself (which is DNA) can contain: (i) a DNA form of any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments of the foregoing sequences, or combinations 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 inclusion of the sequence reduces the expression of the protein or RNA encoded by the cassette in DRG cells of an organism relative to its expression in a target tissue (e.g., neural tissue, e.g., neuronal cells in the brain).

[0104] In some embodiments, the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments of the foregoing sequences, or combinations thereof; or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii), can cause a reduction in the expression of ncRNA or a polypeptide encoded by mRNA in DRG cells compared to the expression of ncRNA or a polypeptide encoded by mRNA that is otherwise identical but lacks sequence (i), (ii), or (iii). For example, an mRNA containing sequence (i), (ii), or (iii) can cause the expression level of the polypeptide encoded by the mRNA in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the polypeptide encoded by an mRNA that is otherwise identical but lacks sequence (i), (ii), or (iii). In these embodiments, the reduction in the expression of the polypeptide in DRG cells, when compared to an mRNA that is otherwise identical but lacks sequence (i), (ii), or (iii), is greater than the reduction in the expression of the polypeptide in target cells. Similarly, an ncRNA containing sequence (i), (ii), or (iii) can cause the expression level in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of an ncRNA that lacks sequence (i), (ii), or (iii) in DRG cells. In these embodiments, the reduction in the expression of the ncRNA in DRG cells, when compared to an ncRNA that is otherwise identical but lacks sequence (i), (ii), or (iii), is greater than the reduction in the expression of the ncRNA in target cells.

[0105] In some embodiments, the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) can cause a reduction in the expression level of the polypeptide encoded by the mRNA, the mRNA itself, or the ncRNA in DRG cells by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the polypeptide, mRNA, or ncRNA of an RNA transcript that is otherwise identical but lacks sequence (i), (ii), or (iii) in DRG cells. In these embodiments, the reduction in the expression of the polypeptide or RNA transcript in DRG cells is greater than the reduction in the expression of the polypeptide in target cells when compared to an RNA transcript that is otherwise identical but lacks sequence (i), (ii), or (iii).

[0106] In some embodiments, the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) sequences that are 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) do not cause a significant decrease in the expression of the polypeptide, mRNA, or ncRNA encoded by the mRNA in the target cell compared to the expression of the polypeptide, mRNA, or ncRNA of an RNA transcript that is otherwise identical but does not have sequence (i), (ii), or (iii) in the target cell. In some embodiments, sequence (i), (ii), or (iii) can cause the expression level of the polypeptide, mRNA, or ncRNA encoded by the mRNA in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the polypeptide, mRNA, or ncRNA of an RNA transcript that is otherwise identical but does not have sequence (i), (ii), or (iii) in the target cell. In these embodiments, the decrease in the expression of the polypeptide in DRG cells, when compared to an RNA transcript that is otherwise identical but does not have sequence (i), (ii), or (iii), is greater than the decrease in the expression of the polypeptide, mRNA, or ncRNA in the target cell.

[0107] In some embodiments, the RNA transcript is a therapeutic RNA transcript. In some aspects, the therapeutic RNA transcript is an mRNA that contains a sequence encoding a polypeptide (e.g., a therapeutic protein), which can be, for example, an intracellular protein, a membrane-bound protein, or a secreted protein. In some embodiments, the therapeutic protein is a protein associated with a neurological disease or disorder, e.g., a protein whose abnormal function (e.g., caused by a genetic mutation or aberration) is associated with a neurological disease or disorder. In further embodiments, the therapeutic RNA transcript contains an ncRNA sequence that targets an endogenous molecule (e.g., a gene, a protein, or an RNA) associated with a neurological disease or disorder.

[0108] Neurological diseases and disorders include those associated with one or more genetic mutations, and those of unknown etiology. In some embodiments, neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X chromosome syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), infantile epilepsy with migratory focal epileptic seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and slow waves in sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0109] Many genetic abnormalities have long been associated with epilepsy, including many of the aforementioned neurological diseases and disorders. Examples of genes affected by these genetic abnormalities (i.e., genes whose activity and / or expression have been 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, 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.

[0110] Thus, in embodiments where the RNA transcript is an mRNA that includes a sequence encoding a therapeutic protein for treating a neurological disease or disorder, the therapeutic protein can be: (i) a functional form of a protein encoded by a gene selected from: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1GABRB3, 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 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 regulates the expression of the gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0111] In some cases, the target cell can be a nerve cell, muscle cell, heart cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In any of these embodiments, the target cell can be a nerve cell, for example, a brain cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in these embodiments, the nerve cell can be a GABAergic cell, for example, a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, dopaminergic neuron, glial cell, ependymal cell, oligodendrocyte, astrocyte, microglial cell, motor neuron, vascular cell, GABAergic neuron, or non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0112] The cassette can be linear, circular, and in some embodiments, the nucleic acid cassette can be a vector, such as a plasmid or viral vector, for example, an adeno-associated virus (AAV) vector or a lentiviral vector. In a specific embodiment, the viral vector can be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, AAV-DJ8, and their hybrids.

[0113] Also provided is a nucleic acid cassette that contains a transgene encoding an RNA transcript, where the RNA contains miRNA binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, mir-183-3p, mir-196a-5p, mir-494-3p, or their complementary sequences. In some embodiments, the RNA may contain binding sites for miRNAs produced from the mir-196b, mir-10b, mir-24-2, mir-183, mir-196a, or mir-494 genes. If the RNA is otherwise naturally occurring, the miRNA binding sites should not be present in the naturally occurring form of the RNA. In some embodiments, the cassette may contain two or more, three or more, or four or more binding sites for miRNAs selected from, for example, mir-196b-5p, mir-10b-5p, mir-24-2-5p, mir-183-3p, mir-196a-5p, mir-494-3p, or their complementary sequences. In some embodiments, the RNA is mRNA, for example, mRNA encoding a therapeutic protein (as described elsewhere herein). In these embodiments, the binding sites can be at any position in the mRNA, particularly in non-coding sequences such as the 3'UTR region, 5'UTR region, introns, or any combination thereof.

[0114] In any embodiment, the nucleic acid cassette can be non-naturally occurring, which means that, for example, the miRNA binding sites in the RNA transcript expressed from the nucleic acid cassette are heterologous to one or more other regions of the RNA transcript. In any embodiment, the nucleic acid cassette can contain a promoter and / or an enhancer. In some embodiments, the nucleic acid cassette can consist of a promoter, a coding sequence, and a terminator, where the promoter, coding sequence, and terminator are operably linked. In these embodiments, the promoter can be heterologous to the coding sequence, which means that the promoter does not drive the expression of the coding sequence in wild-type cells. In any embodiment, the nucleic acid cassette can additionally contain an enhancer.

[0115] In some embodiments, the mRNA can encode a polypeptide, for example, a therapeutic protein, which can be, for example, an intracellular protein, a membrane-bound protein, or a secreted protein.

[0116] In some embodiments, the polypeptide is a therapeutic protein whose altered function (e.g., altered by a genetic mutation) is associated with a neurological disease or disorder. As mentioned above, neurological diseases and disorders include those associated with one or more genetic mutations, as well as those of unknown etiology. Examples of neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or conditions include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, Fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0117] In these embodiments, the therapeutic protein can be: (i) a protein encoded by a gene selected from: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, 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 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0118] In some cases, the target cell can be a nerve cell, muscle cell, heart cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In any of these embodiments, the target cell can be a nerve cell, for example, a brain cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in these embodiments, the nerve cell can be a GABAergic cell, for example, a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, dopaminergic neuron, glial cell, ependymal cell, oligodendrocyte, astrocyte, microglial cell, motor neuron, vascular cell, GABAergic neuron, or non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0119] The cassette can be linear, circular, and in some embodiments, the nucleic acid cassette can be a vector, such as a plasmid or viral vector, for example, an adeno-associated virus (AAV) vector or a lentiviral vector. In a specific embodiment, the viral vector can be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, AAV-DJ8, and their hybrids.

[0120] Also provided is an RNA transcript encoded by the nucleic acid cassette described herein.

[0121] There is also provided a method of reducing the expression of a polypeptide, mRNA or ncRNA encoded by mRNA in DRG relative to the expression of the polypeptide, mRNA or ncRNA in a target tissue. In these embodiments, the method may include constructing a nucleic acid cassette to include a DRG off-target sequence as described herein in the RNA transcript encoded therein. For example, the nucleic acid cassette may be constructed to include in the RNA transcript encoded therein: (i) one of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment or combination thereof of the foregoing sequences; 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). Details of the cassette prepared by this method are described herein. In some embodiments, the method may include introducing the expression cassette as described herein or the RNA encoded thereby into an organism (e.g., a human subject), wherein the inclusion of any one or more of the disclosed DRG off-target sequences reduces the expression of the protein in the DRG cells of the organism relative to the target tissue.

[0122] In any of the embodiments herein, the target cell can be a nerve cell, muscle cell, heart cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell or kidney cell. In some cases, the target cell can be a nerve cell, e.g., a brain cell, brain stem cell, hippocampal cell or cerebellar cell. For example, in some embodiments, the nerve cell is a GABAergic cell, e.g., a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, dopaminergic neuron, glial cell, ependymal cell, oligodendrocyte, astrocyte, microglial cell, motor neuron, vascular cell, GABAergic neuron or non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin) or other CNS cells (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST and VIP).

[0123] In any embodiment, the method may further include administering to a subject a vector (e.g., an AAV or lentiviral vector) encoding the RNA transcript, e.g., wherein the RNA transcript is an mRNA encoding a therapeutic protein. In some embodiments, the method may include administering the RNA transcript to a subject.

[0124] Expression cassette

[0125] The nucleic acid cassette may comprise one or more additional regulatory elements (e.g., promoters, terminators, and / or enhancers, etc.), which induce the expression of the transgene in a specific cell type or a specific cell type category. For example, a cell type-selective regulatory element may induce gene expression in a specific cell type relative to one or more other cell types. Alternatively or in addition, a cell type-selective regulatory element may induce gene expression in a specific cell category relative to one or more other cell categories. In one embodiment, the cell type-selective regulatory element of the present invention enhances gene expression in a specific cell type or a specific cell category. In another embodiment, the cell type-selective regulatory element represses gene expression in a specific cell type or a specific cell category. The cell type-selective regulation of gene expression (e.g., enhancing or repressing gene expression) does not need to affect gene expression only in the target cell type or cell category. Instead, the cell type-selective regulation of gene expression (e.g., enhancing or repressing gene expression) only requires that the gene expression in the target cell type increases or decreases relative to the gene expression in one or more other cell types or cell categories.

[0126] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:1; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0127] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:2; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0128] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:3; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0129] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:4; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0130] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:5; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0131] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 6; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0132] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 7; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0133] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:8; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0134] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:9; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0135] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO:10; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0136] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) at least two different sequences selected from SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0137] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) at least three different sequences selected from SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0138] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) at least four different sequences selected from SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0139] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) at least five different sequences selected from SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0140] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NOs. 1-10 and 43-48, in any order; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0141] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 43; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0142] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 44; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0143] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 45; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0144] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, and the DRG off-target region comprises: (i) SEQ ID NO: 46; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0145] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, the DRG off-target region comprising: (i) SEQ ID NO: 47; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0146] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a DRG off-target region, the DRG off-target region comprising: (i) SEQ ID NO: 48; (ii) a variant, functional fragment, multiple copies, or a combination thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0147] In some embodiments, the nucleic acid cassette can comprise a CNS-selective promoter operably linked to a polynucleotide encoding a therapeutic protein and one or more DRG off-target elements / sequences as disclosed herein. A CNS promoter is a promoter that specifically regulates gene expression in one or more cells of the central nervous system. For example, a CNS-selective promoter can specifically regulate gene expression in one or more neurons or glial cells of the CNS. In one embodiment, the CNS-selective promoter specifically regulates gene expression in one or more neurons or astrocytes. In another embodiment, the CNS-selective promoter specifically regulates gene expression in one or more astrocytes. In certain embodiments, the CNS-selective promoter enhances expression in CNS cells (e.g., neurons or glial cells, such as astrocytes) relative to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, microglia, motor neurons, vascular cells, non-GABAergic neurons, or other CNS cells).

[0148] Examples of CNS-selective promoters include, but are not limited to: the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, the synapsin I promoter, the 67 kDa glutamate decarboxylase (GAD67) promoter, the homeobox D1x5 / 6 promoter, the glutamate receptor 1 (GluR1) promoter, the preprotachykinin 1 (Tac1) promoter, the neuron-specific enolase (NSE) promoter, the dopaminergic receptor 1 (Drd1a) promoter, the MAP1B promoter, the Tα1α-tubulin promoter, the decarboxylase promoter, the dopamine beta-hydroxylase promoter, the NCAM promoter, the HES-5 promoter, the alpha-catenin promoter, the peripherin promoter, the GAP-43 promoter, and the PaqR4 promoter. Suitable promoters are also described, for example, in WO 2018 / 187363, the sequence of which is incorporated herein by reference. Other sequences can be used.

[0149] In some embodiments, the nucleic acid cassette can comprise 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 glutamate 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, relative 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), a GABAergic neuron-selective promoter enhances expression in GABAergic neurons.

[0150] A PV neuron-selective promoter is a promoter that specifically regulates gene expression in PV neurons. For example, a PV neuron-selective promoter enhances expression in PV neurons relative to one or more other CNS cell types.

[0151] In certain embodiments, the neuron-selective promoter can be of human origin or comprise sequences of human origin. In some cases, the promoter can be of murine origin or comprise sequences of murine origin. In some cases, the promoter is non-naturally occurring or comprises non-naturally occurring sequences. In some cases, the sequence of the promoter can be 100% of human origin. In other cases, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the promoter sequence is of human origin. For example, 50% of the promoter sequence can be of human origin and the remaining 50% is not of human origin (e.g., of murine origin or fully synthetic).

[0152] In some embodiments, the therapeutic protein encoded by the mRNA is associated with a neurological disease or disorder. As mentioned above, neurological diseases and disorders include those associated with one or more genetic mutations, as well as those of unknown etiology.

[0153] Examples of neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include, but are not limited to, Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panagiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0154] In these embodiments, the therapeutic protein can be: (i) a protein encoded by a gene selected from: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, 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 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 regulates the expression of the gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor that regulates (e.g., activates or represses) a gene of interest.

[0155] In certain embodiments, the nucleic acid constructs described herein further comprise another regulatory element in addition to a promoter, such as a sequence associated with transcription initiation or termination, an enhancer sequence, and an efficient RNA processing signal. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stabilizing elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, or polyadenylation (polyA) sequences, or combinations thereof. Regulatory elements can modulate gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression. At the RNA level, regulation can occur at the levels of translation (e.g., stabilizing elements that stabilize the mRNA for translation), RNA cleavage, RNA splicing, and / or transcriptional termination. In various embodiments, the regulatory element can recruit transcription factors to the coding region, which increase gene expression selectivity, increase the rate of RNA transcript production, increase the stability of the RNA produced, and / or increase the rate of protein synthesis from the RNA transcript in the cell type of interest.

[0156] In certain embodiments, the nucleic acid cassette can further comprise a polyA sequence. Suitable polyA sequences include, for example, artificial polyA (PA75) of about 75 bp in length (see, for example, WO2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit β-globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 Elb polyA, growth hormone polyA, or PBGD polyA. In certain embodiments, the polyA sequence is positioned downstream of the polynucleotide encoding the therapeutic functional protein in the nucleic acid constructs described herein.

[0157] Liver off-target

[0158] In addition to the above DRG off-target embodiments, the present disclosure also includes liver off-target elements. The provided liver off-target elements can be present in nucleic acid cassettes, RNA molecules (e.g., RNA transcripts), synthetic RNA molecules, etc., as described above for DRG off-target elements. Additionally, methods of using liver off-target elements to reduce the expression and / or activity of a transgene in hepatocytes / tissues are provided. Such methods are similar to the methods detailed above for using DRG off-target elements to reduce the expression and / or activity of a transgene in DRG cells. Thus, all embodiments described herein for DRG off-target elements can be applied to the liver off-target elements described below, it being understood that the tissues / cells off-targeted by the liver off-target elements are live tissues / hepatocytes rather than DRG cells.

[0159] Accordingly, in certain embodiments, a nucleic acid cassette is provided that comprises a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs: 65, 110, and 112; (ii) variants, functional fragments, or combinations thereof of the foregoing sequences; or (iii) a sequence that is at least 80% identical to (i) or (ii). In these embodiments, the sequence reduces the expression of the RNA transcript in hepatocytes. Embodiments utilizing these sequences are described in more detail below. These liver off-target sequences can be employed in any embodiment where off-targeting in the liver is desired.

