Elements for detargeting gene expression in the dorsal root ganglia and / or liver

Abstract

The present disclosure provides sequences that reduce expression of an operably linked transgene in dorsal root ganglion cells and / or liver cells. In some embodiments, the sequences can be used in gene therapy vectors to detarget expression of a therapeutic transgene in the dorsal root ganglion and / or liver cells of a subject.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 410,161, filed September 26, 2022, 63 / 464,772, filed May 8, 2022, 63 / 414,873, filed October 10, 2022, and 63 / 412,119, filed September 30, 2022, which applications are incorporated herein by reference.

[0002] (Incorporation by reference of sequence listing provided as a sequence listing XML file) The Sequence Listing is provided herewith as Sequence Listing XML, ENCO-006WO_SEQ_LIST, created on September 22, 2023, and having a size of 141,924 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

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

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

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

[0006] Provided herein, among other things, are nucleic acid cassettes containing a transgene encoding an RNA transcript, e.g., mRNA, wherein the RNA transcript comprises a sequence of (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii). These sequences reduce transgene expression in cells of the dorsal root ganglion (DRG) relative to other cells or tissues (e.g., other cells in the brain) and can therefore be used in various gene therapy strategies to target cells not present in the DRG.

[0007] Also provided is a nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the sequence of (i) any of SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii), wherein the sequence reduces expression of the RNA transcript in liver cells.

[0008] Also provided is a nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, the therapeutic transgene comprising a first sequence that detargets expression in dorsal root ganglion (DRG) cells and a second sequence that detargets expression in liver cells, wherein addition of the first and second sequences results in decreased expression of the RNA transcript or the polypeptide encoded thereby in DRG and liver cells relative to the target tissue, e.g., GABAergic cells.

[0009] In certain embodiments, the incorporation of one or more of these sequences results in an improved safety profile of the gene therapy by reducing or eliminating toxicity to non-target cells (e.g., DRG and / or liver cells) caused by expression of the transgene in these cells.

[0010] In some embodiments, the nucleic acid cassette is an expression cassette, and the expression cassette may comprise, in operable linkage, a promoter, a coding sequence, one or more detargeting sequences as described above, and a terminator. In some embodiments, at least one sequence present in the expression cassette is heterologous to another of the sequences in the expression cassette. For example, in some embodiments, an expression cassette of the present disclosure comprises a promoter that is heterologous to the operably linked coding sequence. In some embodiments, the expression cassette may further comprise an enhancer and / or an intron.

[0011] As will be apparent, in embodiments in which the RNA transcript encoded by the transgene is an mRNA that encodes a therapeutic protein and includes a detargeting sequence, the mRNA encoded by the transgene includes both the coding sequence for the therapeutic protein and the detargeting sequence, such that the expressed mRNA includes both the coding sequence and the DRG detargeting sequence in the same transcript. Although the mechanism may be unclear, it is understood that the detargeting element may target an RNA transcript, e.g., an mRNA molecule, that includes elements for preferential degradation in non-target cells.

[0012] It should be noted that synthetic RNA molecules comprising the detargeting sequences disclosed herein may also be inactivated in specific tissues (e.g., DRG, liver, or both) when introduced into such cells, e.g., cells of a subject. For example, a synthetic antisense RNA comprising one or more DRG detargeting elements of the present disclosure, one or more liver detargeting elements of the present disclosure, or a combination of both, when administered to a subject, will have reduced activity in the detargeted tissue. No limitation in this regard is intended.

[0013] In some embodiments, the promoter of the expression cassette can be selective for cells in a particular tissue (e.g., a target tissue), but can also drive transgene expression in DRG and / or liver cells. In some embodiments, the promoter is a CNS-selective promoter, e.g., Ca 2+The promoter may be selected from the group consisting of a calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

[0014] In any embodiment, the sequence can be in the 3'UTR, 5'UTR, or intron of the mRNA.

[0015] 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 is selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / 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 expression of a gene from (i).

[0016] In some embodiments, the RNA transcript may comprise a combination of sequences (i), (ii), and (iii).

[0017] Also provided is a vector comprising a cassette as summarized above. The vector can be a plasmid or a viral vector, such as an adeno-associated virus (AAV) or lentiviral vector.

[0018] Also provided are AAV or lentiviral particles or cells containing a cassette as summarized above (which, when packaged, may be in single-stranded form).

[0019] Also provided is an RNA having a sequence characteristic of the RNA encoded by any of the nucleic acid cassettes described herein.

[0020] Various methods are also provided. In some embodiments, the methods can be used to express a protein. In these embodiments, the methods can include introducing an expression cassette or mRNA encoded thereby, as summarized above, into an organism, where the sequence reduces expression of the protein in DRG and / or liver cells of the organism. In further embodiments, where the RNA itself is the active agent (i.e., a non-coding RNA, e.g., microRNA, antisense RNA, etc., as described elsewhere herein), the sequence reduces or eliminates activity of the RNA in DRG and / or liver cells of the organism. In some embodiments, the methods can include administering the expression cassette or mRNA or non-coding RNA to a patient with a neurological disease or disorder. In these embodiments, administering can be systemic or local (e.g., administered locally to brain or CNS tissue, such as by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration methods).In these embodiments, the subject to whom the expression cassette or mRNA or non-coding RNA is administered may be, for example, Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy, encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migratory focal seizures, epileptic myoclonic absence, epileptic encephalopathy of sleep (CSWS), infantile spasms (West syndrome), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures isolated, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy (Douse syndrome), sleep-related hypermotor epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0021] Other features, advantages, and embodiments may become apparent in view of the following description.