[0160] In one embodiment, the present application provides an expression cassette that comprises a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region that comprises: (i) SEQ ID NO: 65; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0161] In one embodiment, the present application provides an expression cassette that comprises a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region that comprises: (i) SEQ ID NO: 110; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequence; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either (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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0162] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region, and the liver off-target region comprises: (i) SEQ ID NO: 112; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for treating a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0163] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region, and the liver off-target region comprises: (i) at least two different sequences selected from SEQ ID NO. 65, 110, and 112; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for treating a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0164] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region, the liver off-target region comprising: (i) at least three different sequences selected from SEQ ID NOs. 65, 110, and 112; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0165] In one embodiment, the present application provides an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding an RNA transcript, wherein the RNA transcript comprises a liver off-target region, the liver off-target region comprising: (i) at least four different sequences selected from SEQ ID NOs. 65, 110, and 112; (ii) variants, functional fragments, multiple copies, or combinations thereof of the foregoing sequences; or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 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 RNA transcript is a therapeutic expression product for a neurological disease or disorder, e.g., an mRNA encoding a therapeutic protein.

[0166] Sequence combination

[0167] As shown in any of the tables below, the above DRG and liver targeting sequences can be combined with each other or with other off-target sequences to produce cassettes that more effectively off-target a single tissue (i.e., DRG or liver) or a combination of tissues (i.e., DRG and liver). In the tables below, "X" indicates the combination of a first sequence on the x-axis and a second sequence on the y-axis, where, in any combination, the combination can contain a single copy of the first sequence, two copies of the first sequence, three copies of the first sequence, four copies of the first sequence, or at least five copies of the first sequence, and independently contains a single copy of the second sequence, two copies of the second sequence, three copies of the second sequence, four copies of the second sequence, or at least five copies of the second sequence.

[0168] Table 1 below shows exemplary combinations of DRG off-target elements (SEQ ID NOs. 1-10 and 43-48) that can be employed herein.

[0169] Table 1

[0170] SEQ ID 1 2 3 4 5 6 7 8 9 10 43 44 45 46 47 48 1 X X X X X X X X X X X X X X X 2 X X X X X X X X X X X X X X X 3 X X X X X X X X X X X X X X X 4 X X X X X X X X X X X X X X X 5 X X X X X X X X X X X X X X X 6 X X X X X X X X X X X X X X X 7 X X X X X X X X X X X X X X X 8 X X X X X X X X X X X X X X X 9 X X X X X X X X X X X X X X X 10 X X X X X X X X X X X X X X X 43 X X X X X X X X X X X X X X X 44 X X X X X X X X X X X X X X X 45 X X X X X X X X X X X X X X X 46 X X X X X X X X X X X X X X X 47 X X X X X X X X X X X X X X X 48 X X X X X X X X X X X X X X X

[0171] The liver off-target elements of SEQ ID NOs. 65, 110, and 112 can be combined with each other or with the liver off-target elements described in PCT / US2023065801 filed on April 14, 2023 (i.e., SEQ ID NOs. 57-62, 64, and 66-71 in that application), and that application is incorporated herein by reference.

[0172] Table 2 below shows exemplary combinations of liver off-target elements that can be employed herein.

[0173] Table 2

[0174]

[0175]

[0176] In any embodiment, the nucleic acid cassette can contain a therapeutic transgene encoding an RNA transcript (e.g., mRNA), where the RNA transcript contains a first sequence and a second sequence, the first sequence off-targeting expression in DRG cells and the second sequence off-targeting expression in hepatocytes. In these embodiments, the first sequence and the second sequence can cause a reduction in the expression of the RNA transcript or the polypeptide encoded thereby (e.g., when the RNA transcript is mRNA) in DRG cells and hepatocytes relative to a target tissue (e.g., nerve cells, such as brain cells, brain stem cells, hippocampal cells, cerebellar cells, or GABAergic cells (e.g., GABAergic cells as cells expressing parvalbumin)).

[0177] In the cassette, the first and second sequences can cause: the expression level of an RNA transcript or a polypeptide encoded thereby (i.e., when the RNA transcript is an mRNA) in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of an RNA transcript or polypeptide of an otherwise identical RNA transcript lacking the first and second sequences in DRG cells; and independently,

[0178] the expression level of an RNA transcript or a polypeptide encoded thereby in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of an RNA transcript or polypeptide of an otherwise identical RNA transcript lacking the first and second sequences in hepatocytes.

[0179] In any of these embodiments, (a) the first sequence can be: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof of the foregoing sequences; or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); and (b) the second sequence is: (iv) any one of SEQ ID NOs. 57-62, 64-71, 110, and 112; (v) a variant, functional fragment, or combination thereof of the foregoing sequences; or (vi) 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 (iv) or (v).

[0180] In some of these embodiments, the first sequence (i), (ii), or (iii) can provide a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, and hsa-mir-494-3p, and the second sequence (iv), (v), or (vi) can provide a binding site for hsa-mir-22-3p, has-mir-1258, hsa-mir-5589-3p, hsa-mir-17-5p, hsa-mir-203a-3p, hsa-mir-122-3p, hsa-mir-93-5p, and hsa-mir-19a-3p.

[0181] In any of these embodiments, the RNA transcript can comprise a combination of sequences selected from Table 3 below, where in Table 3, SEQ ID NOs: 1-10 and 43-48 are off-target for expression in DRG cells, and SEQ ID NOs 57-62, 64-71, 110, and 112 are off-target for expression in hepatocytes. Table 3 below shows exemplary combinations of DRG off-target and liver off-target sequence elements that can be employed herein.

[0182] Table 3

[0183] SEQ ID 1 2 3 4 5 6 7 8 9 10 43 44 45 46 47 48 57 X X X X X X X X X X X X X X X X 58 X X X X X X X X X X X X X X X X 59 X X X X X X X X X X X X X X X X 60 X X X X X X X X X X X X X X X X 61 X X X X X X X X X X X X X X X X 62 X X X X X X X X X X X X X X X X 64 X X X X X X X X X X X X X X X X 65 X X X X X X X X X X X X X X X X 66 X X X X X X X X X X X X X X X X 67 X X X X X X X X X X X X X X X X 68 X X X X X X X X X X X X X X X X 69 X X X X X X X X X X X X X X X X 70 X X X X X X X X X X X X X X X X 71 X X X X X X X X X X X X X X X X 110 X X X X X X X X X X X X X X X X 112 X X X X X X X X X X X X X X X X

[0184] For clarity, the RNA transcript can comprise multiple different DRG off-target elements in combination with one or more liver off-target elements and / or multiple different liver off-target elements in combination with one or more DRG off-target elements, and each of these off-target elements can independently be present in the RNA transcript in one or more (e.g., two, three, four, or five or more) copies. SEQ ID NO: 111 is an example of such a combination, but there are also several other combinations (as illustrated in the experimental section of this disclosure).

[0185] The first and second sequences reduce transgene expression in dorsal root ganglion cells (DRG) and hepatocytes relative to that in target cells (such as nerve cells, e.g., neurons), and thus can be used in a variety of gene therapy strategies targeting cells other than DRG or liver. Reducing transgene expression in DRG cells and hepatocytes relative to that in target cells means that the reduction in transgene expression driven by the DRG and liver off-target sequences disclosed herein is greater in DRG cells and the liver than in target cells. Thus, although a reduction in transgene expression in target cells can be observed in some embodiments, it is less than the reduction in transgene expression observed in DRG cells and hepatocytes. This reduction in expression in DRG and the liver can mitigate or eliminate toxicity and / or axonal pathologies in subjects receiving gene therapy targeting non-DRG cells or tissues (such as nerve cells, e.g., neurons) and non-hepatocytes, thereby improving their safety profile.

[0186] In some embodiments, the DRG and / or liver off-target elements disclosed herein can be used in combination with other sequences having known cell- or tissue-specific off-target activities. For example, the expression cassettes of the present disclosure can encode an RNA transcript that contains one or more of the off-target sequences disclosed herein (e.g., SEQ ID NOs: 1-10, 57-62, and 64-71, alone or in any combination), and also contains one or more sequences having known off-target activities, such as SEQ ID NO: 63, which has liver off-target activity. There is no limitation in this regard.

[0187] Vector

[0188] Expression vectors can be used to deliver nucleic acid molecules to target cells via transfection or transduction. The vector can be an integrating or non-integrating vector, referring to the ability of the vector to integrate the expression cassette or transgene into the host cell genome. Examples of expression vectors include, but are not limited to: (a) non-viral vectors, such as nucleic acid vectors, including linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (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.

[0189] An expression vector can be a linear oligonucleotide or a circular plasmid, and can be delivered to cells via various transfection methods, including physical and chemical methods. Physical methods generally refer to delivery methods that use physical forces to counteract the cell membrane barrier to facilitate intracellular delivery of genetic material. Examples of physical methods include the use of needles, ballistic DNA, electroporation, sonoporation, optoporation, magnetofection, and hydrodynamic transfection. Chemical methods generally refer to methods in which a chemical carrier delivers nucleic acid molecules to cells, and can include inorganic particles, lipid-based carriers, polymer-based carriers, and peptide-based carriers.

[0190] In some embodiments, inorganic particles are used to administer the expression vector to target cells. Inorganic particles can refer to nanoparticles, such as nanoparticles engineered to have various sizes, shapes, and / or porosities to escape from the reticuloendothelial system or to protect encapsulated molecules from degradation. Inorganic nanoparticles can be prepared from metals (e.g., iron, gold, and silver), inorganic salts, or ceramics (e.g., phosphates or carbonates of calcium, magnesium, or silicon). The surfaces of these nanoparticles can be coated to facilitate DNA binding or targeted gene delivery. Magnetic nanoparticles (e.g., superparamagnetic iron oxide), fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g., cylindrical fullerenes), quantum dots, and supramolecular systems can also be used.

[0191] In some embodiments, cationic lipids (e.g., cationic liposomes) are used to administer the expression vector to target cells. Various types of lipids have been investigated for gene delivery, such as lipid nanoemulsions (e.g., a dispersion of an immiscible liquid in another immiscible liquid stabilized by an emulsifier) or solid lipid nanoparticles.

[0192] In some embodiments, an expression vector is administered to a target cell using a peptide-based delivery mediator. The peptide-based delivery mediator can have the advantages of protecting the genetic material to be delivered, targeting specific cell receptors, disrupting the endosomal membrane, and delivering the genetic material to the nucleus. In some embodiments, an expression vector is administered to a target cell using a polymer-based delivery mediator. The polymer-based delivery mediator can include natural proteins, peptides, and / or polysaccharides or synthetic polymers. In one embodiment, the polymer-based delivery mediator includes polyethyleneimine (PEI). PEI can condense DNA into positively charged particles that bind to anionic cell surface residues and are then taken into the cell via endocytosis. In other embodiments, the polymer-based delivery mediator can comprise poly-L-lysine (PLL), poly(DL-lactic acid) (PLA), poly(DL-lactide-co-glycolide) (PLGA), polyornithine, polyarginine, histone, protamine, dendrimer, chitosan, synthetic amino derivatives of dextran, and / or cationic acrylic polymers. In certain embodiments, the polymer-based delivery mediator can include a mixture of polymers (such as PEG and PLL).

[0193] In certain embodiments, the expression vector can be a viral vector suitable for gene therapy. Preferred characteristics of viral gene therapy vectors or gene delivery vectors can include the ability to replicate and purify stably and to high titers; to mediate targeted delivery (e.g., to deliver a transgene specifically to a tissue or organ of interest without widespread dissemination of the vector elsewhere); and to mediate gene delivery and transgene expression without inducing harmful side effects.

[0194] Several types of viruses (e.g., the non-pathogenic parvovirus known as adeno-associated virus) have been engineered for gene therapy purposes by exploiting the viral infection pathway but avoiding subsequent expression of viral genes that may cause replication and toxicity. Such viral vectors can be obtained by deleting all or part of the coding regions from the viral genome but retaining those sequences that are intact (e.g., terminal repeats) and that may be essential for functions such as packaging the vector genome into the viral capsid or integrating the vector nucleic acid (e.g., DNA) into the host chromatin.

[0195] In various embodiments, suitable viral vectors include retroviruses (e.g., type A, B, C, and D viruses), adenoviruses, parvoviruses (e.g., adeno-associated virus or AAV), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Examples of retroviruses include avian leukosis-sarcoma viruses, human T-lymphotropic virus type 1 (HTLV-1), bovine leukemia virus (BLV), lentiviruses, and foamy viruses. Other viruses include, for example, noroviruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Depending on the ability to integrate into the host genome, viral vectors can be divided into two groups - integrative and non-integrative. Oncogenic retroviruses and lentiviruses can integrate into the host cell chromatin, while adenoviruses, adeno-associated viruses, and herpesviruses mainly persist as episomes outside the chromosomes in the nucleus.

[0196] In certain embodiments, a suitable viral vector is a retroviral vector. A retrovirus refers to a virus of the Retroviridae family. Examples of retroviruses include oncogenic retroviruses such as murine leukemia virus (MLV), and lentiviruses such as human immunodeficiency virus 1 (HIV-1). The retroviral genome is single-stranded (ss) RNA, including various genes that may be provided in cis or in trans. For example, the retroviral genome may contain cis-acting sequences such as two long terminal repeats (LTRs), as well as elements for gene expression, reverse transcription, and integration into the host chromosome. Other components include a packaging signal (psi or Ψ) for the specific packaging of RNA into newly formed virions and a polypurine tract (PPT), i.e., the starting site for plus-strand DNA synthesis during reverse transcription. In addition, the retroviral genome may contain the gag, pol, and env genes. The gag gene encodes structural proteins, the pol gene encodes enzymes that accompany the ssRNA and reverse-transcribe the viral RNA into DNA, and the env gene encodes the viral envelope. Generally, gag, pol, and env are provided in trans for viral replication and packaging.

[0197] In certain embodiments, the retroviral vectors provided herein can be lentiviral vectors. At least five serogroups or serotypes of lentiviruses have been identified. Viruses of different serotypes can differentially infect certain cell types and / or hosts. For example, lentiviruses include primate retroviruses and non-primate retroviruses. Primate retroviruses include HIV and simian immunodeficiency virus (SIV). Non-primate retroviruses include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and ovine progressive pneumonia virus. Lentiviruses or lentiviral vectors can be capable of transducing quiescent cells. Like oncoretroviral vectors, lentiviral vectors can be designed based on separating cis-acting sequences from trans-acting sequences.

[0198] 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. It is understood that AAV does not cause human disease and elicits only a mild immune response. AAV vectors can also infect both dividing and quiescent cells without integrating into the host cell genome.

[0199] The AAV genome consists of linear single-stranded DNA that is approximately 4.7 kb in length. The genome contains two open reading frames (ORFs) that are flanked by inverted terminal repeat (ITR) sequences that are approximately 145 bp in length. The ITR consists of a nucleotide sequence at the 5' end (5' ITR) and a nucleotide sequence at the 3' end (3' ITR) that contains palindromic sequences. The ITR acts in cis by folding into a T-shaped hairpin structure through complementary base pairing that serves as a primer during DNA replication initiation for second-strand synthesis. The two open reading frames encode the rep and cap genes that are involved in virion replication and packaging. In one exemplary embodiment, the AAV vector provided herein does not contain the rep and cap genes. Such genes can be provided in trans for the production of virions as further described below.

[0200] In certain embodiments, the AAV vector can include filler nucleic acid. In some embodiments, the filler nucleic acid can encode a green fluorescent protein or an antibiotic resistance gene, such as kanamycin or ampicillin. In certain embodiments, the filler nucleic acid can be located outside of the ITR sequences (e.g., compared to the polynucleotide encoding a therapeutic protein and the regulatory sequences located between the 5' ITR sequence and the 3' ITR sequence).

[0201] There are various AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8. These serotypes differ in their tropism or the cell types they infect. AAV can contain genomes and capsids from multiple serotypes (e.g., pseudotyped). For example, AAV can contain a serotype 2 genome (e.g., ITR) encapsulated in a capsid from serotype 5 or serotype 9. Pseudotyping can improve transduction efficiency and alter tropism.

[0202] In some embodiments, an AAV vector or AAV viral particle or virion can be used to deliver a construct comprising a cell-selective regulatory element operably linked to a polynucleotide encoding a therapeutic functional protein to a certain cell, cell type, or tissue, and can be performed in vivo, ex vivo, or in vitro. In an exemplary embodiment, such an AAV vector is a replication-deficient vector. In some embodiments, the AAV virus is engineered or genetically modified such that it can only replicate and produce virions in the presence of a helper factor.

[0203] In certain embodiments, a viral vector can be selected to produce virions with high infectivity but no selectivity for a particular cell type. In certain embodiments, the viral vector 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 the selective expression of the polynucleotide in the target cell, target cell type, or target tissue, while the one or more regulatory elements repress transgene expression in off-target cells, off-target cell types, or off-target tissues, or confer significantly lower, trace, or statistically lower gene expression in one or more off-target cells, off-target cell types, or off-target tissues.

[0204] In some cases, AAV serotypes that can cross the blood-brain barrier or infect CNS cells are preferred.

[0205] In exemplary embodiments, the present application provides expression vectors that have been designed for delivery by AAV. The AAV can be any serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8, or chimeric, hybrid, or variant AAV. The AAV can also be self-complementary AAV (scAAV), where "self-complementary" AAV is an AAV in which the coding region has been designed to form an intramolecular double-stranded DNA template. Once such a vector infects a cell, the two complementary halves of the scAAV associate to form a double-stranded DNA (dsDNA) unit that is ready to immediately replicate and transcribe, rather than waiting for the synthesis of a second strand to occur under cell-mediated conditions. The design of scAAV vectors is described in a variety of publications, including McCarty et al., Gene Therapy 20018:1248-54.

[0206] 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 as described above, and a 3' ITR. In certain embodiments, the expression vector designed for delivery by AAV comprises a 5' ITR, an enhancer, a promoter, the construct as described above, and a 3' ITR.

[0207] Host cell

[0208] In another aspect, the invention relates to a host cell comprising the nucleic acid cassette as described above. The host cell can be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In an exemplary embodiment, the host cell refers to any cell line that is susceptible to infection by a virus of interest and is suitable for in vitro culture.

[0209] In certain embodiments, the host cells provided herein can be used for ex vivo gene therapy purposes. In such embodiments, these cells are transfected with the nucleic acid molecule or expression cassette as described above and subsequently transplanted into a patient or subject. The transplanted cells can have an autologous, allogeneic, or xenogeneic origin. For clinical applications, cell isolation will generally be performed under good manufacturing practice (GMP) conditions. Prior to transplantation, the cell quality is typically examined and the absence of microbial contaminants or other contaminants is determined, and then pre-treatment, such as radiation and / or immunosuppressive treatment, can be performed. In addition, the host cells can be transplanted together with growth factors to stimulate cell proliferation and / or differentiation.

[0210] In certain embodiments, host cells can be used to deliver ex vivo gene therapy into the CNS. Preferably, the cells are eukaryotic cells, such as mammalian cells, including but not limited to humans, non-human primates (such as apes, chimpanzees, monkeys, and orangutans), domestic animals (including dogs and cats), and livestock (such as horses, cows, pigs, sheep, and goats), or other mammalian species, including but not limited to mice, rats, guinea pigs, rabbits, hamsters, etc. One of ordinary skill in the art will select a more suitable cell based on the patient or subject to be transplanted.

[0211] In certain embodiments, the host cells provided herein can be cells having self-renewal and pluripotency properties, such as stem cells or induced pluripotent stem cells. Stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells (MSCs) are capable of differentiating into at least one of osteoblasts, chondrocytes, adipocytes, or myocytes, 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 MSCs are well known to those of ordinary skill in the art. Induced pluripotent stem cells (also referred to as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from somatic cells.

[0212] Yamanaka et al. induced iPS cells by transferring the Oct3 / 4, Sox2, Klf4, and c-Myc genes into mouse and human fibroblasts and forcing the cells to express these genes (WO 2007 / 069666).

[0213] Thomson et al. subsequently used Nanog and Lin28 in place of Klf4 and c-Myc to produce human iPS cells (WO2008 / 118820).

[0214] In an exemplary embodiment, the host cells provided herein are packaging cells. The cells can be adherent cells or suspension cells. The packaging cells, together with an accessory vector or a viral or DNA construct, provide in trans all of the missing functions required for complete replication and packaging of a viral vector.

[0215] Preferably, the packaging cell is a eukaryotic cell, such as a mammalian cell, including cells of apes, humans, dogs, and rodents. Examples of human cells are PER.C6 cells (WO01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus monkey 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.

[0216] As an alternative to mammalian sources, the cell lines used in the present invention can be derived from avian sources, such as chickens, ducks, geese, quails, or pheasants. Examples of avian cell lines include avian embryonic stem cells (WO01 / 85938 and WO03 / 076601), immortalized duck retinal cells (WO2005 / 042728), and cells derived from avian embryonic stem cells, including chicken cells (WO2006 / 108846) or duck cells, such as the EB66 cell line (WO2008 / 129058 and WO2008 / 142124).

[0217] In another embodiment, the host cell is an insect cell, such as SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB-Sf21), MG1 cells (BTI-TN-MG1), or High Five TM cells (BTI-TN-5B1-4).

[0218] In certain embodiments, the host cells provided herein contain a nucleic acid construct (e.g., a plasmid) carrying a recombinant AAV vector / genome containing the cassette as described above, and may further contain one or more additional nucleic acid constructs, such as: (i) a nucleic acid construct encoding the rep gene and the cap gene but not carrying the ITR sequence (e.g., an AAV helper plasmid); and / or (ii) a nucleic acid construct providing the adenovirus functions necessary for AAV replication (e.g., a plasmid). In an exemplary embodiment, the host cells provided herein contain: i) the nucleic acid construct or expression vector as described above; ii) a nucleic acid construct encoding the AAV rep gene and the cap gene that does not carry the ITR sequence; iii) a nucleic acid construct containing adenovirus helper genes (described further below).

[0219] 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 cells provided herein comprise: i) a nucleic acid molecule or expression cassette, and ii) a plasmid encoding the AAV rep gene and the cap gene, which does not carry ITR sequences and further comprises adenovirus helper genes. Alternative methods are known. For example, the rep gene, the cap gene, and the adenovirus helper genes need not be located on the same plasmid, but can be provided on different plasmids, or the rep gene and the cap gene can be provided on a plasmid different from the adenovirus helper genes.

[0220] In certain embodiments, host cells suitable for large - scale production of AAV vectors are insect cells that can be co - infected with 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 respectively express the AAV rep, AAV cap, and the AAV vector to be packaged. The recombinant baculovirus vectors will provide the viral helper gene functions required for viral replication and / or packaging.

[0221] Further guidance on constructing and producing viral vectors for gene therapy according to the present invention can be found in the following literature: Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Volume 737, Merten and Al-Rubeai (editors); 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 (editor), 2010 Springer-Verlag; pp. 143-170; Adeno-Associated Virus: Methods and Protocols. R.O. Snyder and P. Moulllier (editors), 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. Chilion and A. Bosch (editors); Third Edition, 2014 Humana Press (Springer).

[0222] Virion and method for producing virion

[0223] In certain embodiments, the present application provides virus particles comprising a viral vector. The terms "virus particle" and "virion" are used interchangeably herein and refer to infectious and generally replication-defective virus particles that contain a viral genome (e.g., a viral expression vector) packaged within a capsid, which, depending on the case, for example for retroviruses, may be a lipid envelope surrounding the capsid. A "capsid" refers to the structure in which the viral genome is packaged. The capsid is composed of a number of oligomeric structural subunits that are made of protein. For example, AAV has an icosahedral capsid formed by the interaction of the following three capsid proteins: VP1, VP2, and VP3. In one embodiment, the virions provided herein are recombinant AAV virions or rAAV virions obtained by packaging an AAV vector within a protein coat.

[0224] In certain embodiments, the recombinant AAV virions provided herein can be prepared by encapsidating an AAV genome derived from a specific AAV serotype within virus particles formed by the native Cap proteins of the AAV corresponding to the same specific serotype. In other embodiments, the AAV virus particles provided herein comprise a viral vector containing ITRs of a given AAV serotype packaged into proteins from a different serotype. See, for example, Bunning H et al., J Gene Med 2008; 10:717-733. For example, a viral vector having ITRs of a given AAV serotype can be packaged into: a) virus particles composed of capsid proteins derived from the same or different AAV serotypes (e.g., AAV2 ITRs and AAV9 capsid proteins; AAV2 ITRs and AAV8 capsid proteins; etc.); b) mosaic virus particles composed of a mixture of capsid proteins from different AAV serotypes or mutants (e.g., AAV2 ITRs with AAV1 and AAV9 capsid proteins); c) chimeric virus particles composed of capsid proteins truncated by domain swapping between different AAV serotypes or variants (e.g., AAV2 ITRs with an AAV8 capsid protein having an AAV9 domain); or d) targeted virus particles engineered to display a selective binding domain that is capable of strict interaction with a target cell-specific receptor (e.g., AAV5 ITRs with an AAV9 capsid protein genetically truncated by insertion of a peptide ligand; or an AAV9 capsid protein with a peptide ligand conjugated to the capsid surface rather than genetically modified).

[0225] Those skilled in the art should understand that the AAV virions provided herein may contain capsid proteins of any AAV serotype. In one embodiment, the viral particles contain capsid proteins of AAV serotypes 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 particles contain the nucleic acid construct of the present invention, wherein the 5' TTR sequence and the 3' TTR sequence of the nucleic acid construct are of the AAV2 serotype, and the capsid protein is of the AAV9 serotype.

[0226] Many methods are known in the art for producing rAAV virions, including transfection, stable cell line production, and infectious hybrid virus production systems, which include 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 producing rAAV viral particles require: 1) a suitable host cell, including, for example, cell lines derived from humans, such as HeLa, A549, or 293 cells, or cell lines derived from insects, such as SF-9 (for baculovirus production systems); 2) suitable helper virus functions, which are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide helper functions; 3) the AAV rep gene and the cap gene, as well as the gene products; 4) a transgene flanked by AAV ITR sequences; and 5) a suitable culture medium and culture medium components for supporting rAAV production.

[0227] In various embodiments, the host cells described herein comprise the following three components: (1) the rep gene and the cap gene, (2) genes providing helper functions, and (3) a transgene flanked by ITRs. The AAV rep gene, the AAV cap gene, and the genes providing helper functions can be introduced into cells by incorporating the genes into a vector (such as a plasmid) and then introducing the vector into the host cells. The rep gene, the cap gene, and the helper function genes can be incorporated into the same plasmid or different plasmids. In a preferred embodiment, the AAV rep gene and the cap gene are incorporated into one plasmid, and the genes providing helper functions are incorporated into another plasmid. The various plasmids (e.g., containing the AAV rep gene and cap gene, helper functions, or transgene) for generating host cells for virion production can be introduced into cells by using any suitable method well known in the art. Examples of transfection methods include, but are not limited to: calcium phosphate co-precipitation, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and biolistic transfection. In certain embodiments, the plasmids providing the rep gene and cap gene, helper functions, and transgene can be introduced into cells simultaneously. In another embodiment, the plasmids providing the rep gene and cap gene and helper functions can be introduced into cells before or after introducing the plasmid containing the transgene. In an exemplary embodiment, cells are transfected simultaneously with the following three plasmids (e.g., triple transfection method): (1) a plasmid containing the transgene, (2) a plasmid containing the AAV rep gene and cap gene, and (3) a plasmid containing the genes providing helper functions. Exemplary host cells can be 293, A549, or HeLa cells.

[0228] In other embodiments, one or more of (1) the AAV rep and cap genes, (2) the genes providing helper functions, and (3) the transgene (e.g., a PV-selective regulatory element operably linked to a polynucleotide encoding a therapeutic protein disclosed herein) can be carried by the packaging cell, in episomal form and / or in the form integrated into the genome of the packaging cell. In one embodiment, the host cell can be a packaging cell in which the AAV rep and cap genes and the helper functions are stably maintained, 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, 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 can be a packaging cell in which the helper functions are stably maintained, 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 production cell line stably transfected with the rep and cap genes, the helper functions, and the transgene sequence. Exemplary packaging cells and production cells can be derived from 293, A549, or HeLa cells.

[0229] In another embodiment, the production cell line is an insect cell line (typically Sf9 cells) infected with a baculovirus expression vector providing the Rep protein and the Cap protein. This system does not require adenovirus helper genes (Ayuso E et al., Curr. Gene Ther. 2010, 10:423-436).

[0230] 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 the functional activities of the cap protein include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote packaging of AAV DNA into the capsid (i.e., encapsidation), bind to cell receptors, and promote entry of the virion into the host cell. In principle, any Cap protein can be used in the context of the present invention.

[0231] It has been reported that Cap proteins affect the host tropism, cell, tissue, or organ specificity, receptor utilization, infection efficiency, and immunogenicity of AAV viruses.

[0232] Thus, the AAV cap used in rAAV can be selected taking into account, for example, the species of the subject (e.g., human or non-human), the immune status of the subject, the subject's suitability for long-term or short-term treatment, or a particular therapeutic application (e.g., treating a particular disease or disorder, or delivering to a particular cell, tissue, or organ). In certain embodiments, the cap protein is derived from an AAV selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9 serotypes. In one exemplary embodiment, the cap protein is derived from AAV9.

[0233] In some embodiments, the AAV Cap used in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV caps or its encoding nucleic acid. In some embodiments, the AAV cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the aforementioned AAV caps, or has a higher degree of similarity.

[0234] In some embodiments, the AAV cap is chimeric, comprising domains from two, three, four, or more of the aforementioned AAV caps. In some embodiments, the AAV cap is a mosaic of VP1, VP2, and VP3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned caps.

[0235] In some embodiments, the AAV cap used in the rAAV virion is engineered to contain a heterologous sequence or other modification. For example, a peptide or protein sequence conferring selective targeting or immune evasion can be engineered into the cap protein.

[0236] Alternatively or in addition, the cap can be chemically modified such that the surface of the rAAV is polyethylene glycolated (i.e., PEGylated), which can promote immune evasion. The cap protein can also be mutagenized (e.g., to remove its native receptor binding, or to mask immunogenic epitopes).

[0237] As used herein, the term "rep protein" refers to a polypeptide having at least one functional activity of a native AAV rep protein (e.g., rep 40, 52, 68, 78). Examples of functional activities of the rep protein include any activity associated with the physiological function of the protein, including promoting DNA replication by recognizing, binding to, and cleaving the AAV origin of DNA replication, as well as DNA helicase activity. Additional functions include regulating transcription from an AAV (or other heterologous) promoter, and site-specific integration of AAV DNA into the host chromosome. In one specific embodiment, the AAV rep gene can be from serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrh10; more preferably from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9.

[0238] In some embodiments, the AAV rep protein used in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV reps or their encoding nucleic acids. In some embodiments, the AAV rep is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the aforementioned AAV reps, or has a higher degree of similarity.

[0239] As used herein, the phrase "helper function" or "helper gene" refers to viral proteins upon which AAV depends for replication. Helper functions include those proteins required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and AAV capsid assembly. The virus-based helper functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except 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, UL8, UL52, and UL29, as well as the herpesvirus polymerase. In a preferred embodiment, the proteins upon which AAV depends for replication are derived from adenovirus.

[0240] In some embodiments, the viral proteins upon which AAV depends for replication used in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned viral proteins or their encoding nucleic acids. In some embodiments, the viral protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the aforementioned viral proteins, or has a higher degree of similarity.

[0241] Methods for assaying the functions of the cap protein, rep protein, and viral proteins on which AAV depends for replication are well known in the art.

[0242] Host cells for expressing a transgene of interest can be grown under conditions sufficient to assemble AAV virions. In certain embodiments, the host cells are grown for a suitable period of time to facilitate AAV virion assembly and release of the virions into the culture medium. Generally, the cells can be 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 faster, depending on the culture conditions and the particular host cells used), the production levels are generally significantly reduced. Generally, the culture time is measured from the point of virus production. For example, in the case of AAV, virus production typically begins when helper virus functions are supplied in a suitable host cell as described herein. Generally, the cells are harvested about 48 hours to about 100 hours, preferably about 48 hours to about 96 hours, preferably about 72 hours to about 96 hours, preferably about 68 hours to about 72 hours after helper virus infection (or after virus production begins).

[0243] rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for different lengths of time, etc.) suitable for the particular host cells utilized. rAAV production cultures include adherent-dependent cultures, which can be cultured in suitable adherent-dependent vessels such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. rAAV vector production cultures can also comprise host cells adapted to suspension, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems.

[0244] Suitable media known in the art can be used to produce rAAV virions. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), each of which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV production medium is capable of being supplemented with serum or serum-derived recombinant proteins at levels of 0.5%-20% (v / v or w / v). Alternatively, rAAV vectors can be produced under serum-free conditions, which can also be referred to as media that are free of products derived from animals.

[0245] After culturing host cells to permit AAV virion production, the resulting virions can be harvested and then purified. In certain embodiments, AAV virions can be (1) obtained from these host cells by lysing the host cells in the production culture, and / or (2) obtained from the culture medium of the cells after a period of transfection (preferably 72 hours). rAAV virions can be harvested from the production culture in the spent medium, provided that the cells are cultured under conditions that cause the rAAV virions to be released from the intact cells into the medium (see, e.g., 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.