[0022] Those skilled in the art will understand that the following drawings are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0023] [Figure 1A]FIG. 1 shows a flow chart of the DRG detargeting regulatory element (RE) library design used in the Examples. [Figure 1B] Figure 1 shows a scatter plot depicting expression levels in the brain versus dorsal root ganglia (DRG) for a library of test constructs. Relative transcript abundance (Log2) for brain (y-axis) and DRG tissue (x-axis) normalized to AAV library abundance and a constitutive control sequence is shown. The relative expression of each sequence element in the library is shown compared to the average of promoters paired with random control sequences. Each data point represents a unique sequence screened. Control constructs with randomized sequences in the detargeting region are indicated by light circles (within solid boxes). The dashed area depicted on the scatter plot indicates the brain versus DRG expression pattern used to select candidate DRG detargeting elements. Examples of candidate DRG detargeting elements are indicated by dark circles. [Figure 2] Graphs of the log2 fold change in EGFP expression in the cortex, DRG, and hippocampus of mice injected with (1) an AAV9 vector in which the EGFP transcript expressed by the vector contains four copies of the binding site for hsa-mir-183-3p (4 × 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 four copies of the binding site for hsa-mir-196b-5p (4 × SEQ ID NO:1, provided herein as SEQ ID NO:43), compared to an AAV9 construct without a detargeting element. Values ​​used for calculations are the average EGFP-KASH transcript levels per μg of total RNA (normalized to VCN / diploid genome). [Figure 3]Representative IHC images showing EGFP-KASH expression in cortex / hippocampus (top row) and lumbar DRG (bottom row) tissues from mice injected with AAV9 vectors in which the EGFP transcripts expressed by the vectors (1) have no detargeting element in the detargeting region (no detargeting), (2) have tetrameric miR-183-3p binding sites in the detargeting region (4 × SEQ ID NO: 4), or (3) have tetrameric miR-196b-5p binding sites in the detargeting region (4 × SEQ ID NO: 1). (Image scale is provided in the leftmost image.) [Figure 4] For each treatment arm (n=5; dots on the graph represent individual animals), the mean and standard deviation (SD) of the percent EGFP-positive nuclei from two cortical regions (top graph) and four DRG regions (bottom panel) of brain tissue are shown. An unmanipulated negative control (UNM) was included in each experiment (no AAV9 vector injection). Treatment arms contained AAV9 vectors in which the EGFP transcripts expressed by the vector either had no detargeting element in the detargeted region (no detargeting) or had the indicated detargeting element in the detargeted region (provided as SEQ ID NOs). [Figure 5] Examples of representative IHC images are provided showing EGFP-KASH expression in the brain and DRG from four mice from Figure 4: a control mouse (no detargeting element) injected with an AAV9 vector in which the EGFP transcript expressed by the vector does not have a detargeting element in the detargeted region, and three mice each injected with a different AAV9 vector in which the EGFP transcript expressed by the vector has the indicated detargeting element in the detargeted region (provided as sequence numbers). [Figure 6A]After intravenous administration to mice at P1, AAV9 carrying a pan-neuronal promoter and a control UTR (control) or a candidate DRG detargeting UTR (SEQ ID NO: 46, containing four copies of the hsa-mir-10b-5p binding site, and SEQ ID NO: 48, containing two copies of the hsa-mir-196b-5p binding site and two copies of the hsa-mir-10b-5p binding site) was administered intravenously. Figure 6A shows GFP%+ nuclei in the DRG (left panel) and brain (right panel). Figure 6B shows representative images of mouse samples stained for AAV transgene expression from a control vector lacking a detargeting element in the detargeting region and a vector in which the detargeting region of the transcript contains four copies of hsa-mir-10b-5p (SEQ ID NO: 46; the same mouse is shown in Figure 6A). A spike-in AAV vector carrying a myc-tagged mCherry gene under the control of a pan-neuronal promoter was added to the controls to assess biodistribution. These experiments demonstrate that hsa-mir-10b-5p binding sites in the detargeted region of an RNA transcript (in this case, the 3'UTR) substantially reduce off-target expression in the DRG while maintaining CNS expression. Individual validation of lead DRG detargeting elements in mice showed a 5- to 16-fold reduction in DRG expression, without a reduction in brain expression. [Figure 6B]After intravenous administration to mice at P1, AAV9 carrying a pan-neuronal promoter and a control UTR (control) or a candidate DRG detargeting UTR (SEQ ID NO: 46, containing four copies of the hsa-mir-10b-5p binding site, and SEQ ID NO: 48, containing two copies of the hsa-mir-196b-5p binding site and two copies of the hsa-mir-10b-5p binding site) was administered intravenously. Figure 6A shows GFP%+ nuclei in the DRG (left panel) and brain (right panel). Figure 6B shows representative images of mouse samples stained for AAV transgene expression from a control vector lacking a detargeting element in the detargeting region and a vector in which the detargeting region of the transcript contains four copies of hsa-mir-10b-5p (SEQ ID NO: 46; the same mouse is shown in Figure 6A). A spike-in AAV vector carrying a myc-tagged mCherry gene under the control of a pan-neuronal promoter was added to the controls to assess biodistribution. These experiments demonstrate that hsa-mir-10b-5p binding sites in the detargeted region of an RNA transcript (in this case, the 3'UTR) substantially reduce off-target expression in the DRG while maintaining CNS expression. Individual validation of lead DRG detargeting elements in mice showed a 5- to 16-fold reduction in DRG expression, without a reduction in brain expression. [Figure 7A] The DRG detargeting element in the AAV9 transcript rescued protein overexpression in DRG in mice (Figure 7A shows IHC, and Figure 7B shows intensity values) without affecting brain expression in mice (Figure 7C shows IHC, and Figure 7D shows intensity values). The 4xhsa-mir-10b-5p DRG detargeting element (SEQ ID NO: 46) was evaluated in an AAV9 vector carrying a neuronal transgene under the control of a pan-neuronal promoter. IHC analysis after ICV administration in mice at P1 showed that the DRG detargeting element rescued protein overexpression in DRG (i.e., reduced it to the level seen in control vehicle injections) without altering expression in the brain compartment. [Figure 7B]The DRG detargeting element in the AAV9 transcript rescued protein overexpression in DRG in mice (Figure 7A shows IHC, and Figure 7B shows intensity values) without affecting brain expression in mice (Figure 7C shows IHC, and Figure 7D shows intensity values). The 4xhsa-mir-10b-5p DRG detargeting element (SEQ ID NO: 46) was evaluated in an AAV9 vector carrying a neuronal transgene under the control of a pan-neuronal promoter. IHC analysis after ICV administration in mice at P1 showed that the DRG detargeting element rescued protein overexpression in DRG (i.e., reduced it to the level seen in control vehicle injections) without altering expression in the brain compartment. [Figure 7C] The DRG detargeting element in the AAV9 transcript rescued protein overexpression in DRG in mice (Figure 7A shows IHC, and Figure 7B shows intensity values) without affecting brain expression in mice (Figure 7C shows IHC, and Figure 7D shows intensity values). The 4xhsa-mir-10b-5p DRG detargeting element (SEQ ID NO: 46) was evaluated in an AAV9 vector carrying a neuronal transgene under the control of a pan-neuronal promoter. IHC analysis after ICV administration in mice at P1 showed that the DRG detargeting element rescued protein overexpression in DRG (i.e., reduced it to the level seen in control vehicle injections) without altering expression in the brain compartment. [Figure 7D]The DRG detargeting element in the AAV9 transcript rescued protein overexpression in DRG in mice (Figure 7A shows IHC, and Figure 7B shows intensity values) without affecting brain expression in mice (Figure 7C shows IHC, and Figure 7D shows intensity values). The 4xhsa-mir-10b-5p DRG detargeting element (SEQ ID NO: 46) was evaluated in an AAV9 vector carrying a neuronal transgene under the control of a pan-neuronal promoter. IHC analysis after ICV administration in mice at P1 showed that the DRG detargeting element rescued protein overexpression in DRG (i.e., reduced it to the level seen in control vehicle injections) without altering expression in the brain compartment. [Figure 8A]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 8B]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 8C]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 8D]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 8E]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 8F]Rescue of protein overexpression in NHP DRG tissue by detargeting elements. Figure 8A shows the experimental design of an NHP study to evaluate the DRG detargeting element (SEQ ID NO: 46) in NHPs. NHPs were intravenously injected with vehicle (Group 1), an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene (Group 3), or an AAV with a pan-neuronal promoter driving the expression of an mRNA transcript encoding a neuronal gene further comprising SEQ ID NO: 46 (DRG detargeting sequence) in the 3' UTR (Group 5). Figure 8B shows molecular analysis of AAV transcript expression between Group 3 (promoter, indicated as "P" in the figure) and Group 5 (P + SEQ ID NO: 46). AAV-driven transcript expression in the brain and DRG compartments was assessed by RT-ddPCR analysis using vector-specific primers / probes. Total RNA expression was normalized to the AAV genome copy number in each compartment and expressed as a fold change relative to the promoter-only condition. Although no differences in AAV-driven transgene expression were detected in the forebrain and midbrain, a >10-fold decrease in AAV-mediated transcript expression was observed across the DRG compartments (cervical, thoracic, lumbar, and sacral) analyzed. Figure 8C shows vector biodistribution in the DRG compartments of the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord. Expression in the DRG was primarily seen in sacral DRG tissue. Transgene-driven protein expression (Figure 8D) and endogenously driven protein expression (Figure 8E) by MSD-EEISA (using reagents specific for either the transgene-encoded protein or the endogenous protein) are shown in the cervical (C), thoracic (T), lumbar (L), and sacral (S) spinal cord DRG compartments. This figure shows that the presence of the DRG detargeting element SEQ ID NO:46 (containing four copies of the hsa-mir-10b-5p binding site) in the mRNA transcript reduces expression of the transgene-encoded protein but does not significantly affect expression of the endogenous gene. IHC staining for both the transgene-encoded and endogenous proteins in the sacral (S) DRG compartment is shown in Figure 8F.This figure shows that without the DRG detargeting element in the transcript, protein expression was significantly higher in sacral DRG tissue (middle panel) compared to endogenous levels (left panel). Inclusion of a binding site for the DRG detargeting element, hsa-mir-10b-5p, reduced the level of protein expression to endogenous levels (right panel). [Figure 9] Scatter plot showing the log2 change in brain vs. liver activity for a screen of liver detargeting elements. Constructs selected as liver detargeting elements are shown in dark boxes. Control sequences (no liver detargeting) are shown in light boxes. [Figure 10] 1 is a graph showing the results of an ELISA assay comparing a construct whose expressed transcript does not have a detargeting element (no element control) with a construct whose expressed transcript contains the indicated liver detargeting element. SEQ ID NO: 113 is the positive control sequence. [Figure 11A]Representative images showing the in vivo expression of various constructs in the brain and liver using a CNS-specific promoter are shown. Figure 11A shows representative images of brain and liver expression of myc-tagged transgenes from mice (i) treated with a control vector expressing a transcript (encoding a myc-tagged protein) without a detargeting element (upper panel; no detargeting) and mice (ii) treated with a vector expressing a transcript (encoding a myc-tagged protein) containing SEQ ID NO: 66 in its detargeting region (lower panel). The AAV vector in Figure 11A was delivered via tail vein injection. Figure 11B shows (i) mice injected into the ICM with a control vector expressing a transcript (encoding a myc-tagged protein) under the control of a pan-neuronal promoter and lacking the detargeting element (top panel; no detargeting), and (ii) mice treated with a vector expressing a transcript (encoding a myc-tagged protein) under the control of a pan-neuronal promoter and containing SEQ ID NO: 110 in its detargeting region (middle panel; SEQ ID NO: 110 contains two copies of the hsa-mir-19a-3p binding site (SEQ ID NO: 65), hsa-mir- Representative images of brain and liver expression of myc-tagged transgenes from mice treated with a vector expressing (iii) a transcript (encoding a myc-tagged protein) containing SEQ ID NO: 112 in its detargeted region under the control of a pan-neuronal promoter (lower panel; SEQ ID NO: 112 contains three copies of the hsa-mir-122-3p binding site (SEQ ID NO: 62)), and (iv) two copies of the 1258-5p binding site (SEQ ID NO: 58) and two copies of the hsa-mir-17-5p binding site (SEQ ID NO: 60)). [Figure 11B]Representative images showing the in vivo expression of various constructs in the brain and liver using a CNS-specific promoter are shown. Figure 11A shows representative images of brain and liver expression of myc-tagged transgenes from mice (i) treated with a control vector expressing a transcript (encoding a myc-tagged protein) without a detargeting element (upper panel; no detargeting) and mice (ii) treated with a vector expressing a transcript (encoding a myc-tagged protein) containing SEQ ID NO: 66 in its detargeting region (lower panel). The AAV vector in Figure 11A was delivered via tail vein injection. Figure 11B shows (i) mice injected into the ICM with a control vector expressing a transcript (encoding a myc-tagged protein) under the control of a pan-neuronal promoter and lacking the detargeting element (top panel; no detargeting), and (ii) mice treated with a vector expressing a transcript (encoding a myc-tagged protein) under the control of a pan-neuronal promoter and containing SEQ ID NO: 110 in its detargeting region (middle panel; SEQ ID NO: 110 contains two copies of the hsa-mir-19a-3p binding site (SEQ ID NO: 65), hsa-mir- Representative images of brain and liver expression of myc-tagged transgenes from mice treated with a vector expressing (iii) a transcript (encoding a myc-tagged protein) containing SEQ ID NO: 112 in its detargeted region under the control of a pan-neuronal promoter (lower panel; SEQ ID NO: 112 contains three copies of the hsa-mir-122-3p binding site (SEQ ID NO: 62)), and (iv) two copies of the 1258-5p binding site (SEQ ID NO: 58) and two copies of the hsa-mir-17-5p binding site (SEQ ID NO: 60)). [Figure 12A]Figure 12A shows the fold change in myc-positive cells present in brain tissue (Figure 12A) and liver tissue (Figure 12B) from the mouse in Figure 11B and from mice injected ICM with an AAV vector expressing myc-tagged proteins without detargeting elements under the control of the ubiquitous chicken beta-actin promoter ("CBA"). The liver detargeting 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 beta-actin promoter ("CBA") (*** and **** indicate significant differences in expression). [Figure 12B] Figure 12A shows the fold change in myc-positive cells present in brain tissue (Figure 12A) and liver tissue (Figure 12B) from the mouse in Figure 11B and from mice injected ICM with an AAV vector expressing myc-tagged proteins without detargeting elements under the control of the ubiquitous chicken beta-actin promoter ("CBA"). The liver detargeting 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 beta-actin promoter ("CBA") (*** and **** indicate significant differences in expression). [Figure 13] 1 is a graph showing the relative expression (log2 fold change) in the liver for several different liver detargeting elements in NHPs. Data and averages for two different NHP animals (animal 1 and animal 2) are shown. SEQ ID NO: 55 is a random control. [Figure 14]