[0246] After harvest, the rAAV virions can be purified. As used herein, the term "purified" includes preparations of rAAV virions that are free of at least some of the other components that are naturally present in or may be present in the initial preparation of rAAV virions. Thus, for example, purified rAAV virions can be prepared by enrichment from a source mixture such as a culture lysate or a production culture supernatant using separation techniques. Enrichment can be measured in a variety of ways, such as by the ratio of DNase-resistant particles (DRP) or genomic copies (gc) present in solution, or by infectivity, or it can be measured relative to a second potentially interfering substance present in the source mixture such as a contaminant, including production culture contaminants or process contaminants, including helper virus, culture medium components, etc.

[0247] In certain embodiments, the rAAV production culture harvest can be clarified to remove host cell debris. In some embodiments, the production culture harvest can be clarified using a variety of standard techniques such as centrifugation or filtration through a filter with a pore size of 0.2 µm or greater (e.g., a cellulose acetate filter or a series of depth filters).

[0248] In certain embodiments, the rAAV production culture harvest is further treated with Benzonase TM to digest any high molecular weight DNA present in the production culture. In some embodiments, the Benzonase TM digestion is carried out under standard conditions, e.g., a final concentration of 1 to 2.5 units / ml of Benzonase TM , at a temperature in the range of room temperature to 37 °C for a period of 30 minutes to several hours.

[0249] In certain embodiments, one or more of the following purification steps can be used to isolate or purify rAAV virions: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating rAAV particles; capture of rAAV by hydroxyapatite chromatography; helper virus heat inactivation; capture of rAAV by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and capture of rAAV by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different sequences. Methods for purifying rAAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143.

[0250] In certain embodiments, the purified AAV virions can be dialyzed against PBS and stored at -80 °C after filtration. Using linearized plasmid DNA as a standard curve, the titer of viral genomes can be determined by quantitative PCR (see, e.g., Lock M et al., Hum. Gene Ther. 2010;21:1273-1285).

[0251] Pharmaceutical composition

[0252] In certain embodiments, the present application provides a composition comprising the above nucleic acid cassette (e.g., expression cassette, e.g., rAAV comprising an expression cassette) or RNA encoded thereby (e.g., mRNA or ncRNA) and a pharmaceutically acceptable carrier. (Note that the RNA can be synthetically produced and thus does not have to be produced via transcription of the nucleic acid cassette described herein). In some embodiments, virions comprising the cassette and a pharmaceutically acceptable carrier are provided. In an exemplary embodiment, such a composition is suitable for gene therapy applications. The pharmaceutical composition is preferably sterile and stable under the manufacturing and storage conditions. Sterile solutions can be achieved, for example, by filtration through sterile filtration membranes.

[0253] The acceptable carriers and excipients in the pharmaceutical composition are preferably non-toxic to the recipient at the doses and concentrations employed. Acceptable carriers and excipients can include buffering agents such as phosphates, citrates, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, cetyltrimethylammonium 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 composition of the present disclosure can be administered parenterally in the form of an injectable preparation.

[0254] A sterile solution or any pharmaceutically acceptable liquid can be used as a vehicle to formulate the pharmaceutical composition for injection. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water and normal saline.

[0255] The pharmaceutical composition of the present disclosure can be prepared in microcapsules such as hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate microcapsules. The pharmaceutical composition of the present disclosure can also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. The pharmaceutical composition for gene therapy can be in an acceptable diluent or can contain a sustained-release matrix in which a gene delivery vehicle is embedded.

[0256] The pharmaceutical composition provided herein can be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intracerebral parenchymal administration, intrathecal administration, intracisternal administration, intraventricular administration, or intraperitoneal administration. The pharmaceutical composition can also be formulated for nasal, spray, oral, aerosol, rectal, or vaginal administration, or can also be administered via nasal, spray, oral, aerosol, rectal, or vaginal. In one embodiment, the pharmaceutical composition provided herein is administered to the CNS or cerebrospinal fluid (CSF), for example, by intracerebral parenchymal injection, intrathecal injection, intracisternal injection, or intraventricular injection. The tissue target can be specific, for example, CNS-specific, or can be a combination of several tissues (e.g., muscle tissue and CNS tissue). Exemplary tissues or other targets can include liver, skeletal muscle, cardiac muscle, adipose deposits, kidney, lung, vascular endothelium, epithelium, hematopoietic cells, cancer cells, CNS, and / or CSF. In a preferred embodiment, the pharmaceutical composition provided herein is administered to the CNS or CSF by injection (e.g., by intracerebral parenchymal injection, intrathecal injection, intracisternal injection, or intraventricular injection). One or more of these methods can be used to administer the pharmaceutical composition of the present disclosure.

[0257] In certain embodiments, the pharmaceutical compositions provided herein comprise an “effective amount” or “therapeutically effective amount”. As used herein, such an amount refers to an amount that is effective at the dosage and for the period of time necessary to achieve the desired therapeutic result.

[0258] The dosage of the pharmaceutical compositions of the present disclosure depends on a variety of factors, including the route of administration, the disease to be 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. Generally, the dosage can be an amount effective to treat the disease without inducing significant toxicity. In certain embodiments, the pharmaceutical composition can be in unit dosage form as needed.

[0259] The pharmaceutical compositions of the present disclosure can be administered to a subject in need thereof when medically necessary. In one exemplary embodiment, a single administration is sufficient. In one embodiment, the pharmaceutical composition is suitable for human subjects and is administered by intracerebral parenchymal injection, intrathecal injection, cisterna magna injection, or intraventricular injection. In one embodiment, the pharmaceutical composition is delivered via a bolus injection through a peripheral vein. In other embodiments, the pharmaceutical composition is delivered via an infusion through a peripheral vein.

[0260] In another aspect, the present application also provides a kit that contains in one or more containers a nucleic acid molecule, a vector, a host cell, a virion, or a pharmaceutical composition as described herein. The kit can include instructions or packaging materials that describe how to administer the nucleic acid molecule, vector, host cell, or virion contained within the kit to a patient. The containers of the kit can be made of any suitable material, such as glass, plastic, metal, etc., and can have any suitable size, shape, or configuration. In certain embodiments, the kit can include one or more ampoules or syringes that contain a nucleic acid molecule, a vector, a host cell, a virion, or a pharmaceutical composition in a suitable liquid or solution form.

[0261] Treatment method

[0262] 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 diseases, such as neurological diseases. In some embodiments, the chemical, protein or nucleic acid molecule of the present invention can be used to treat or improve one or more symptoms associated with gene mutations or low-expression or non-expression genes in a subject. In certain embodiments, the treatment can be treating the subject via gene therapy, wherein the gene therapy is directly administered to the subject in need thereof (e.g., directly administered to the CNS of the subject), or systemically administered via injection and / or infusion. The therapy can be formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intracerebral parenchymal administration, intrathecal administration, cisterna magna administration, intraventricular administration or intraperitoneal administration, or administered via the nose, spray, mouth, aerosol, rectum or vagina, e.g., by intracerebral parenchymal injection, intrathecal injection, cisterna magna injection or intraventricular injection. The tissue target can be specific, e.g., CNS-specific, or can be a combination of several tissues.

[0263] In any embodiment herein, the target cell can be a nerve cell, muscle cell, heart cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell or kidney cell. In some cases, the target cell can be a nerve cell, e.g., a brain cell, brain stem cell, hippocampal cell or cerebellar cell. For example, in some embodiments, the nerve cell is a GABAergic cell, e.g., a cell expressing parvalbumin. In some cases, the target cell can be a CNS cell, such as an excitatory neuron, dopaminergic neuron, glial cell, ependymal cell, oligodendrocyte, astrocyte, microglial cell, motor neuron, vascular cell, GABAergic neuron or non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or other CNS cells (e.g., a CNS cell type that has never expressed any of PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST and VIP).

[0264] In any embodiment, the therapy of the present invention can be used to increase the production or expression of a target protein in cells such as GABA neurons or parvalbumin neurons.

[0265] In certain embodiments, the treatments provided herein do not cause adverse reactions in a subject. Treatment with the nucleic acid molecules, expression vectors, pharmaceutical compositions, or virions described herein can cause fewer or less severe adverse reactions in a subject compared to treatment with a similar gene therapy containing the same transgene linked to a non-albumin neuron-selective regulatory element.

[0266] Sequence listing

[0267] Table 4 below provides certain sequences that may be referred to in other parts of the present disclosure.

[0268] SEQ ID NO: 1-10 and 43-48 are DRG off-target sequences that may be present in the RNA transcripts encoded in the nucleic acid cassettes of the present disclosure.

[0269] SEQ ID NOs 57-62, 64-71, 110, and 112 are liver off-target sequences that may be present in the RNA transcripts encoded in the nucleic acid cassettes of the present disclosure.

[0270] Table 4 also provides the RNA sequences (which may be present in the cassette itself) and the DNA forms of certain miRNAs and pre-miRNAs that can bind to those RNA sequences.

[0271] Also listed in Table 4 are additional sequences (including sequences of controls and some exemplary combinations of sequences that cause off-target expression in both DRG cells and hepatocytes).

[0272] Table 4

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Embodiment

[0283] Embodiments of the present disclosure include nucleic acid cassettes that encode a transgenic construct comprising one or more DRG off-target elements, one or more liver off-target elements, or both; RNA transcripts derived therefrom; and isolated and / or synthetic RNA molecules comprising one or more DRG off-target elements, one or more liver off-target elements, or both. Embodiments also include methods of making and using such nucleic acid cassettes and RNA molecules, including using the nucleic acid cassettes and RNA molecules as therapeutic agents. The following embodiments are intended to further delineate specific aspects of the present disclosure but are not intended to limit its scope.

[0284] Embodiment 1. A nucleic acid cassette comprising a therapeutic transgenic construct that encodes an RNA transcript, wherein the RNA transcript comprises the sequence of: (i) any one of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof of the sequence; or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0285] Embodiment 2. The nucleic acid cassette according to Embodiment 1, wherein the RNA transcript comprises a sequence of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 1-10 and 43-48, the sequence reducing expression in dorsal root ganglion (DRG) cells.

[0286] Embodiment 3. The nucleic acid cassette according to Embodiment 1 or 2, wherein the RNA transcript further comprises a second sequence of (i), (ii), or (iii).

[0287] Embodiment 4. The nucleic acid cassette according to Embodiment 3, wherein the RNA transcript further comprises a third sequence of (i), (ii), or (iii).

[0288] Embodiment 5. The nucleic acid cassette according to Embodiment 4, wherein the RNA transcript further comprises a fourth sequence of (i), (ii), or (iii).

[0289] Embodiment 6. The nucleic acid cassette according to any one of Embodiments 1-5, wherein the RNA transcript comprises two or more copies of the sequence of (i), (ii), or (iii).

[0290] Embodiment 7. The nucleic acid cassette according to Embodiment 6, wherein the RNA transcript comprises three or more copies of the sequence of (i), (ii), or (iii).

[0291] Embodiment 8. The nucleic acid cassette according to embodiment 7, wherein the RNA transcript comprises four or more copies of the following sequences: (i), (ii), or (iii).

[0292] Embodiment 9. The nucleic acid cassette according to embodiment 8, wherein the RNA transcript comprises five or more copies of the following sequences: (i), (ii), or (iii).

[0293] Embodiment 6. The nucleic acid cassette according to any one of embodiments 1 to 5, wherein the sequence (i), (ii), or (iii) provides a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, and hsa-mir-494-3p.

[0294] Embodiment 11. The nucleic acid cassette according to any one of embodiments 1 to 10, wherein the RNA transcript is mRNA, optionally wherein the sequence (i), (ii), or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

[0295] Embodiment 12. The nucleic acid cassette according to embodiment 11, wherein the sequence (i), (ii), or (iii) is located in the 3'UTR region of the mRNA.

[0296] Embodiment 13. The nucleic acid cassette according to embodiment 11, wherein the sequence (i), (ii), or (iii) is located in the 5'UTR region of the mRNA.

[0297] Embodiment 14. The nucleic acid cassette according to embodiment 11, wherein the sequence (i), (ii), or (iii) is located in the intron of the mRNA.

[0298] Embodiment 15. The nucleic acid cassette according to any one of embodiments 1 to 14, wherein the nucleic acid cassette is non-naturally occurring.

[0299] Embodiment 16. The nucleic acid cassette according to any one of embodiments 1 to 15, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0300] Embodiment 17. The nucleic acid cassette according to Embodiment 16, 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0301] Embodiment 18. The nucleic acid cassette according to any one of Embodiments 1 to 17, wherein the nucleic acid cassette comprises an enhancer.

[0302] Embodiment 19. The nucleic acid cassette according to any one of Embodiments 1 to 18, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder, optionally wherein the RNA transcript is an mRNA encoding a therapeutic protein associated with the neurological disease or disorder.

[0303] Embodiment 20. The nucleic acid cassette of embodiment 19, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0304] Embodiment 21. The nucleic acid cassette according to embodiment 19 or 20, wherein the therapeutic protein encoded by the mRNA is selected from: (i) a protein encoded by a gene selected from: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0305] Embodiment 22. The nucleic acid cassette according to any one of embodiments 1 to 21, wherein:

[0306] (a) the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a variant, functional fragment or combination thereof of the sequence; or

[0307] (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);

[0310] (b) the nucleic acid cassette comprises a CNS-selective promoter; and

[0311] (c) the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0312] Embodiment 23. The nucleic acid cassette according to any one of Embodiments 1 to 22, wherein:

[0313] (a) the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a variant, functional fragment, or a combination thereof of the sequence; or

[0314] (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); and

[0317] (b) the nucleic acid cassette comprises a promoter selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter,

[0318] 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter,

[0319] glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter,

[0320] neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a)

[0321] promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; optionally

[0322] (c) wherein the therapeutic RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is encoded by a gene selected from:

[0323] ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A,

[0324] CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4,

[0325] CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13,

[0326] FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD,

[0327] GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2,

[0328] KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2,

[0329] MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1,

[0330] PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A,

[0331] SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1,

[0332] SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1,

[0333] SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0334] Embodiment 24. The nucleic acid cassette according to any one of Embodiments 1 to 23, wherein the sequence (i), (ii), or (iii) causes the expression of the RNA transcript in DRG cells to be reduced compared to the expression of the RNA transcript that is otherwise identical but lacks the sequence (i), (ii), or (iii); when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) causes the expression of the polypeptide encoded by the mRNA in DRG cells to be reduced compared to the expression of the polypeptide of the mRNA that is otherwise identical but lacks the sequence (i), (ii), or (iii).

[0335] Embodiment 25. The nucleic acid cassette according to Embodiment 24, wherein the sequence (i), (ii), or (iii) causes the expression level of the RNA transcript and / or the polypeptide encoded by the mRNA in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the RNA transcript or the polypeptide of the RNA transcript that is otherwise identical but lacks the sequence (i), (ii), or (iii).

[0336] Embodiment 26. The nucleic acid cassette according to embodiment 24 or 25, wherein the expression level of the RNA transcript and / or the polypeptide encoded by the mRNA in DRG cells caused by the sequence (i), (ii), or (iii) 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 the expression level of the RNA transcript and / or the polypeptide in DRG cells of an RNA transcript that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0337] Embodiment 27. The nucleic acid cassette according to any one of embodiments 1 to 26, wherein the sequence (i), (ii), or (iii) does not cause a significant decrease in the expression of the RNA transcript in the target cell compared to the expression of an RNA transcript that is otherwise identical but does not have the sequence (i), (ii), or (iii); when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) does not cause a significant decrease in the expression of the polypeptide encoded by the mRNA in the target cell compared to the expression of the polypeptide of an mRNA that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0338] Embodiment 28. The nucleic acid cassette according to embodiment 27, wherein the sequence (i), (ii), or (iii) does not cause a decrease in the expression of the RNA transcript and / or the polypeptide encoded thereby (when the RNA transcript is mRNA) in the target cell compared to the expression of the polypeptide of an RNA transcript that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0339] Embodiment 29. The nucleic acid cassette according to embodiment 27, wherein the expression level of the RNA transcript and / or the polypeptide encoded thereby in the target cell caused by the sequence (i), (ii), or (iii) is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the polypeptide of an RNA transcript that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0340] Embodiment 30. The nucleic acid cassette according to any one of Embodiments 27 to 29, wherein the target cell is a nerve cell.

[0341] Embodiment 31. The nucleic acid cassette according to Embodiment 30, wherein the nerve cell is a brain cell, a neural stem cell, a hippocampal cell, or a cerebellar cell.

[0342] Embodiment 32. The nucleic acid cassette according to Embodiment 31, wherein the nerve cell is a GABAergic cell.

[0343] Embodiment 33. The nucleic acid cassette according to Embodiment 32, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0344] Embodiment 34. The nucleic acid cassette according to any one of Embodiments 1 to 33, wherein the nucleic acid cassette is a linear construct or a vector.

[0345] Embodiment 35. The nucleic acid cassette according to Embodiment 34, wherein the vector is a plasmid.

[0346] Embodiment 36. The nucleic acid cassette according to Embodiment 34, wherein the vector is a viral vector.