[0023] Figure 1 shows a schematic diagram of the design and construction of a combinatorial AAV library using selected DRG and liver detargeting elements of the present disclosure. The AAV vector structure is shown at the top and includes a promoter operably linked to a transgene containing a 3'UTR with a detargeting region, with three positions (positions 1, 2, and 3) into which a detargeting element or control / benchmark element may be inserted. All permutations of elements were present in the AAV library. The sequence numbers of the selected DRG, liver, and control / benchmark elements are shown in the table in both DNA and RNA form (DNA is the sequence in the AAV vector, and RNA is the sequence in the mRNA transcript expressed from the vector). [Figure 15] Figure 1 shows scatter plots of the expression patterns of individual vectors in an AAV library in mouse tissues. The top panel compares log2 brain expression activity (y-axis) versus log2 DRG expression activity (x-axis), and the bottom panel compares log2 brain expression activity (y-axis) versus log2 liver expression activity (x-axis). In the top panel, highlighted vector dots contain one or more of the top DRG detargeting elements. In the bottom panel, highlighted vector dots contain one or more of the top liver detargeting elements. In both panels, dark vector dots contain only the neutral control sequence. This data demonstrates that DRG and liver detargeting elements are highly effective at reducing transgene expression in their respective tissues while maintaining expression in brain tissue. [Figure 16] This figure shows the effect of individual elements in an AAV combinatorial library on transgene expression in DRG, liver, and forebrain tissues in mice injected with the combinatorial library. These values ​​were determined by recovering transcribed libraries from DRG, liver, and brain tissue compartments using RNA / AAV DNA amplicon sequencing and NGS quantification for differential expression. A regression-based model of tissue expression revealed the top contributing elements across thousands of data points / instances. Data are presented as log2 fold change in the specified tissue with and without the detargeting element, identified by SEQ ID NO: on the X-axis. Each SEQ ID NO: represents an RNA sequence present in the transcript in the detargeted region of the expressed transcript, except for SEQ ID NO: 55, which is the DNA sequence of a random control element in the AAV vector library. SEQ ID NO: 119 is the DRG detargeting benchmark, and SEQ ID NO: 113 is the liver detargeting benchmark. Modeling results identified the top 3'UTR sequence elements associated with selective DRG and / or liver detargeting while maintaining expression in the brain. [Figure 17]The combined detargeting sequence elements show a range of detargeting in both the liver and DRG (scatter plot, upper panel). Candidate combinatorial detargeting regions that show strong detargeting in both the DRG and liver were identified (circles highlighted in dark colors in the scatter plot). These candidate combinatorial detargeting regions contain at least one DRG and at least one liver detargeting element in the detargeting region of the 3'UTR. The selected combinatorial detargeting elements were shown to have no negative effect on transgene expression in brain tissue (lower graph). The graph shows the log2 fold change in the designated tissues with and without the combinatorial detargeting elements identified by three SEQ ID NOs on the x-axis. The three SEQ ID NOs represent RNA sequences present in the transcripts in the detargeting region of the expressed transcript, except for SEQ ID NOs: 55 and 56 in the last column, which are the DNA sequences of random control elements in the AAV vector library. [Figure 18] Combinatorial analysis for multiplex validation of DRG detargeting sequences in NHPs. NHPs (n=2) were administered the combinatorial AAV library designed as shown in Figure 14 by intracerebroventricular (ICV) injection. DRG detargeting coefficients were determined from each tissue: DRG (sacral compartment) and brain (hippocampal compartment) from highly transduced samples as described in Figure 14. Figure 18 shows a regression-based model of differential expression to explain the detargeting contribution of each element across all points / instances in the recovered library. SEQ ID NOs. on the x-axis represent RNA sequences present in transcripts in the detargeted region of the expressed transcript, except for SEQ ID NOs. 55 and 56 in the last two columns, which are DNA sequences of random control elements in the AAV vector library. [Figure 19] 19 shows the correlation between the effect of elements in the combinatorial AAV library on expression in brain, DRG, and liver tissues between mice and NHPs. As shown in Figure 19, the Pearson correlation between the mouse expression coefficient and the NHP expression coefficient for each of the detargeted elements was 0.86 for brain expression, 0.91 for liver expression, and 0.79 for DRG expression.