[0347] Embodiment 37. The nucleic acid cassette according to Embodiment 36, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0348] Embodiment 38. The nucleic acid cassette according to Embodiment 37, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0349] Embodiment 39. The nucleic acid cassette according to Embodiment 37 or 38, wherein the AAV is scAAV.

[0350] Embodiment 40. The nucleic acid cassette according to Embodiment 36, wherein the viral vector is a lentiviral vector.

[0351] Embodiment 41. An RNA having a sequence encoded by the nucleic acid cassette according to any one of Embodiments 1 to 40.

[0352] Embodiment 42. A nucleic acid cassette, the nucleic acid cassette comprising a transgene encoding an RNA transcript, wherein the RNA transcript is a therapeutic RNA transcript, for example, an mRNA encoding a therapeutic protein, and comprising binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0353] Embodiment 43. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising binding sites for two or more miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0354] Embodiment 44. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising binding sites for three or more miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0355] Embodiment 45. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising two binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0356] Embodiment 46. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising three binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0357] Embodiment 47. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising four binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p or their complementary sequences.

[0358] Embodiment 48. The nucleic acid cassette according to Embodiment 42, the nucleic acid cassette comprising more than four binding sites for miRNAs selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p and mir-494-3p or their complementary sequences.

[0359] Embodiment 49. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is mir-196b-5p.

[0360] Embodiment 50. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is mir-10b-5p.

[0361] Embodiment 51. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is mir-24-2-5p.

[0362] Embodiment 52. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is hsa-mir-183-3p.

[0363] Embodiment 53. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is mir-196a-5p.

[0364] Embodiment 54. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the miRNA is mir-494-3p.

[0365] Embodiment 55. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the RNA transcript contains multiple copies of the same miRNA binding site.

[0366] Embodiment 56. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the RNA transcript contains multiple different miRNA binding sites.

[0367] Embodiment 57. The nucleic acid cassette according to any one of Embodiments 42 to 48, wherein the RNA transcript contains multiple copies of the same miRNA binding site and multiple different miRNA binding sites.

[0368] Embodiment 58. The nucleic acid cassette according to any one of Embodiments 42 to 57, wherein the RNA transcript further contains a sequence having at least 10 contiguous nucleotides of any one of SEQ ID NOs. 1-10 and 43-48, the sequence reducing expression in DRG cells.

[0369] Embodiment 59. The nucleic acid cassette according to any one of embodiments 42 to 57, wherein the RNA transcript further comprises at least two sequences having at least 20 contiguous nucleotides of any one of SEQ ID NOs. 1-10 and 43-48, and the sequences reduce expression in DRG cells.

[0370] Embodiment 60. The nucleic acid cassette according to any one of embodiments 42 to 59, wherein the RNA transcript is mRNA, and the miRNA binding site is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

[0371] Embodiment 61. The nucleic acid cassette according to embodiment 60, wherein the miRNA binding site is located in the 3'UTR region of the mRNA.

[0372] Embodiment 62. The nucleic acid cassette according to embodiment 60, wherein the miRNA binding site is located in the 5'UTR region of the mRNA.

[0373] Embodiment 63. The nucleic acid cassette according to embodiment 60, wherein the miRNA binding site is located in the intron of the mRNA.

[0374] Embodiment 64. The nucleic acid cassette according to any one of embodiments 42 to 63, wherein the nucleic acid cassette is non-naturally occurring.

[0375] Embodiment 65. The nucleic acid cassette according to any one of embodiments 42 to 64, wherein the nucleic acid cassette comprises a promoter.

[0376] Embodiment 66. The nucleic acid cassette according to any one of embodiments 42 to 65, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0377] Embodiment 67. The nucleic acid cassette according to Embodiment 66, 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0378] Embodiment 68. The nucleic acid cassette according to any one of Embodiments 42 to 67, wherein the nucleic acid cassette comprises an enhancer.

[0379] Embodiment 69. The nucleic acid cassette according to any one of Embodiments 42 to 68, wherein the therapeutic RNA transcript is for treating a neurological disease or disorder, optionally wherein the therapeutic RNA transcript is an mRNA encoding a therapeutic protein associated with the neurological disease or disorder.

[0380] Embodiment 70. The nucleic acid cassette according to Embodiment 69, wherein the therapeutic protein is selected from: (i) a protein encoded by a gene selected from: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0381] Embodiment 71. The nucleic acid cassette according to any one of Embodiments 42 to 70, wherein:

[0382] (a) the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48;

[0383] (ii) a variant, functional fragment, or combination thereof of the sequence; or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to

[0384] (i) or (ii);

[0387] (b) the nucleic acid cassette comprises a CNS-selective promoter; and

[0388] (c) the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0389] Embodiment 72. The nucleic acid cassette according to Embodiment 71, wherein:

[0390] (a) the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments thereof, or combinations thereof; or (iii) sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii); and

[0391] (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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; optionally

[0392] (c) wherein the RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0393] Embodiment 73. The nucleic acid cassette according to any one of Embodiments 42 to 72, wherein the miRNA binding site causes the expression of the RNA transcript and / or the polypeptide encoded thereby (when the RNA transcript is an mRNA) in DRG cells to be reduced compared to the expression of the polypeptide of an RNA transcript that is otherwise identical but lacks the miRNA binding site in DRG cells.

[0394] Embodiment 74. The nucleic acid cassette according to any one of Embodiments 42 to 73, wherein the miRNA binding site causes the expression level of the RNA transcript and / or the polypeptide encoded thereby in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5 or at most 1 / 10 compared to the expression level of the polypeptide of an RNA transcript that is otherwise identical but lacks the miRNA binding site in DRG cells.

[0395] Embodiment 75. The nucleic acid cassette according to any one of Embodiments 42 to 74, wherein the miRNA binding site causes the expression level of the polypeptide encoded by the RNA transcript in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the polypeptide of an otherwise identical RNA transcript lacking the miRNA binding site in DRG cells.

[0396] Embodiment 76. The nucleic acid cassette according to any one of Embodiments 42 to 75, wherein the sequence (i), (ii) or (iii) does not cause the expression of the RNA transcript in target cells to be significantly reduced compared to the expression of an otherwise identical RNA transcript lacking the sequence (i), (ii) or (iii) in target cells; when the RNA transcript is mRNA, the sequence (i), (ii) or (iii) does not cause the expression of the polypeptide encoded by the mRNA in target cells to be significantly reduced compared to the expression of the polypeptide of an otherwise identical mRNA lacking the sequence (i), (ii) or (iii) in target cells.

[0397] Embodiment 77. The nucleic acid cassette according to Embodiment 76, wherein the sequence (i), (ii) or (iii) does not reduce the expression of the RNA transcript in target cells compared to the expression of an otherwise identical RNA transcript lacking the sequence (i), (ii) or (iii) in target cells; when the RNA transcript is mRNA, the sequence (i), (ii) or (iii) does not reduce the expression of the polypeptide encoded by the mRNA in the target cells compared to the expression of the polypeptide of an otherwise identical mRNA lacking the sequence (i), (ii) or (iii) in the target cells.

[0398] Embodiment 78. The nucleic acid cassette according to Embodiment 76, wherein the expression level of the RNA transcript in the target cell caused by the sequence (i), (ii), or (iii) is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the RNA transcript in the target cell of an mRNA that is otherwise identical but lacks the sequence (i), (ii), or (iii); when the RNA transcript is an mRNA, the expression level of the polypeptide encoded by the mRNA in the target cell caused by the sequence (i), (ii), or (iii) is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the polypeptide in the target cell of an mRNA that is otherwise identical but lacks the sequence (i), (ii), or (iii).

[0399] Embodiment 79. The nucleic acid cassette according to any one of Embodiments 42 to 78, wherein the target cell is a nerve cell.

[0400] Embodiment 80. The nucleic acid cassette according to Embodiment 79, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0401] Embodiment 81. The nucleic acid cassette according to Embodiment 80, wherein the nerve cell is a GABAergic cell.

[0402] Embodiment 82. The nucleic acid cassette according to Embodiment 81, wherein the GABAergic cell is a cell expressing parvalbumin.

[0403] Embodiment 83. The nucleic acid cassette according to any one of Embodiments 42 to 82, wherein the nucleic acid cassette is a linear construct or a vector.

[0404] Embodiment 84. The nucleic acid cassette according to Embodiment 83, wherein the vector is a plasmid.

[0405] Embodiment 85. The nucleic acid cassette according to Embodiment 83, wherein the vector is a viral vector.

[0406] Embodiment 86. The nucleic acid cassette according to Embodiment 85, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0407] Embodiment 87. The nucleic acid cassette according to Embodiment 86, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0408] Embodiment 88. The nucleic acid cassette according to Embodiment 86 or 87, wherein the AAV is scAAV.

[0409] Embodiment 89. The nucleic acid cassette according to Embodiment 85, wherein the viral vector is a lentiviral vector.

[0410] Embodiment 90. An RNA encoded by the nucleic acid cassette according to any one of Embodiments 42 to 89.

[0411] Embodiment 91. The nucleic acid cassette according to any one of Embodiments 42 to 89, wherein the RNA transcript is an mRNA encoding a polypeptide.

[0412] Embodiment 92. The nucleic acid cassette according to Embodiment 91, wherein the polypeptide is a therapeutic protein.

[0413] Embodiment 93. An RNA having a sequence encoded by the nucleic acid cassette according to any one of Embodiments 42 to 92.

[0414] Embodiment 94. A method of reducing the dorsal root ganglion (DRG) expression of a therapeutic RNA transcript or a therapeutic protein encoded by the therapeutic RNA transcript (i.e., when the therapeutic RNA transcript is mRNA) while maintaining the expression of the therapeutic RNA transcript or the therapeutic protein in target cells, the method comprising: comprising the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of the sequences; or (iii) sequences that are 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) in the therapeutic RNA transcript.

[0415] Embodiment 95. The method according to Embodiment 94, wherein the therapeutic RNA transcript further comprises a second sequence (i), (ii), or (iii).

[0416] Embodiment 96. The method according to Embodiment 94, wherein the therapeutic RNA transcript further comprises a third sequence (i), (ii), or (iii).

[0417] Embodiment 97. The method according to embodiment 94, wherein the therapeutic RNA transcript further comprises a fourth sequence (i), (ii), or (iii).

[0418] Embodiment 98. The method according to embodiment 94, wherein the therapeutic RNA transcript comprises five or more sequences (i), (ii), or (iii).

[0419] Embodiment 99. The method according to any one of embodiments 94 to 98, wherein the therapeutic RNA transcript comprises two or more copies of the following sequences: (i), (ii), or (iii).

[0420] Embodiment 100. The method according to any one of embodiments 94 to 99, wherein the therapeutic RNA transcript comprises three or more copies of the following sequences: (i), (ii), or (iii).

[0421] Embodiment 101. The method according to any one of embodiments 94 to 100, wherein the therapeutic RNA transcript comprises four or more copies of the following sequences: (i), (ii), or (iii).

[0422] Embodiment 102. The method according to any one of embodiments 94 to 101, wherein the therapeutic RNA transcript comprises five or more copies of the following sequences: (i), (ii), or (iii).

[0423] Embodiment 103. The method according to any one of embodiments 94 to 102, wherein the therapeutic RNA transcript comprises at least 10 contiguous nucleotides having any one of SEQ ID NOs: 1-10 and 43-48, and the contiguous nucleotides reduce expression in DRG cells.

[0424] Embodiment 104. The method according to any one of embodiments 94 to 103, wherein the therapeutic RNA transcript is mRNA, and the sequence (i), (ii), or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

[0425] Embodiment 105. The method according to embodiment 104, wherein the sequence (i), (ii), or (iii) is located in the 3'UTR region of the mRNA.

[0426] Embodiment 106. The method according to embodiment 104, wherein the sequence (i), (ii), or (iii) is located in the 5'UTR region of the mRNA.

[0427] Embodiment 107. The method according to embodiment 104, wherein the sequence (i), (ii), or (iii) is located in an intron of the mRNA.

[0428] Embodiment 108. The method according to any one of embodiments 94 to 107, wherein the method comprises administering to a subject a nucleic acid cassette encoding the therapeutic RNA transcript.

[0429] Embodiment 109. The method according to any one of embodiments 94 to 108, wherein the administration is systemic administration.

[0430] Embodiment 110. The method according to any one of embodiments 94 to 108, wherein the administration is local administration.

[0431] Embodiment 111. The method according to embodiment 110, wherein the nucleic acid is locally administered to the brain or CNS tissue.

[0432] Embodiment 112. The method according to embodiment 110 or 111, wherein the administration is carried out by intracerebral, intrathecal, cisterna magna, intraventricular, or intracranial administration.

[0433] Embodiment 113. The method according to any one of embodiments 94 to 112, wherein the therapeutic RNA transcript is used for treating a neurological disease or disorder.

[0434] Embodiment 114. The method according to Embodiment 113, wherein the RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0435] Embodiment 115. The method according to any one of Embodiments 108 to 114, wherein the subject has a neurological disease or disorder.

[0436] Embodiment 116. The method of embodiment 115, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central-temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep ( CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0437] Embodiment 117. The method of any one of Embodiments 108 to 116, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0438] Embodiment 118. A method according to embodiment 117, wherein the CNS-selective promoter is selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-interconnectin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter.

[0439] Embodiment 119. The method of any one of Embodiments 108 to 118, wherein:

[0440] (a) The therapeutic RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of said sequences; or (iii) sequences that are 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);

[0443] (b) The nucleic acid cassette comprises a CNS-selective promoter; and

[0444] (c) The therapeutic RNA transcript is for treating a neurological disease or disorder.

[0445] Embodiment 120. The method according to Embodiment 119, wherein:

[0446] (a) The therapeutic RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) variants, functional fragments, or combinations thereof of said sequences; or (iii) sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); and

[0449] (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,

[0450] 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter,

[0451] glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter,

[0452] neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a)

[0453] promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; optionally

[0454] (c) wherein the therapeutic RNA transcript is mRNA, and wherein the mRNA encodes a therapeutic protein encoded by a gene selected from the following:

[0455] ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2,

[0456] CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1,

[0457] FOLR1, FOXG1, GAB RAI, GABRB3, GABRD, GABRG2,

[0458] GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C,

[0459] Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A,

[0460] SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1,

[0461] TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0462] Embodiment 121. The method according to any one of embodiments 94 to 120, wherein the sequence (i), (ii), or (iii) causes the expression level of the RNA transcript in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the RNA transcript in DRG cells that is otherwise identical but does not have the sequence (i), (ii), or (iii); when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) causes the expression level of the protein encoded by the mRNA in DRG cells to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the protein in DRG cells from an mRNA that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0463] Embodiment 122. The method according to any one of embodiments 94 to 121, wherein the sequence (i), (ii), or (iii) causes the expression level of the RNA transcript in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the RNA transcript in DRG cells that is otherwise identical but does not have the sequence (i), (ii), or (iii)

[0464] ; when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) causes the expression level of the protein encoded by the mRNA in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the protein in DRG cells from an mRNA that is otherwise identical but does not have the sequence (i), (ii), or (iii).

[0465] Embodiment 123. The method according to any one of Embodiments 94 to 122, wherein the sequence (i), (ii), or (iii) does not cause the expression of the RNA transcript in the target cell to be significantly reduced compared to the expression of the RNA transcript in the target cell that is otherwise identical but does not have the sequence (i), (ii), or (iii); when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) does not cause the expression of the protein encoded by the mRNA in the target cell to be significantly reduced compared to the expression of the protein in the target cell that is otherwise identical but does not have the sequence (i), (ii), or (iii) of the mRNA.

[0466] Embodiment 124. The method according to Embodiment 123, wherein the sequence (i), (ii), or (iii) does not cause the expression of the RNA transcript and / or the protein encoded thereby (i.e., when the RNA transcript is mRNA) in the target cell to be reduced compared to the expression of the RNA transcript and / or the protein in the target cell that is otherwise identical but does not have the sequence (i), (ii), or (iii) of the mRNA.

[0467] Embodiment 125. The method according to any one of Embodiments 94 to 124, wherein the sequence (i), (ii), or (iii) causes the expression level of the RNA transcript in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the RNA transcript in the target cell that is otherwise identical but does not have the sequence (i), (ii), or (iii); when the RNA transcript is mRNA, the sequence (i), (ii), or (iii) causes the expression level of the protein encoded by the mRNA in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the protein in the target cell that is otherwise identical but does not have the sequence (i), (ii), or (iii) of the mRNA.

[0468] Embodiment 126. The method according to any one of Embodiments 124 to 125, wherein the target cell is a nerve cell.

[0469] Embodiment 127. The method according to Embodiment 126, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell or a cerebellar cell.

[0470] Embodiment 128. The method according to Embodiment 127, wherein the nerve cell is a GABAergic cell.