[0024] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0025] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all serotypes, subtypes, and both naturally occurring and recombinant forms, except where 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 hybrids thereof (i.e., chimeric AAV vectors), as well as avian, bovine, canine, equine, primate, non-primate, 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 in public databases such as GenBank. As used herein, "rAAV vector" refers to an AAV vector containing a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeat (ITR) sequences. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV). See, e.g., Raj et al., Expert Rev Hematol. 2011 Oct;4(5):539-549. "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector.When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as a "rAAV viral particle" or simply "rAAV particle." AAV can contain genome components and capsids from multiple serotypes (e.g., pseudotyped vectors). For example, an AAV can contain a serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotyped vectors can demonstrate improved transduction efficiency as well as modified tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred. In some embodiments, the recombinant AAV vector is AAV1, AAV8, AAV9, AAVDJ, or a chimeric AAV containing features of two or more of these serotypes. In various embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector. In certain embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector and comprises heterologous nucleic acid flanked by ITRs from an AAV serotype other than AAV 9. In certain embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector and comprises heterologous nucleic acid flanked by ITRs of AAV serotype 2 (i.e., ITR2).

[0026] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or 2 or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0027] In any of the embodiments described herein, "comprising" can be interchanged with "consisting essentially of" or "consisting of." For example, embodiments in which particular elements are included using the open-ended term "comprising" encompass embodiments in which the elements are included using the more restrictive term "consisting essentially of" or "consisting of."

[0028] The terms "determine," "measure," "assess," "estimate," "assay," "analyze," and their grammatical equivalents are used interchangeably herein to refer to any form of measurement, including determining whether an element is present (e.g., detecting). These terms can include both quantitative and / or qualitative determinations. Estimation can be relative or absolute.

[0029] The term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or non-coding RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide contains introns or splice sites, e.g., is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0030] "Expression cassette" refers to a nucleic acid molecule containing one or more regulatory elements operably linked to a sequence to be expressed as an RNA transcript, including mRNA (i.e., an RNA molecule that contains a coding sequence (e.g., one or more genes) for expression of a protein) or non-coding RNA.

[0031] A "transgene" refers to a portion of a nucleic acid cassette designed to be expressed in a cell. In some embodiments, a transgene encodes an RNA transcript, e.g., an mRNA, or a non-coding RNA, e.g., an antisense RNA. In some embodiments, a transgene of the present disclosure encodes a therapeutic cargo, e.g., a therapeutic protein or therapeutic RNA, and also includes one or more DRG and / or liver detargeting sequences / elements to reduce expression of the transgene in DRG and / or liver cells.