[0471] Embodiment 129. The method according to Embodiment 128, wherein the GABAergic cell is a cell expressing parvalbumin.

[0472] Embodiment 130. The method according to any one of Embodiments 94 to 129, wherein the RNA transcript is expressed from a nucleic acid cassette.

[0473] Embodiment 131. The method according to Embodiment 130, wherein the nucleic acid cassette is a linear construct.

[0474] Embodiment 132. The method according to Embodiment 130, wherein the nucleic acid cassette is a vector.

[0475] Embodiment 133. The method according to Embodiment 132, wherein the vector is a plasmid.

[0476] Embodiment 134. The method according to Embodiment 132, wherein the vector is a viral vector.

[0477] Embodiment 135. The method according to Embodiment 134, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0478] Embodiment 136. The method according to Embodiment 135, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV-DJ.

[0479] Embodiment 137. The method according to Embodiment 135 or 136, wherein the AAV is scAAV.

[0480] Embodiment 138. The method according to Embodiment 134, wherein the viral vector is a lentiviral vector.

[0481] Embodiment 139. The method according to any one of Embodiments 132 to 138, wherein the method comprises administering the vector to a subject.

[0482] Embodiment 140. The method according to any one of Embodiments 132 to 138, the method further comprises administering the vector to a subject.

[0483] Embodiment 141. A nucleic acid cassette, the nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the following sequences: (i) any one of SEQ ID NO.65, 110, and 112; (ii) variants, functional fragments, or combinations thereof of the sequences; or (iii) sequences that are 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).

[0484] Embodiment 142. The nucleic acid cassette according to Embodiment 141, wherein the sequence reduces the expression of the RNA transcript in hepatocytes.

[0485] Embodiment 143. The nucleic acid cassette according to Embodiment 141 or 142, wherein the RNA transcript further comprises a second sequence (i), (ii), or (iii).

[0486] Embodiment 144. The nucleic acid cassette according to Embodiment 143, wherein the RNA transcript further comprises a third sequence (i), (ii), or (iii).

[0487] Embodiment 145. The nucleic acid cassette according to Embodiment 144, wherein the RNA transcript further comprises a fourth sequence (i), (ii), or (iii).

[0488] Embodiment 146. The nucleic acid cassette according to any one of Embodiments 141 to 145, wherein the RNA transcript comprises two or more copies of the following sequences: (i), (ii), or (iii).

[0489] Embodiment 147. The nucleic acid cassette according to Embodiment 146, wherein the RNA transcript comprises three or more copies of the following sequences: (i), (ii), or (iii).

[0490] Embodiment 148. The nucleic acid cassette according to Embodiment 147, wherein the RNA transcript comprises four or more copies of the following sequences: (i), (ii), or (iii).

[0491] Embodiment 149. The nucleic acid cassette according to Embodiment 148, wherein the RNA transcript comprises five or more copies of the following sequences: (i), (ii), or (iii).

[0492] Embodiment 150. The nucleic acid cassette according to any one of Embodiments 141 to 149, wherein the RNA transcript is mRNA, and wherein the sequence is located in one or more of the following: the 3' UTR region of the mRNA, the 5' UTR region of the mRNA, or an intron of the mRNA.

[0493] Embodiment 151. The nucleic acid cassette according to Embodiment 150, wherein the sequence is located in the 3' UTR region of the mRNA.

[0494] Embodiment 152. The nucleic acid cassette according to Embodiment 150, wherein the sequence is located in the 5' UTR region of the mRNA.

[0495] Embodiment 153. The nucleic acid cassette according to Embodiment 150, wherein the sequence is located in an intron of the mRNA.

[0496] Embodiment 154. The nucleic acid cassette according to any one of Embodiments 141 to 153, wherein the nucleic acid cassette is non-naturally occurring.

[0497] Embodiment 155. The nucleic acid cassette according to any one of Embodiments 141 to 154, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0498] Embodiment 156. The nucleic acid cassette according to Embodiment 155, 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0499] Embodiment 157. The nucleic acid cassette according to any one of Embodiments 141 to 156, wherein the nucleic acid cassette comprises an enhancer.

[0500] Embodiment 158. The nucleic acid cassette according to any one of Embodiments 141 to 157, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0501] Embodiment 159. The nucleic acid cassette of embodiment 158, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with persistent spikes and slow waves during sleep waves (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0502] Embodiment 160. The nucleic acid cassette according to Embodiment 158 or 159, wherein the therapeutic RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0503] Embodiment 161. The nucleic acid cassette according to any one of Embodiments 141 to 160, wherein:

[0504] (a) the RNA transcript comprises the sequence;

[0505] (b) the nucleic acid cassette comprises a CNS-selective promoter; and

[0506] (c) the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0507] Embodiment 162. The nucleic acid cassette according to any one of Embodiments 141 to 161, wherein:

[0508] (a) the RNA transcript comprises the sequence; and

[0509] (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,

[0510] 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter,

[0511] glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter,

[0512] neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a)

[0513] promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; optionally

[0514] (c) wherein the RNA transcript is mRNA, wherein the mRNA encodes a therapeutic protein, the therapeutic protein being encoded by a gene selected from:

[0515] ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2,

[0516] CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1,

[0517] FOLR1, FOXG1, GAB RAI, GABRB3, GABRD, GABRG2,

[0518] GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C,

[0519] Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A,

[0520] SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1,

[0521] 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 regulates the expression of the gene from (i).

[0522] Embodiment 163. The nucleic acid cassette according to any one of Embodiments 141 to 162, wherein the sequence causes the expression of the RNA transcript and / or the polypeptide encoded thereby (i.e., when the RNA transcript is mRNA) in hepatocytes to be reduced compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the sequence in hepatocytes.

[0523] Embodiment 164. The nucleic acid cassette according to Embodiment 163, wherein the expression level of the RNA transcript and / or the polypeptide encoded thereby in hepatocytes is reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the sequence in hepatocytes.

[0524] Embodiment 165. The nucleic acid cassette according to Embodiment 163 or 164, wherein the expression level of the RNA transcript and / or the polypeptide encoded thereby in hepatocytes is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the sequence in hepatocytes.

[0525] Embodiment 166. The nucleic acid cassette according to any one of Embodiments 141 to 165, wherein the sequence does not cause the expression of the RNA transcript and / or the polypeptide encoded thereby in the target cell to be significantly reduced compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the sequence in the target cell.

[0526] Embodiment 167. The nucleic acid cassette according to Embodiment 166, wherein the sequence does not reduce the expression of the RNA transcript and / or the polypeptide encoded thereby in the target cell compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the sequence in the target cell.

[0527] Embodiment 168. The nucleic acid cassette according to Embodiment 167, wherein the sequence causes the expression level of the RNA transcript and / or the polypeptide encoded thereby in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the RNA transcript and / or the polypeptide in the target cell of an otherwise identical RNA transcript without the sequence.

[0528] Embodiment 169. The nucleic acid cassette according to any one of Embodiments 166 to 168, wherein the target cell is a nerve cell.

[0529] Embodiment 170. The nucleic acid cassette according to Embodiment 169, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell or a cerebellar cell.

[0530] Embodiment 171. The nucleic acid cassette according to Embodiment 169, wherein the nerve cell is a GABAergic cell.

[0531] Embodiment 172. The nucleic acid cassette according to Embodiment 170, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0532] Embodiment 173. The nucleic acid cassette according to any one of Embodiments 141 to 172, wherein the nucleic acid cassette is a linear construct or a vector.

[0533] Embodiment 174. The nucleic acid cassette according to Embodiment 173, wherein the vector is a plasmid.

[0534] Embodiment 175. The nucleic acid cassette according to Embodiment 173, wherein the vector is a viral vector.

[0535] Embodiment 176. The nucleic acid cassette according to Embodiment 175, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0536] Embodiment 177. The nucleic acid cassette according to Embodiment 176, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV-DJ.

[0537] Embodiment 178. The nucleic acid cassette according to Embodiment 176 or 177, wherein the AAV is scAAV.

[0538] Embodiment 179. The nucleic acid cassette according to Embodiment 173, wherein the viral vector is a lentiviral vector.

[0539] Embodiment 180. An RNA having a sequence encoded by the nucleic acid cassette according to any one of Embodiments 141 to 180.

[0540] Embodiment 181. A method of reducing hepatic expression of a therapeutic RNA transcript and / or a protein encoded thereby (i.e., when the RNA transcript is an mRNA) while maintaining expression of the RNA transcript and / or the protein in a target tissue, the method comprising: comprising the following sequences: (i) any one of SEQ ID NO.SEQ ID NO. 65, 110, and 112; (ii) variants, functional fragments thereof, or combinations thereof; or (iii) sequences that are 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).

[0541] Embodiment 182. The method according to Embodiment 181, wherein when the RNA transcript is an mRNA, the sequence is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or an intron of the mRNA.

[0542] Embodiment 183. The method according to Embodiment 182, wherein the sequence is located in the 3'UTR region of the mRNA.

[0543] Embodiment 184. The method according to Embodiment 182, wherein the sequence is located in the 5'UTR region of the mRNA.

[0544] Embodiment 185. The method according to Embodiment 182, wherein the sequence is located in an intron of the mRNA.

[0545] Embodiment 186. The method according to any one of Embodiments 181 to 185, wherein the method comprises administering to a subject a nucleic acid cassette encoding the RNA transcript.

[0546] Embodiment 187. The method according to Embodiment 186, wherein the administration is systemic administration.

[0547] Embodiment 188. The method according to Embodiment 186, wherein the administration is local administration.

[0548] Embodiment 189. The method according to embodiment 188, wherein the nucleic acid is topically administered to the brain or CNS tissue.

[0549] Embodiment 190. The method according to embodiment 188 or 189, wherein the administration is carried out by intracerebral, intrathecal, cisterna magna, intraventricular or intracranial administration.

[0550] Embodiment 191. The method according to any one of embodiments 181 to 190, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0551] Embodiment 192. The method according to embodiment 191, wherein the therapeutic RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0552] Embodiment 193. The method according to any one of embodiments 186 to 193, wherein the subject has a neurological disease or disorder.

[0553] Embodiment 194. The method of embodiment 186, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central-temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep ( CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0554] Embodiment 195. The method of any one of Embodiments 181 to 194, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0555] Embodiment 196. A method according to embodiment 195, wherein the CNS-selective promoter is selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, pretachykininogen 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter.

[0556] Embodiment 197. The method of any one of Embodiments 181 to 196, wherein:

[0557] (a) The RNA transcript comprises the sequence;

[0558] (b) The nucleic acid cassette comprises a CNS-selective promoter; and

[0559] (c) The RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0560] Embodiment 198. The method according to any one of Embodiments 181 to 197, wherein:

[0561] (a) The RNA transcript comprises the sequence; and

[0562] (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,

[0563] 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter,

[0564] glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter,

[0565] neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a)

[0566] promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; optionally

[0567] (c) wherein the RNA transcript is mRNA that encodes a therapeutic protein, and the therapeutic protein is encoded by a gene selected from the following: ALDH7A1,

[0568] ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2,

[0569] CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1,

[0570] FOXG1, GAB RAI, GABRB3, GAB RD, GABRG2, GRIN2A,

[0571] GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1,

[0572] KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1,

[0573] NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN,

[0574] SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3,

[0575] SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i);

[0576] (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0577] Embodiment 199. The method according to any one of Embodiments 181 to 198, wherein the inclusion of the sequence causes the expression level of the RNA transcript and / or the protein encoded thereby (i.e., when the RNA transcript is mRNA) in hepatocytes to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5 or at most 1 / 10 compared to the expression level of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but without the sequence in hepatocytes.

[0578] Embodiment 200. The method according to any one of Embodiments 181 to 199, wherein the inclusion of the sequence causes the expression level of the RNA transcript and / or the protein encoded thereby in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but without the sequence.

[0579] Embodiment 201. The method according to any one of Embodiments 181 to 200, wherein the sequence does not cause the expression of the RNA transcript and / or the protein encoded thereby in the target cell to be significantly reduced compared to the expression of the RNA transcript and / or the protein of an RNA transcript that is otherwise identical but lacks the sequence in the target cell.

[0580] Embodiment 202. The method according to Embodiment 201, wherein the sequence does not cause the expression of the RNA transcript and / or the protein encoded thereby in the target cell to be reduced compared to the expression of the RNA transcript and / or the protein of an RNA transcript that is otherwise identical but lacks the sequence in the target cell.

[0581] Embodiment 203. The method according to any one of Embodiments 181 to 202, wherein the expression level of the RNA transcript and / or the protein encoded thereby in the target cell by the sequence is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the RNA transcript and / or the protein of an RNA transcript that is otherwise identical but lacks the sequence in the target cell.

[0582] Embodiment 204. The method according to any one of Embodiments 201 to 203, wherein the target cell is a nerve cell.

[0583] Embodiment 205. The method according to Embodiment 204, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell or a cerebellar cell.

[0584] Embodiment 206. The method according to Embodiment 205, wherein the nerve cell is a GABAergic cell.

[0585] Embodiment 207. The method according to Embodiment 206, wherein the GABAergic cell is a cell expressing parvalbumin.

[0586] Embodiment 208. The method according to any one of Embodiments 181 to 207, wherein the RNA transcript is expressed from a nucleic acid cassette.

[0587] Embodiment 209. The method according to Embodiment 208, wherein the nucleic acid cassette is a linear construct.

[0588] Embodiment 210. The method according to Embodiment 208 or 209, wherein the nucleic acid cassette is a vector.

[0589] Embodiment 211. The method according to Embodiment 210, wherein the vector is a plasmid.

[0590] Embodiment 212. The method according to Embodiment 210, wherein the vector is a viral vector.

[0591] Embodiment 213. The method according to Embodiment 212, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0592] Embodiment 214. The method according to Embodiment 213, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV-DJ.

[0593] Embodiment 215. The method according to Embodiment 213 or 214, wherein the AAV is scAAV.

[0594] Embodiment 216. The method according to Embodiment 212, wherein the viral vector is a lentiviral vector.

[0595] Embodiment 217. The method according to any one of Embodiments 210 to 216, wherein the method comprises administering the vector to a subject.

[0596] Embodiment 218. The method according to any one of Embodiments 181 to 207, wherein the method comprises administering the RNA to a subject.

[0597] Embodiment 219. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, the RNA transcript comprising a first sequence and a second sequence, the first sequence causing off-target expression in dorsal root ganglion (DRG) cells, and the second sequence causing off-target expression in hepatocytes.

[0598] Embodiment 220. The nucleic acid cassette according to Embodiment 219, wherein the first sequence and the second sequence cause a decrease in the expression of the RNA transcript or the polypeptide encoded thereby (i.e., when the RNA transcript is mRNA) in DRG cells and hepatocytes relative to the target tissue.

[0599] Embodiment 221. The nucleic acid cassette according to Embodiment 219 or 220, wherein the first sequence and the second sequence cause:

[0600] The expression level of the RNA transcript and / or the polypeptide encoded thereby (i.e., when the RNA transcript is mRNA) in DRG cells is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to the expression level of the RNA transcript or polypeptide of an RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in DRG cells; and independently,

[0601] The expression level of the RNA transcript or the polypeptide encoded thereby in hepatocytes is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to the expression level of the RNA transcript or polypeptide of an RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in hepatocytes.

[0602] Embodiment 222. The nucleic acid cassette according to any one of Embodiments 219 to 221, wherein:

[0603] (a) The first sequence is: (i) any one of SEQ ID NOs. 1-10 and 43-48;

[0604] (ii) a variant, functional fragment or combination thereof of the sequence; or (iii) a sequence that is

[0605] at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (i) or (ii); and

[0608] (b) The second sequence is: (iv) any one of SEQ ID NOs. 57-62, 64-71, 110 and 112; (v) a variant, functional fragment or combination thereof of the sequence; or

[0609] (vi) 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 (iv) or (v).

[0612] Embodiment 222A: The nucleic acid cassette according to any one of Embodiments 219 to 222, wherein the first sequence (i), (ii), or (iii) provides a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p, and hsa-mir-494-3p, and the second sequence (iv), (v), or (vi) provides a binding site for hsa-mir-22-3p, has-mir-1258, hsa-mir-5589-3p, hsa-mir-17-5p, hsa-mir-203a-3p, hsa-mir-122-3p, hsa-mir-93-5p, and hsa-mir-19a-3p.

[0613] Embodiment 223. The nucleic acid cassette according to any one of Embodiments 219 to 222A, wherein:

[0614] the first sequence comprises at least 15 contiguous nucleotides having any one of SEQ ID NOs. 1-10 and 43-48, and the contiguous nucleotides reduce expression in dorsal root ganglion (DRG) cells; and

[0615] the second sequence comprises at least 15 contiguous nucleotides having any one of SEQ ID NOs. 57-62, 64-71, 110, and 112, and the contiguous nucleotides reduce expression in hepatocytes.