[0032] "RNA transcript" refers to an RNA molecule transcribed from a template, such as an RNA molecule transcribed from an expression cassette described herein. The RNA transcript expressed from the expression cassette described herein can be in any desired form, including mRNA that encodes a polypeptide / protein, or non-coding RNA (ncRNA), also known as RNA that exerts a desired function without being used as a template for protein expression. Examples of ncRNAs include, but are not limited to, microRNAs (microRNAs, miRNAs, or miRs), primary microRNAs (pri-miRNAs or pri-miRs), pre-microRNAs (pre-miRNAs, pre-miRNAs, or pre-miRs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), antisense RNAs (asRNAs), transfer RNAs (tRNAs), long non-coding RNAs (lncRNAs), short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), ribozymes, and CRISPR guide RNAs (gRNAs).

[0033] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition described herein that is sufficient to affect the intended application, including but not limited to, disease treatment, as defined below. A therapeutically effective amount may vary depending on the intended therapeutic application (intracellular or in vivo), or the subject and disease state being treated, such as the subject's weight and age, the severity of the disease state, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a specific response in target cells. The specific dose will vary depending on the particular composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which it is delivered.

[0034] A "fragment" of a nucleotide or peptide sequence is meant to refer to a sequence that is shorter than what is considered to be the "full length" sequence.

[0035] A "functional fragment" of a DNA, RNA, or protein sequence refers to a biologically active fragment of a sequence that is shorter than a full-length or reference DNA, RNA, or protein sequence, but that retains at least one biological activity (either functional or structural) that is substantially similar to the biological activity of the full-length or reference DNA, RNA, or protein sequence. For example, a "functional fragment" can be a fragment of a sequence disclosed herein that reduces expression of a transgene to which it is operably linked in DRG and / or liver cells.

[0036] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0037] As used herein, the term "human-derived" refers to a sequence found in the human genome (or human genome build), or a sequence homologous thereto. A homologous sequence can be a sequence having a region with at least 80% sequence identity (e.g., as measured by BLAST) compared to a region of the human genome. For example, a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a human sequence is considered to be of human origin. In some cases, a regulatory element contains human-derived and non-human-derived sequences, such that the entire 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.

[0038] The term "in vitro" refers to an event that occurs outside a subject's body. For example, an in vitro assay includes any assay that is performed outside of a subject. In vitro assays include cell-based assays in which live or dead cells are used. In vitro assays also include cell-free assays in which no intact cells are used.

[0039] The term "in vivo" refers to events that take place inside a subject's body.

[0040] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally, at a chromosomal location that is different from its natural chromosomal location, or contains only the coding sequence.

[0041] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents refer to the juxtaposition of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., which are in a relationship permitting them to operate in the expected manner. For example, a regulatory element, which may include a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

[0042] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation or composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0043] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered.

[0044] 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). Regulatory elements include, but are not limited to, promoters, enhancers, repressors, silencers, insulator sequences, introns, UTRs, inverted terminal repeat (ITR) sequences, long terminal repeats (LTRs), stability elements, miRNA target sites, post-translational response elements, or polyA sequences, or combinations thereof. Regulatory elements can function at the DNA and / or RNA level, for example, by regulating gene expression at the transcriptional, late transcriptional, or translational phases of gene expression; by regulating the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination; by recruiting transcription factors to the coding region that increase gene expression; by increasing the rate at which RNA transcripts are produced, increasing the stability of the RNA produced, and / or increasing the rate of protein synthesis from RNA transcripts; and / or by preventing RNA degradation and / or increasing its stability to promote protein synthesis. In exemplary embodiments, regulatory elements refer to enhancers, repressors, promoters, or combinations thereof, particularly enhancer-promoter combinations or repressor-promoter combinations. In exemplary embodiments, the regulatory elements are derived from human sequences.

[0045] Generally, "sequence identity" or "sequence homology," which may be used interchangeably, refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity," also referred to as "percent homology." The percent identity to a reference sequence (e.g., a nucleic acid or amino acid sequence) can be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence multiplied by 100. Conservative substitutions are not considered matches when determining the number of matches for sequence identity. It will be understood that if the length of a first sequence (A) is not equal to the length of a second sequence (B), the percent identity of an A:B sequence will be different from the percent identity of a B:A sequence. Sequence alignment, such as for purposes of estimating percent identity, can be performed by any suitable alignment algorithm or program, including, but not limited to, the Needleman-Wunsch algorithm, the BLAST algorithm, the Smith-Waterman algorithm (see, e.g., EMBOSS Water aligner), and the Clustal Omega alignment program (F. Sievers et al., Mol Sys Biol. 7:539 (2011)). Optimal alignment can be estimated using any suitable parameters of the selected algorithm, including the default parameters. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).

[0046] The terms "subject" and "individual" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. The methods described herein may be useful in human therapy, veterinary applications, and / or preclinical research in animal models of a disease or condition.

[0047] As used herein, the terms "treat," "treatment," "therapy," and the like refer to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, palliating, delaying, or slowing the progression, reducing the impact or symptoms, preventing the onset, preventing recurrence, inhibiting the onset of a disease or disorder, ameliorating, or obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, e.g., a therapeutic benefit and / or a prophylactic benefit. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to or at risk of developing the disease, but who has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., halting its development; and (c) relieving the disease, i.e., causing regression of the disease. A therapeutic benefit includes eradicating or ameliorating the underlying disease being treated. Therapeutic benefit is also achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disease, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disease. In some cases, for prophylactic benefit, the composition is administered to a subject at risk of developing a particular disease or reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed. The methods of the present disclosure can be used in any mammal. In some cases, treatment can result in a reduction or cessation of symptoms. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0048] A "variant" of a nucleotide sequence refers to a sequence that has genetic modifications or mutations compared to the most common wild-type DNA sequence (e.g., a cDNA or sequence referenced by its GenBank accession number) or a designated reference sequence. A variant may be shorter than the reference sequence and / or may have one or more mutations relative to the reference sequence. In some cases, a variant may have a nucleotide sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to the reference sequence.

[0049] As used herein, a "vector" refers to a nucleic acid molecule that can be used to mediate the delivery of another nucleic acid molecule to which it has been linked into a cell where it can be replicated or expressed. The term includes vectors as self-replicating nucleic acid structures as well as vectors that have integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Other examples of vectors include plasmids and viral vectors.

[0050] As used herein, a "target cell" generally refers to a cell in which expression of the RNA or protein product of a nucleic acid cassette is desired. A non-target cell is a cell in which expression of the RNA or protein product of a nucleic acid is undesirable. As used herein, "detargeting" generally refers to reducing expression in non-target cells.

[0051] Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art, and the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and recombinant DNA technology that are within the knowledge of those skilled in the art. DETAILED DESCRIPTION OF THE INVENTION

[0052] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0053] The upper and lower limits of a range may independently be included in the range and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0054] 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 described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0055] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" includes a plurality of such proteins, a reference to a "nucleic acid" includes a reference to one or more nucleic acids and equivalents thereof known to those of skill in the art, and so forth. It is further noted that the claims may be drafted to exclude optional elements. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0056] It is recognized that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments according to the present invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination were individually and explicitly disclosed herein.