[0616] Embodiment 224. The nucleic acid cassette according to any one of Embodiments 219 to 223, wherein the RNA transcript comprises at least two copies of any one of sequences (i) to (vi).

[0617] Embodiment 225. The nucleic acid cassette according to Embodiment 224, wherein the RNA transcript comprises at least three or at least four copies of any one of sequences (i) to (vi).

[0618] Embodiment 226. The nucleic acid according to any one of Embodiments 219 to 225, wherein the RNA transcript comprises a combination of sequences selected from Table 3.

[0619] Embodiment 227. The nucleic acid cassette according to any one of embodiments 219 to 227, wherein the RNA transcript is mRNA, and wherein the first sequence and the second sequence are independently located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

[0620] Embodiment 228. The nucleic acid cassette according to embodiment 227, wherein the first sequence and the second sequence are located in the 3'UTR region of the mRNA.

[0621] Embodiment 229. The nucleic acid cassette according to embodiment 227, wherein the first sequence and the second sequence are located in the 5'UTR region of the mRNA.

[0622] Embodiment 230. The nucleic acid cassette according to embodiment 227, wherein the first sequence and the second sequence are located in the intron of the mRNA.

[0623] Embodiment 231. The nucleic acid cassette according to any one of embodiments 219 to 230, wherein the nucleic acid cassette is non-naturally occurring.

[0624] Embodiment 232. The nucleic acid cassette according to any one of embodiments 219 to 231, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0625] Embodiment 233. The nucleic acid cassette according to embodiment 232, wherein the CNS-selective promoter is selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

[0626] Embodiment 234. The nucleic acid cassette according to any one of embodiments 219 to 233, wherein the nucleic acid cassette comprises an enhancer.

[0627] Embodiment 235. The nucleic acid cassette according to any one of embodiments 219 to 234, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0628] Embodiment 236. The nucleic acid cassette of embodiment 235, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with persistent spikes and slow waves during sleep waves (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0629] Embodiment 237. The nucleic acid cassette according to Embodiment 235 or 236, wherein the RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0630] Embodiment 238. The nucleic acid cassette according to any one of Embodiments 219 to 237, wherein:

[0631] the nucleic acid cassette comprises a CNS-selective promoter; and

[0632] the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0633] Embodiment 239. The nucleic acid cassette according to any one of Embodiments 219 to 238, wherein:

[0634] 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; optionally

[0635] wherein the RNA transcript is mRNA, the mRNA encodes a therapeutic protein, and the therapeutic protein is encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GAB RAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

[0636] Embodiment 240. The nucleic acid cassette according to any one of embodiments 219 to 239, wherein the first sequence and the second sequence cause the expression of the RNA transcript and / or the polypeptide encoded thereby (i.e., when the RNA transcript is mRNA) in DRG cells and hepatocytes to be reduced compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in DRG cells and hepatocytes.

[0637] Embodiment 241. The nucleic acid cassette according to embodiment 240, wherein the first sequence and the second sequence cause the expression level of the RNA transcript and / or the polypeptide encoded thereby in DRG cells and hepatocytes to be reduced to at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in DRG cells and hepatocytes.

[0638] Embodiment 242. The nucleic acid cassette according to embodiment 240 or 241, wherein the first sequence and the second sequence cause the expression level of the RNA transcript and / or the polypeptide encoded thereby in DRG cells and hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in DRG cells and hepatocytes.

[0639] Embodiment 243. The nucleic acid cassette according to any one of embodiments 219 to 242, wherein the first sequence and the second sequence do not cause the expression of the RNA transcript and / or the polypeptide encoded thereby in the target cells to be significantly reduced compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in the target cells.

[0640] Embodiment 244. The nucleic acid cassette according to embodiment 243, wherein the first sequence and the second sequence do not cause the expression of the RNA transcript and / or the polypeptide encoded thereby in the target cells to be reduced compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in the target cells.

[0641] Embodiment 245. The nucleic acid cassette according to any one of Embodiments 219 to 244, wherein the expression level of the RNA transcript and / or the polypeptide encoded thereby in the target cell caused by the first sequence and the second sequence is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the RNA transcript and / or the polypeptide in the target cell of an RNA transcript that is otherwise identical but does not have the first sequence and the sequence.

[0642] Embodiment 246. The nucleic acid cassette according to any one of Embodiments 243 to 245, wherein the target cell is a nerve cell.

[0643] Embodiment 247. The nucleic acid cassette according to Embodiment 246, wherein the nerve cell is a brain cell, a neural stem cell, a hippocampal cell or a cerebellar cell.

[0644] Embodiment 248. The nucleic acid cassette according to Embodiment 247, wherein the nerve cell is a GABAergic cell.

[0645] Embodiment 249. The nucleic acid cassette according to Embodiment 248, wherein the GABAergic cell is a cell expressing parvalbumin.

[0646] Embodiment 250. The nucleic acid cassette according to any one of Embodiments 219 to 249, wherein the nucleic acid cassette is a linear construct or a vector.

[0647] Embodiment 251. The nucleic acid cassette according to Embodiment 250, wherein the vector is a plasmid.

[0648] Embodiment 252. The nucleic acid cassette according to Embodiment 250, wherein the vector is a viral vector.

[0649] Embodiment 253. The nucleic acid cassette according to Embodiment 252, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0650] Embodiment 254. The nucleic acid cassette according to Embodiment 253, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV-DJ.

[0651] Embodiment 255. The nucleic acid cassette according to Embodiment 253 or 254, wherein the AAV is scAAV.

[0652] Embodiment 256. The nucleic acid cassette according to Embodiment 252, wherein the viral vector is a lentiviral vector.

[0653] Embodiment 257. An RNA transcript having a sequence encoded by the nucleic acid cassette according to any one of Embodiments 219 to 256.

[0654] Embodiment 258. A method of reducing the dorsal root ganglion (DRG) and liver expression of a therapeutic RNA transcript and / or a protein encoded thereby (i.e., when the RNA transcript is an mRNA) while maintaining the expression of the RNA transcript and / or the protein in a target tissue, the method comprising adding a first sequence and a second sequence to the RNA transcript, wherein the first sequence causes off-target expression in DRG cells and the second sequence causes off-target expression in hepatocytes.

[0655] Embodiment 259. The method according to Embodiment 219 or 220, wherein the first sequence and the second sequence cause:

[0656] The expression level of the RNA transcript and / or the polypeptide encoded thereby in DRG cells is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in DRG cells; and independently,

[0657] The expression level of the RNA transcript and / or the polypeptide encoded thereby in hepatocytes is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript or polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in hepatocytes.

[0658] Embodiment 260. The method according to Embodiment 258 or 258, wherein the RNA transcript is encoded by the nucleic acid cassette according to any one of Embodiments 219 to 256.

[0659] Embodiment 261. A method for expressing a therapeutic protein, the method comprising:

[0660] The nucleic acid cassette of any one of embodiments 219 to 256 is administered to the subject.

[0661] Embodiment 262. The method of embodiment 261, wherein the administration is systemic administration.

[0662] Embodiment 263. The method of embodiment 261, wherein the administration is topical administration.

[0663] Embodiment 264. The method of Embodiment 263, wherein the nucleic acid box is administered locally to the brain or CNS tissue.

[0664] Embodiment 265. The method of embodiment 263 or 264, wherein the administration is carried out by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0665] Embodiment 266. A method according to any one of Embodiments 261 to 265, wherein the subject suffers from a neurological disease or disorder.

[0666] Embodiment 267. The method of embodiment 266, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central-temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep ( CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0667] Embodiment 268. The method according to any one of embodiments 261 to 267, wherein the RNA transcript and / or the protein encoded thereby (i.e., when the RNA transcript is mRNA) is expressed in DRG cells and hepatocytes at a level that is at most 2 / 3, at most 1 / 2, at most 1 / 5, or at most 1 / 10 compared to the expression of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in DRG cells and hepatocytes.

[0668] Embodiment 269. The method according to any one of embodiments 261 to 268, wherein the RNA transcript and / or the protein encoded thereby is expressed in DRG cells and 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 the expression level of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in DRG cells and hepatocytes.

[0669] Embodiment 270. The method according to any one of embodiments 261 to 269, wherein the expression of the RNA transcript and / or the protein encoded thereby in the target cells is not reduced compared to the expression of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in the target cells.

[0670] Embodiment 271. The method according to any one of embodiments 261 to 270, wherein the expression of the RNA transcript and / or the protein encoded thereby in the target cells is not reduced compared to the expression of the RNA transcript and / or the protein of the RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in the target cells.

[0671] Embodiment 272. The method according to any one of embodiments 261 to 271, wherein the RNA transcript and / or the protein encoded thereby (i.e., when the RNA transcript is mRNA) is expressed in the target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the RNA transcript and / or the protein in the target cell of an RNA transcript that is otherwise identical but lacks the first sequence and the second sequence.

[0672] Embodiment 273. The method according to any one of embodiments 270 to 272, wherein the target cell is a nerve cell.

[0673] Embodiment 274. The method according to embodiment 273, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell or a cerebellar cell.

[0674] Embodiment 275. The method according to embodiment 274, wherein the nerve cell is a GABAergic cell.

[0675] Embodiment 276. The method according to embodiment 275, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0676] Example

[0677] The following examples are given to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present invention. It is not intended to limit the scope of what the inventors regard as their invention, nor is it intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, e.g.: bp, base pair; kb, kilobase; pl, picoliter; s, second; min, minute; h, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; i.m., intramuscular; i.p., intraperitoneal; s.c., subcutaneous; etc.

[0678] Example 1

[0679] Screening of DRG off-target elements

[0680] Preparation of a candidate DRG off-target element library:Figure 1A A flow chart showing the design of a DRG off-target regulatory element (RE) library is shown. Candidate dorsal root ganglion (DRG) off-target elements are selected from annotated 3' untranslated regions (3'UTRs) of genes with the following characteristics: (i) low expression in DRG; (ii) high expression in neural tissues (e.g., cortex, hippocampus); and (iii) similar expression patterns between humans and mice. The 3'UTRs are screened as discrete overlapping regions (or "tiles").

[0681] Additional candidate DRG off-target elements designed to serve as miRNA binding sites are selected based on the following criteria: (i) high expression of miRNA in DRG; and (ii) low expression in another tissue (e.g., other neural tissues, including cortex and hippocampus). Seventeen candidate DRG off-target miRNA binding sites are included in the screen.

[0682] An adeno-associated virus (AAV) genomic plasmid library is generated to screen the selected candidate DRG off-target elements. The AAV genomic plasmid used to generate the library includes 5' and 3' ITR regions (SEQ ID NO: 30 and 31, respectively), which flank an expression cassette that includes the following operably linked elements (in 5'-3' order): EF1α promoter, EGFP-KASH transgene, an off-target region with a barcode (described below), WPRE, and a human growth hormone (hGH) polyA signal sequence. The AAV genomic plasmid also contains a restriction enzyme site that is positioned to allow cloning of the candidate DRG off-target sequence and associated barcode into the off-target region.

[0683] Candidate DRG off-target miRNA binding sites are present in the off-target region of the library construct in the following ways: (i) individually; (ii) as 2, 3, or 4 tandem repeats of the same miRNA binding site; and (iii) as a combinatorial tetramer of 4 different miRNA binding sites. In constructs (ii) and (iii), the miRNA binding sites are separated by a 22bp spacer sequence. The library contains five replicate samples of each construct. Each construct (including each replicate of the five replicate constructs) includes a unique barcode sequence for identification in downstream analysis. The library contains approximately 10,000 unique combinatorial tetramers (constructs in (iii) above), each unique combinatorial tetramer being approximately 225bp in length.

[0684] Candidate DRG off-target elements from the 3'UTR (or "tiles") are screened individually, i.e., one candidate DRG off-target tile is present in the off-target region of the library construct. The library contains five replicates of each construct, each replicate including a unique barcode sequence for identification in downstream analysis. For cloning, the sequences of each candidate DRG off-target tile are examined for the presence of restriction enzyme sites necessary for downstream cloning, and those sequences containing such sites are discarded. Each remaining candidate DRG off-target tile is designated as a barcode element and synthesized (Twist Biosciences) with flanking sequences for cloning into the AAV genomic plasmid backbone.

[0685] Representative negative control AAV genomic plasmids were also generated that completely lacked off-target regions or contained random sequences not selected as candidate DRG off-target elements. These negative control plasmids included all other functional regions of the AAV genomic plasmid, including the barcode that allowed their downstream identification as negative controls.

[0686] The candidate DRG off-target library (including both miRNA-based and 3'UTR-based off-target regions) was transformed into electrocompetent E. coli cells, amplified in 200 mL of LB liquid medium, and harvested for plasmid purification (Qiagen Plasmid Plus Maxi kit) to generate an AAV genomic plasmid library preparation. A portion of the transformed E. coli cells was plated on agar plates to form single colonies, harvested, and subjected to Sanger sequencing to verify the fidelity of the cloning process. The negative control AAV genomic plasmids were incorporated into the AAV genomic plasmid library preparation to produce the final AAV genomic plasmid library for AAV vector production.

[0687] AAV library preparation: According to industry standards, AAV9 vectors are produced in adherent HEK293T cells in DMEM + 10% FBS (fetal bovine serum). Briefly, the cells are triple-transfected using PEI-MAX with: (i) the candidate DRG off-target element AAV genomic plasmid library (as described above); (ii) the Rep / Cap plasmid; and (iii) pALD-X80 (adenovirus helper plasmid). AAV is harvested from HEK293T cells, purified from the lysate using an iodixanol ultracentrifugation separation gradient, further refined using an anion exchange column, and subsequently concentrated and formulated in a PBS-based buffer.

[0688] AAV Library Injection: Female C57BL / 6J mice (The Jackson Laboratory) at the time of pregnancy were obtained and housed on-site to acclimate to the new environment until they were grouped. After birth, the pups (n = 6) were injected with the AAV library by intrathecal (IT) injection on postnatal day 1 (P1). Each P1 mouse received 3.74E11 gc / animal, with a single dose of 6 μL. The pups were nursed by their mother, then given a standard chow diet ad libitum and placed on a 12-hour light / 12-hour dark light cycle. The pups were housed as a litter in the breeding room, weaned at day 21, and sacrificed at 4 weeks of age for sample collection and analysis.

[0689] Sample Collection. Tissues from the injected mice were collected directly into cold RNAlater (Sigma Aldrich). The tissues collected included the forebrain, midbrain, hindbrain, spinal cord, and all L1-L6 dorsal root ganglion (DRG) pairs. The samples were stored in RNAlater at 4°C for 24 hours and then transferred to -80°C until further processing.

[0690] RNA and cDNA Generation: Using a Kingfisher (Thermo Fisher Scientific) automated system, mRNA was isolated from the collected tissues using the Dynabeads mRNA "direct" purification kit (Invitrogcn), which uses oligo dT residues bound to the surface of Dynabeads to isolate mRNA. 16 μL or up to 5 μg was used as input for a 40 μL cDNA reaction. cDNA was generated by reverse transcription using the SuperScript IV VILO kit (Thermo Fisher Scientific).

[0691] Amplicon Generation: Using a set of universal primers targeting all elements in the library, amplicons derived from the reporter gene were amplified via PCR. For each brain tissue sample, there were three technical replicates, and for each DRG sample, there were four technical replicates at the start of the first amplicon PCR step. The number of cycles for amplicon PCR was first optimized using qPCR. Step 1 PCR amplified the 3' UTR region of the reporter gene mRNA under optimized conditions. DNA obtained from both the AAV library (the library injected into the mice) and the AAV genomic plasmid library (used to prepare the AAV library) was used as the template for Step 1 PCR as a control in the sequencing run (see below). All technical replicate samples for each biological replicate were manually pooled together in equimolar amounts as the final sequencing library pool.

[0692] Amplicon sequencing: Each sample was combined in equimolar amounts into the final sequencing pool. The sequencing pool also contained amplicons from the AAV genomic plasmid library (for preparing the AAV library) and amplicons from the AAV library itself. The molar concentration of the final library was calculated based on the Bioanalyzer trace of high-sensitivity D1000 Tapestation (Agilent 4200) dsDNA, in combination with the HS dsDNA Qubit fluorometric quantification results. The sample was then combined with PhiX at a molar ratio of 60:40 to diversify the sample pool. The diversified library pool was further diluted and prepared according to the instructions of the Nextseq 2000 P3 200Cycles kit (Illumina)...

Claims

1. A nucleic acid cassette, the nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the following sequences: (i) SEQ ID NO.2 and any one of SEQ ID NO.1, 3-10 or 43-48; (ii) variants, functional fragments or combinations thereof of the sequences; or (iii) sequences that are at least 80% identical to (i) or (ii).