[0057] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0058] Aspects of the present disclosure provide nucleic acid molecules comprising one or more DRG detargeting elements, one or more liver detargeting elements, or a combination of both. In certain embodiments, the nucleic acid molecule is an RNA molecule further comprising a heterologous RNA sequence, e.g., a protein-coding RNA sequence or noncoding RNA (ncRNA). In some of these embodiments, the heterologous RNA is, for example, a therapeutic RNA encoding a therapeutic protein or ncRNA with a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule further comprising a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript comprising one or more detargeting elements and a heterologous RNA sequence. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript comprising one or more detargeting elements and a heterologous RNA sequence. As described in detail herein and summarized above, the presence of one or more DRG detargeting elements in an RNA molecule comprising 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 comprising a heterologous RNA sequence without one or more DRG detargeting elements. Similarly, the presence of one or more liver detargeting elements in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in liver cells, e.g., liver cells of a subject, compared to an RNA molecule comprising the heterologous RNA sequence without one or more DRG detargeting elements. Furthermore, the presence of one or more DRG detargeting elements and one or more liver detargeting elements in an RNA molecule having a heterologous RNA sequence reduces the activity of the heterologous RNA sequence in DRG cells and liver cells, e.g., DRG cells and liver cells of a subject, compared to an RNA molecule comprising the heterologous RNA sequence without one or more DRG detargeting elements and liver detargeting elements.

[0059] Aspects of the present disclosure provide nucleic acid molecules comprising one or more regions that hybridize under physiological conditions (e.g., in a subject's cells) with 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 a detargeting element of the present disclosure). These hybridizing 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., a protein-coding RNA sequence or a non-coding RNA (ncRNA). In some of these embodiments, the heterologous RNA is, for example, a therapeutic RNA encoding a therapeutic protein or ncRNA with 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 containing one or more binding sites. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript comprising one or more detargeting elements and a heterologous RNA sequence. As described in detail herein and summarized above, the presence of one or more regions in an RNA molecule comprising a heterologous RNA sequence that hybridize under physiological conditions with 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 reduces the activity of the heterologous RNA sequence in DRG cells, e.g., DRG cells of a subject, compared to an RNA molecule comprising a heterologous RNA sequence that does not have one or more hybridization regions. Thus, aspects of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize under physiological conditions with hsa-mir-196b-5p (SEQ ID NO: 21). Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-10b-5p (SEQ ID NO: 22) under physiological conditions.Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-24-2-5p (SEQ ID NO: 23) under physiological conditions. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-183-3p (SEQ ID NO: 24) under physiological conditions. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-196a-5p (SEQ ID NO: 25) under physiological conditions. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-494-3p (SEQ ID NO: 26) under physiological conditions.

[0060] In certain aspects, the present disclosure provides nucleic acid molecules comprising 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 a detargeting element of the present disclosure). In certain embodiments, the nucleic acid molecule is an RNA molecule further comprising a heterologous RNA sequence, e.g., a protein-coding RNA sequence or a non-coding RNA (ncRNA). In some of these embodiments, the heterologous RNA is, for example, a therapeutic RNA encoding a therapeutic protein or ncRNA with a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule further comprising a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript comprising one or more binding sites. In some embodiments, the DNA molecule is a nucleic acid cassette designed to express an RNA transcript comprising one or more detargeting elements and a heterologous RNA sequence. As described in detail herein and summarized 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 comprising the heterologous RNA sequence lacking one or more binding sites. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more binding sites for hsa-mir-196b-5p. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more binding sites for hsa-mir-10b-5p. Accordingly, embodiments of the present disclosure include nucleic acid molecules comprising one or more binding sites for hsa-mir-24-2-5p. Accordingly, embodiments of the present disclosure include nucleic acid molecules that contain one or more binding sites for hsa-mir-183-3p. Accordingly, embodiments of the present disclosure include nucleic acid molecules that contain one or more binding sites for hsa-mir-196a-5p.Accordingly, aspects of the present disclosure include nucleic acid molecules that contain one or more binding sites for hsa-mir-494-3p.

[0061] Aspects of the present disclosure provide nucleic acid molecules comprising one or more regions that hybridize to hsa-mir-19a-3p (SEQ ID NO: 95) (which is the detargeting element of the present disclosure) under physiological conditions. Aspects of the present disclosure provide nucleic acid molecules comprising one or more binding sites for hsa-mir-19a-3p. In certain embodiments, the nucleic acid molecule is an RNA molecule further comprising a heterologous RNA sequence, e.g., a protein-coding RNA sequence or a non-coding RNA (ncRNA). In some of these embodiments, the heterologous RNA is, for example, a therapeutic RNA encoding a therapeutic protein or ncRNA with a desired therapeutic function. In some embodiments, the nucleic acid molecule is a DNA molecule further comprising a heterologous DNA sequence, e.g., a DNA sequence that can be used as a template to generate an RNA transcript comprising 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 comprising one or more hsa-mir-19a-3p binding sites and a heterologous RNA sequence. As described in detail herein and summarized 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 liver cells, e.g., liver cells of a subject, compared to an RNA molecule comprising the heterologous RNA sequence that does not have one or more hsa-mir-19a-3p binding sites.

[0062] In some embodiments, nucleic acid molecules of the present disclosure comprise one or more of the above hybridizing regions / miRNA binding sites, in any combination, with one or more additional hybridizing regions / miRNA binding sites that detarget, e.g., to a cell or tissue of interest, e.g., as described herein. In some embodiments, nucleic acid molecules of the present disclosure comprise one or more of the above hybridizing regions and / or miRNA binding sites, in any combination, with one or more additional detargeting elements to DRG and / or liver, e.g., as described herein. No limitation in this regard is intended.

[0063] As summarized above, the present disclosure describes a nucleic acid cassette comprising a transgene encoding an RNA, wherein the RNA comprises (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. The transgene may encode an mRNA or a non-coding RNA, such as a pri-miRNA, pre-miRNA, or miRNA, an antisense RNA, a short non-coding RNA, a long non-coding RNA, a snoRNA, snRNA, tRNA, or rRNA. In some cases, the nucleic acid cassette is a transgene encoding an mRNA, wherein the mRNA comprises (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a functional fragment thereof, or (iii) a sequence at least 80% identical to (i) or (ii), or any combination thereof. These sequences reduce transgene expression in dorsal root ganglion cells (DRG) compared to target cells (neuronal cells, e.g., neurons), and can therefore be used in various gene therapy strategies that target cells not present in DRG. Reducing transgene expression in DRG cells compared to target cells means that the reduction in transgene expression driven by the DRG detargeting sequences disclosed herein is greater in DRG cells than in target cells. Thus, in certain embodiments, a reduction in transgene expression in target cells may be observed, but it is less than that observed in DRG cells. This reduction in expression in DRG can reduce or eliminate DRG toxicity and / or axonal damage in subjects undergoing gene therapy targeting non-DRG cells or tissues, such as neural cells, e.g., neurons, thereby improving their safety profile.