2. The nucleic acid cassette according to claim 1, wherein the RNA transcript comprises a sequence of at least 15 contiguous nucleotides having any one of SEQ ID NO.1-10 and 43-48, the sequence reducing expression in dorsal root ganglion (DRG) cells.

3. The nucleic acid cassette according to claim 1 or 2, wherein the RNA transcript further comprises a second, third, fourth or fifth or more sequences (i), (ii) or (iii).

4. The nucleic acid cassette according to any one of claims 1 to 3, wherein the RNA transcript comprises two or more, three or more, four or more, or five or more copies of the following sequences: (i), (ii) or (iii).

5. The nucleic acid cassette according to any one of claims 1 to 4, wherein the sequence (i), (ii) or (iii) provides a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p and hsa-mir-494-3p.

6. The nucleic acid cassette according to any one of claims 1 to 5, wherein the RNA transcript is mRNA, and the sequence (i), (ii) or (iii) is located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

7. The nucleic acid cassette according to any one of claims 1 to 6, wherein the nucleic acid cassette comprises a CNS-selective promoter.

8. The nucleic acid cassette according to claim 7, 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter.

9. The nucleic acid cassette of any one of claims 1 to 8, wherein the RNA transcript is used to treat a neurological disease or disorder.

10. The nucleic acid cassette of claim 9, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CS WS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

11. The nucleic acid cassette according to claim 9 or 10, wherein the RNA transcript is mRNA, and the mRNA encodes a therapeutic protein selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

12. The nucleic acid cassette according to any one of claims 1 to 11, wherein: (a) the RNA transcript is mRNA, and the mRNA comprises the following sequences: (i) any one of SEQ ID NO.1-10 and 43-48; (ii) a variant, functional fragment or combination thereof of the sequence; or (iii) a sequence having at least 80% identity with (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of: Ca2+ / calmodulin-dependent kinase subunit α (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α- internexin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; and (c) The mRNA encodes a therapeutic protein, which is encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

13. The nucleic acid cassette according to any one of claims 1 to 12, wherein the sequence (i), (ii) or (iii) causes the expression of the RNA transcript and / or the polypeptide encoded by the RNA transcript in DRG cells to be reduced compared to the expression of the RNA transcript and / or the polypeptide of the RNA transcript from other aspects that are equivalent but without the sequence (i), (ii) or (iii).

14. The nucleic acid cassette according to any one of claims 1 to 13, wherein the sequence (i), (ii) or (iii) causes the expression level of the RNA transcript and / or the polypeptide encoded by the RNA transcript in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript and / or the polypeptide of the RNA transcript from other aspects that are equivalent but without the sequence (i), (ii) or (iii).

15. The nucleic acid cassette according to any one of claims 1 to 14, wherein the sequence (i), (ii) or (iii) does not cause the expression of the RNA transcript and / or the polypeptide encoded by the RNA transcript in the target cell to be reduced as compared to the expression of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript that does not have the sequence (i), (ii), or (iii).

16. The nucleic acid cassette according to any one of claims 1 to 15, wherein the sequence (i), (ii) or (iii) causes the expression level of the RNA transcript and / or the polypeptide encoded by the RNA transcript in the target cell to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript that does not have the sequence (i), (ii), or (iii).

17. The nucleic acid cassette according to any one of claims 15 to 16, wherein the target cell is a nerve cell.

18. The nucleic acid cassette according to claim 17, wherein the nerve cell is a brain cell, a neural stem cell, a hippocampal cell or a cerebellar cell.

19. The nucleic acid cassette according to any one of claims 1 to 18, wherein the nucleic acid cassette is a viral vector.

20. The nucleic acid cassette according to claim 19, wherein the viral vector is an adeno-associated virus (AAV) vector.

21. An RNA having a sequence encoded by the nucleic acid cassette according to any one of claims 1 to 20.

22. A method for reducing the dorsal root ganglion (DRG) expression of an RNA transcript and / or a therapeutic protein encoded thereby, the method comprising: comprising the following sequences: (i) any one of SEQ ID NOs: 1-10 and 43-48; (ii) variants, functional fragments or combinations thereof of the sequence; or (iii) a sequence that is at least 80% identical to (i) or (ii) in the RNA transcript.

23. The method according to claim 22, wherein the sequence causes: the expression level of the RNA transcript and / or the polypeptide encoded thereby in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to the expression level of the RNA transcript and / or polypeptide of an otherwise identical RNA transcript that does not have the sequence.

24. The method according to claim 22 or 23, wherein the RNA transcript is encoded by the nucleic acid cassette according to any one of claims 1 to 20.

25. A method for expressing a therapeutic RNA transcript, the method include: The nucleic acid cassette of any one of claims 1 to 20 is administered to a subject.

26. The method of claim 25, wherein the administration is systemic administration.

27. The method of claim 25, wherein the administration is topical administration.

28. The method of claim 27, wherein the nucleic acid cassette is administered locally into the brain or CNS tissue.

29. The method of claim 27 or 28, wherein the administration is performed by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

30. The method of any one of claims 25 to 29, wherein the subject suffers from a neurological disease or disorder.

31. The method of claim 30, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSW S), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

32. The method of any one of claims 25 to 31, wherein the therapeutic RNA transcript is an mRNA, wherein the protein encoded by the mRNA is expressed in DRG cells 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 the expression level of the protein in DRG cells from an otherwise identical mRNA but lacking the sequence.

33. The method according to any one of claims 25 to 32, wherein the expression of the therapeutic RNA transcript and / or the protein encoded thereby in the target cell is not reduced as compared to the expression of the therapeutic RNA transcript and / or the protein in the target cell of an RNA transcript that is otherwise identical but lacks said sequence.

34. The method according to any one of claims 25 to 33, wherein the therapeutic RNA transcript and / or the protein encoded thereby is expressed in the target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the therapeutic RNA transcript and / or the protein in the target cell of a therapeutic RNA transcript that is otherwise identical but lacks said sequence.

35. The method according to any one of claims 33 to 34, wherein the target cell is a nerve cell.

36. The method according to claim 35, wherein the nerve cell is a brain cell, a neural stem cell, a hippocampal cell or a cerebellar cell.

37. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the following sequences: (i) any one of SEQ ID NOs: 65, 110 and 112; (ii) variants, functional fragments or combinations thereof of said sequences; or (iii) a sequence that is at least 80% identical to (i) or (ii), wherein said sequence reduces the expression of the mRNA in hepatocytes.

38. The nucleic acid cassette according to claim 37, wherein the nucleic acid cassette comprises a CNS-selective promoter.

39. The nucleic acid cassette according to claim 38, wherein the CNS-selective promoter is selected from the group consisting of: Ca 2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter.

40. The nucleic acid cassette according to any one of claims 37 to 39, wherein the RNA transcript is for treating a neurological disease or disorder.

41. The nucleic acid cassette of claim 40, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (C The patient was diagnosed with SWS, infantile spasms (SWS), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gastau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

42. The nucleic acid cassette according to claim 40 or 41, wherein the RNA transcript is mRNA, and the mRNA encodes a therapeutic protein selected from: (i) a protein encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

43. The nucleic acid cassette according to any one of claims 37 to 42, wherein: (a) the RNA transcript is mRNA, and the mRNA contains the sequence; (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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter and PaqR4 promoter; and (c) The mRNA encodes a therapeutic protein, which is encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

44. The nucleic acid cassette according to any one of claims 37 to 43, wherein the sequence causes the expression level of the RNA transcript and / or the protein encoded thereby in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the expression level of the RNA transcript or protein of an otherwise identical RNA transcript without the sequence in hepatocytes.

45. The nucleic acid cassette according to any one of claims 37 to 44, wherein the sequence causes the expression level of the RNA transcript and / or the protein encoded thereby in target cells to be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the RNA transcript or protein of an otherwise identical RNA transcript without the sequence in target cells.

46. The nucleic acid cassette according to claims 44 to 45, wherein the target cell is a nerve cell.

47. The nucleic acid cassette according to any one of claims 37 to 46, wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.

48. An mRNA having a sequence encoded by the nucleic acid cassette according to any one of claims 37 to 47.

49. A method for reducing hepatic expression of a therapeutic RNA transcript and / or a protein encoded thereby, the method comprising: comprising the following sequences (i) any one of SEQ ID NO.65, 110 and 112; (ii) variants, functional fragments thereof, or combinations thereof; or (iii) a sequence that is at least 80% identical to (i) or (ii) in the RNA transcript.

50. The method according to claim 49, wherein the method comprises administering to a subject a nucleic acid cassette encoding the RNA transcript.

51. The method according to claim 50, wherein the administration is systemic administration.

52. The method according to claim 50, wherein the administration is local administration.

53. The method according to claim 52, wherein the nucleic acid is locally administered to the brain or CNS tissue.

54. The method according to any one of claims 49 to 53, wherein the RNA transcript is mRNA, and wherein the mRNA encodes a therapeutic protein associated with a neurological disease or disorder.

55. The method according to any one of claims 50 to 54, wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.

56. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, the RNA transcript comprising a first sequence that causes off-target expression in dorsal root ganglion (DRG) cells and a second sequence that causes off-target expression in hepatocytes.

57. The nucleic acid cassette according to claim 56, wherein the first sequence and the second sequence cause a reduction in the expression of the RNA transcript and / or the polypeptide encoded thereby in DRG cells and hepatocytes relative to the target tissue.

58. The nucleic acid cassette according to claim 56 or 57, wherein the first sequence and the second sequence cause: the expression level of the RNA transcript or the polypeptide encoded thereby in DRG cells is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript or the polypeptide of an otherwise identical RNA transcript without the first sequence and the second sequence in DRG cells; and independently, The expression level of the RNA transcript or the polypeptide encoded thereby in hepatocytes is reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the RNA transcript or polypeptide of an RNA transcript that is otherwise identical but lacks the first sequence and the second sequence in hepatocytes.

59. The nucleic acid according to any one of claims 56 to 58, wherein the RNA transcript comprises a combination of sequences selected from Table 1.

60. The nucleic acid cassette according to any one of claims 56 to 59, wherein: (a) The first sequence is: (i) any one of SEQ ID NOs. 1-10 and 43-48; (ii) a variant, functional fragment or combination thereof of the sequence; or (iii) a sequence that is at least 80% identical to (i) or (ii); and (b) The second sequence is: (iv) any one of SEQ ID NOs. 57-62, 64-71, 110 and 112; (v) a variant, functional fragment or combination thereof of the sequence; or (vi) a sequence that is at least 80% identical to (iv) or (v).

61. The nucleic acid cassette according to any one of claims 56 to 60, wherein the first sequence (i), (ii) or (iii) provides a binding site for one or more of hsa-mir-196b-5p, hsa-mir-10b-5p, hsa-mir-24-2-5p, hsa-mir-183-3p, hsa-mir-196a-5p and hsa-mir-494-3p, and the second sequence (iv), (v) or (vi) provides a binding site for hsa-mir-22-3p, has-mir-1258, hsa-mir-5589-3p, hsa-mir-17-5p, hsa-mir-203a-3p, hsa-mir-122-3p, hsa-mir-93-5p and hsa-mir-19a-3p.

62. The nucleic acid cassette according to any one of claims 56 to 61, wherein the RNA transcript comprises at least two, at least three or at least four copies of any one of the sequences (i) to (vi).

63. The nucleic acid cassette according to any one of claims 56 to 62, wherein the RNA transcript is mRNA, and the first sequence and the second sequence are independently located in one or more of the following: the 3'UTR region of the mRNA, the 5'UTR region of the mRNA, or the intron of the mRNA.

64. The nucleic acid cassette according to any one of claims 56 to 63, wherein the nucleic acid cassette comprises a CNS-selective promoter.

65. The nucleic acid cassette of claim 64, wherein the CNS-selective promoter is selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamate decarboxylase (GAD67) promoter, a homeobox D1x5 / 6 promoter, a glutamate receptor 1 (GluR1) promoter, a preprotachykinin 1 (Tac1) promoter, a neuron-specific enolase (NSE) promoter, a dopaminergic receptor 1 (Drd1a) promoter, a MAP1B promoter, a Tα1α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, a NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

66. The nucleic acid cassette of any one of claims 56 to 65, wherein the RNA transcript is used to treat a neurological disease or disorder.

67. The nucleic acid cassette of claim 66, wherein the neurological disease or disorder is Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (C The patient was diagnosed with SWS, infantile spasms (SWS), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gastau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

68. The nucleic acid cassette according to claim 66 or 67, wherein the RNA transcript is mRNA, and wherein the mRNA encodes a therapeutic protein selected from: (i) a protein encoded by a gene selected from the group consisting of: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

69. The nucleic acid cassette according to any one of claims 56 to 68, wherein: 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 glutamate decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1α-tubulin promoter, decarboxylase promoter, dopamine beta-hydroxylase promoter, NCAM promoter, HES-5 promoter, alpha-catenin promoter, peripherin promoter, GAP-43 promoter, and PaqR4 promoter; and The RNA transcript is an mRNA encoding a therapeutic protein, and the therapeutic protein is encoded by a gene selected from the following: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity with (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates the expression of the gene from (i).

70. The nucleic acid cassette according to any one of claims 56 to 69, wherein the expression level of the RNA transcript or the polypeptide encoded thereby in the target cell caused by the first sequence and the second sequence is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression level of the RNA transcript or polypeptide in the target cell of an otherwise identical RNA transcript without the first sequence and the sequence.

71. The nucleic acid cassette according to claim 70, wherein the target cell is a nerve cell.

72. The nucleic acid cassette according to claim 71, wherein the nerve cell is a brain cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

73. The nucleic acid cassette according to any one of claims 56 to 72, wherein the nucleic acid cassette is a viral vector.

74. The nucleic acid cassette according to claim 73, wherein the viral vector is an adeno-associated virus (AAV) vector.

75. An RNA having a sequence encoded by the nucleic acid cassette according to any one of claims 56 to 74.

76. A method of reducing the dorsal root ganglion (DRG) and liver expression of a therapeutic RNA transcript and / or a protein encoded thereby, the method comprising adding a first sequence and a second sequence to the therapeutic RNA transcript, wherein the first sequence causes off-target expression in DRG cells and the second sequence causes off-target expression in hepatocytes.

77. The method of claim 76, wherein the first sequence and the second sequence cause: the expression level of the therapeutic RNA transcript and / or the polypeptide encoded thereby in DRG cells to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the therapeutic RNA transcript or polypeptide of a therapeutic RNA transcript that is otherwise identical but does not have the first sequence and the second sequence in DRG cells; and independently, the expression level of the therapeutic RNA transcript and / or the polypeptide encoded thereby in hepatocytes to be reduced by at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to the expression level of the therapeutic RNA transcript or polypeptide of a therapeutic RNA transcript that is otherwise identical but does not have the first sequence and the second sequence in hepatocytes.

78. The method of claims 76 to 77, wherein the therapeutic RNA transcript is encoded by the nucleic acid cassette of any one of claims 56 to 73.

79. A method for expressing a therapeutic RNA transcript, the method comprising: administering to the subject the nucleic acid cassette of any one of claims 56 to 74.

80. The method of claim 79, wherein the administration is systemic administration.

81. The method of claim 79, wherein the administration is local administration.

82. The method of claim 79, wherein the nucleic acid cassette is locally administered to the brain or CNS tissue.

83. The method of any one of claims 79 to 82, wherein the subject has a neurological disease or disorder.

84. The method of claim 83, wherein the subject suffers from Alpers-Hartenloch syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's disease medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign motor epilepsy), early myoclonic encephalopathy (EME), eyelid myoclonic epilepsy (Jevons syndrome), epilepsy of infancy with migrating focal seizures, myoclonic absence epilepsy, epileptic encephalopathy with sustained spikes and waves during sleep (CSW S), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Klevner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy of infancy, Ohtahara syndrome, Panayotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and nonfamilial neonatal seizures, self-limited late-onset occipital lobe epilepsy, Gasteau syndrome, epilepsy with generalized tonic-clonic seizures only, hereditary epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Dozer syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

85. The method of any one of claims 79 to 84, wherein the therapeutic RNA transcript and / or the protein encoded thereby is expressed in DRG cells and 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 the expression level of the therapeutic RNA transcript or protein in DRG cells and hepatocytes from otherwise identical mRNA but lacking the first sequence and the second sequence.

86. The method of any one of claims 79 to 85, wherein the therapeutic RNA transcript and / or the protein encoded thereby is expressed in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the expression level of the therapeutic RNA transcript or protein in a target cell from an otherwise identical therapeutic RNA transcript but without the first and second sequences.

87. The method according to claim 86, wherein the target cell is a nerve cell.

88. The method according to claim 87, wherein the nerve cell is a brain cell, a neural stem cell, a hippocampal cell or a cerebellar cell.

89. The method according to claim 88, wherein the nerve cell is a GABAergic cell.

90. The method according to claim 89, wherein the GABAergic cell is a cell expressing parvalbumin.

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