[0064] The present disclosure further provides RNA molecules having sequence characteristics of the RNA encoded by any of the nucleic acid cassettes described herein. In certain embodiments, the RNA is modified to increase its stability and / or activity when administered to a subject, for example, as a pharmaceutical composition. RNA compositions find use in a variety of therapeutic modalities delivered using a wide range of viral and non-viral delivery systems, including polymeric materials, ionizable lipids, membrane-permeable and zwitterionic lipids, nanoparticles, and dendrimers (see, for example, Kowalski et al., "Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery," Molecular Therapy 2019, Vol. 27 (4), pp. 710-728; and Paunovska et al., "Drug delivery systems for RNA therapeutics," Nature Reviews Genetics 2022, Vol. 23, pp. 265-280).

[0065] The RNA (e.g., mRNA or ncRNA) encoded by the transgene of the nucleic acid cassette can include any combination of two, three, four, or five or more of the sequences. For example, an RNA comprising a sequence of (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a variant or functional fragment thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii), can further comprise a second sequence of (i), (ii), or (iii), a third sequence of (i), (ii), or (iii), a fourth sequence of (i), (ii), or (iii), and / or five or more sequences of (i), (ii), or (iii). In any embodiment, the nucleic acid cassette may include two or more copies (e.g., two, three, four, five, or more than five copies) of the sequence of (i), (ii), or (iii).

[0066] In certain embodiments, the RNA encoded by the transgene of the nucleic acid cassette may comprise (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant or functional fragment thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0067] In any embodiment, the sequence may be, for example, in the 3' UTR, 5' UTR, or intron of an mRNA. If an mRNA contains more than one of the sequences, the sequences may be in different portions of the mRNA. However, in many embodiments, the sequence is in the 3' UTR of an mRNA. In these embodiments, the sequence of (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) may be located in one or more of the 3' UTR region of an mRNA, the 5' UTR of an mRNA, or an intron of an mRNA.

[0068] Any nucleic acid described herein may be non-naturally occurring, and the term "non-naturally occurring" refers to a composition that does not occur in nature. In some embodiments, a non-naturally occurring nucleic acid comprises a contiguous, uninterrupted nucleotide sequence not found in nature, i.e., it differs from any nucleic acid in its natural state (i.e., it 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 one another if they are derived from separate genomic regions that are not found as a contiguous, uninterrupted nucleic acid sequence in their natural state. For example, in some embodiments, a nucleic acid cassette can be composed of a promoter, a coding sequence, a sequence encoding a DRG detargeting element (as disclosed herein), and a terminator, wherein the promoter, coding sequence, sequence encoding the DRG detargeting element, and terminator are operably linked. In these embodiments, at least one of these elements is heterologous to another of the elements. For example, a coding sequence can be heterologous to a sequence encoding a DRG detargeting element, meaning that the sequence encoding the DRG detargeting element is not operably linked in the same manner as the coding sequence in a wild-type cell. In any embodiment, the nucleic acid cassette may additionally comprise an enhancer.

[0069] In any embodiment, the RNA encoded by the transgene of the nucleic acid cassette can comprise a functional fragment of any of SEQ ID NOS: 1-10 and 43-48, where the functional fragment reduces expression of the RNA to which it is operably linked in the DRG. Additionally, the functional fragment may or may not contain mismatches, e.g., 1, 2, 3, 4, or more mismatches, to SEQ ID NOS: 1-8.

[0070] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in 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, a functional fragment of SEQ ID NO: 1 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 1. A functional fragment can start at any nucleotide in SEQ ID NO: 1 that allows for its complete representation in SEQ ID NO: 1.

[0071] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in SEQ ID NO:2 that is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides in length. In certain embodiments, a functional fragment of SEQ ID NO:2 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO:2. A functional fragment may start at any nucleotide in SEQ ID NO:2 that allows for its complete representation in SEQ ID NO:2.

[0072] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in 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, a functional fragment of SEQ ID NO: 3 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 3. A functional fragment may start at any nucleotide in SEQ ID NO: 3 that allows for its complete representation in SEQ ID NO: 3.

[0073] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in 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, a functional fragment of SEQ ID NO:4 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO:4. A functional fragment may start at any nucleotide in SEQ ID NO:4 that allows for its complete representation in SEQ ID NO:4.

[0074] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in 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, a functional fragment of SEQ ID NO: 5 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 5. A functional fragment may start at any nucleotide in SEQ ID NO: 5 that allows for its complete representation in SEQ ID NO: 5.

[0075] In certain embodiments, a functional fragment comprises any contiguous stretch of nucleotides in 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, a functional fragment of SEQ ID NO: 6 comprises one, two, three, or four mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 6. A functional fragment can start at any nucleotide in SEQ ID NO: 6 that allows for its complete representation in SEQ ID NO: 6.

[0076] In certain embodiments, the functional fragment is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least Also 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127,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, 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 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 In certain embodiments, a functional fragment of SEQ ID NO:7 or SEQ ID NO:8 comprises any contiguous stretch of nucleotides in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 that is 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 length.or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches compared to the corresponding contiguous stretch of nucleotides in SEQ ID NO: 10. A functional fragment can start at any nucleotide in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0077] In some embodiments, the RNA may contain an miRNA-binding site for an miRNA 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 complements. In certain embodiments, the RNA may contain one or more binding sites for an miRNA. In these embodiments, the RNA may contain 6, 7, 8, 9, or 10 contiguous nucleotides that potentially base-pair with the seed region (located at the 5' end of the miRNA) of an miRNA such as 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. In some embodiments, the RNA may contain 6, 7, 8, 9, or 10 contiguous nucleotides at the 3' end of any of SEQ ID NOS: 1-6 that potentially base-pair with the seed region (located at the 5' end of the miRNA) of a miRNA, such as 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. One or more binding sites for the miRNA may comprise any of SEQ ID NOS: 1-6. In some embodiments, the sequence may be identical to SEQ ID NOS: 1-6 except for, for example, having one, two, three, or four mismatches compared to SEQ ID NOS: 1-6.

[0078] Specifically, sequences (i), (ii), or (iii) may provide binding sites 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.

[0079] As will be apparent, the nucleic acid cassette itself (which is DNA) can comprise (i) any of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii), wherein the inclusion of the sequence reduces expression of the protein or RNA encoded by the cassette in DRG cells within an organism compared to neuronal cells within a target tissue, e.g., neural tissue, e.g., the brain.

[0080] In some embodiments, a sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) may result in decreased expression of a polypeptide encoded by the ncRNA or mRNA in DRG cells compared to expression of the polypeptide encoded by the ncRNA or mRNA in DRG cells from an otherwise equivalent ncRNA or mRNA that does not contain the sequence of (i), (ii), or (iii). For example, an mRNA comprising the sequence (i), (ii), or (iii) may result in a reduction in expression of the polypeptide encoded by the mRNA in DRG cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold greater than the expression of the polypeptide in DRG cells from an otherwise equivalent mRNA that does not comprise the sequence (i), (ii), or (iii). In these embodiments, the reduction in polypeptide expression in DRG cells is greater than the reduction in polypeptide expression in target cells when compared to an otherwise equivalent mRNA that does not comprise the sequence (i), (ii), or (iii). Similarly, an ncRNA comprising the sequence (i), (ii), or (iii) may have a reduction in expression in DRG cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold greater than the expression of an ncRNA that does not comprise the sequence (i), (ii), or (iii). In these embodiments, the reduction in expression of the ncRNA in DRG cells is greater than the reduction in expression of the ncRNA in target cells when compared to an equivalent ncRNA that does not contain sequence (i), (ii), or (iii).

[0081] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) is expressed as a polypeptide, mRNA, or nRNA in DRG cells from an otherwise equivalent RNA transcript that does not contain the sequence of (i), (ii), or (iii). This may result in reduced expression of the polypeptide encoded by the mRNA, the mRNA itself, or the ncRNA in DRG cells at a level at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than the expression of the cRNA. In these embodiments, the reduction in expression of the polypeptide or RNA transcript in DRG cells is greater than the reduction in expression of the polypeptide in target cells when compared to an equivalent RNA transcript that does not contain the sequence of (i), (ii), or (iii).

[0082] In some embodiments, the sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii) does not result in a significant decrease in expression of a polypeptide, mRNA, or ncRNA encoded by the mRNA in a target cell compared to expression of the polypeptide, mRNA, or ncRNA in the target cell from an otherwise equivalent RNA transcript that does not include the sequence of (i), (ii), or (iii). In some embodiments, the sequence of (i), (ii), or (iii) can result in expression of a polypeptide, mRNA, or ncRNA encoded by the mRNA in a target cell at a level that is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the polypeptide, mRNA, or ncRNA in the target cell from an otherwise equivalent RNA transcript that does not contain the sequence of (i), (ii), or (iii). In these embodiments, the reduction in polypeptide expression in DRG cells is greater than the reduction in polypeptide, mRNA, or ncRNA expression in the target cell when compared to an otherwise equivalent RNA transcript that does not contain the sequence of (i), (ii), or (iii).

[0083] In some embodiments, the RNA transcript is a therapeutic RNA transcript. In some aspects, the therapeutic RNA transcript is an mRNA comprising a sequence encoding a polypeptide, e.g., a therapeutic protein, which may be, for example, intracellular, membrane-bound, or secreted. In some embodiments, the therapeutic protein is associated with a neurological disease or disorder, e.g., a protein whose abnormal function (e.g., resulting from a genetic mutation or abnormality) is associated with a neurological disease or disorder. In further aspects, the therapeutic RNA transcript comprises an ncRNA sequence that targets an endogenous molecule, e.g., a gene, protein, or RNA, associated with a neurological disease or disorder.

[0084] Neurological diseases and disorders include those associated with one or more genetic mutations and those with unknown etiology, hi some embodiments, neurological diseases and disorders include conditions associated with epileptic seizures, neurodegenerative disorders, and / or neurodevelopmental disorders. Examples of neurological diseases or disorders include Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic lid myoclonus Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep (CSWS), infantile spasms (West syndrome). group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures isolated, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douse syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0085] Several genetic abnormalities are associated with epilepsy, including many of the neurological diseases and disorders mentioned above. Examples of genes affected by these genetic abnormalities, i.e., genes whose activity and / or expression are altered by genetic mutations, include ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, These include 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.

[0086] Thus, in embodiments where the RNA transcript is an mRNA comprising a sequence encoding a therapeutic protein for the treatment of a neurological disease or disorder, the therapeutic protein may be expressed by: (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2 , GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Mi Oclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, S (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 expression of a gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0087] In some cases, the target cell may be a neuron, muscle cell, cardiac cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In any of these embodiments, the target cell may be a neuron, such as a cerebral cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in these embodiments, the neuron may be a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron, or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0088] The cassette may be linear or circular, and in some embodiments, the nucleic acid cassette may be a plasmid or a viral vector, such as an adeno-associated viral (AAV) vector or a lentiviral vector. In certain embodiments, the viral vector may be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8, and hybrids thereof.

[0089] Also provided is a nucleic acid cassette comprising a transgene encoding an RNA transcript, wherein the RNA comprises an miRNA binding site for an miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, mir-183-3p, mir-196a-5p, mir-494-3p, or complements thereof. In some embodiments, the RNA may comprise a binding site for an miRNA produced from the mir-196b, mir-10b, mir-24-2, mir-183, mir-196a, or mir-494 gene. The miRNA binding site should not be in the naturally occurring version of the RNA if the RNA is otherwise naturally occurring. In some embodiments, the cassette may contain two or more, three or more, or four or more binding sites for an miRNA selected from, for example, mir-196b-5p, mir-10b-5p, mir-24-2-5p, and mir-183-3p, mir-196a-5p, mir-494-3p, or their complements. In some embodiments, the RNA is an mRNA, e.g., an mRNA encoding a therapeutic protein (as described elsewhere herein). In these embodiments, the binding site may be anywhere within the mRNA, particularly within non-coding sequences such as the 3' UTR region, 5' UTR, introns, or any combination thereof.

[0090] In any embodiment, the nucleic acid cassette may not be naturally occurring, meaning, for example, that the miRNA binding site in the RNA transcript expressed from the nucleic acid cassette is heterologous to one or more other regions of the RNA transcript. In any embodiment, the nucleic acid cassette may include a promoter and / or an enhancer. In some embodiments, the nucleic acid cassette may be comprised of a promoter, a coding sequence, and a terminator, wherein the promoter, coding sequence, and terminator are operably linked. In these embodiments, the promoter may be heterologous to the coding sequence, meaning that the promoter does not drive expression of the coding sequence in wild-type cells. In any embodiment, the nucleic acid cassette may additionally include an enhancer.

[0091] In some embodiments, the mRNA may encode a polypeptide, e.g., a therapeutic protein, which may be, for example, intracellular, membrane-bound, or secreted.

[0092] In some embodiments, the polypeptide is a therapeutic protein whose altered function (e.g., due to a genetic mutation) is associated with a neurological disease or disorder. As described above, neurological diseases and disorders include those associated with one or more genetic mutations and those with 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 disorders include Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic lid myoclonus Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep (CSWS), infantile spasms (West syndrome). group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures isolated, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douse syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0093] In these embodiments, the therapeutic protein is selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, 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 expression of a gene from (i). The transcription factor encoded by the mRNA can be an engineered transcription factor or a naturally occurring transcription factor.

[0094] In some cases, the target cell may be a neuron, muscle cell, cardiac cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In any of these embodiments, the target cell may be a neuron, such as a cerebral cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in these embodiments, the neuron may be a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron, or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0095] The cassette may be linear or circular, and in some embodiments, the nucleic acid cassette may be a plasmid or a viral vector, such as an adeno-associated viral (AAV) vector or a lentiviral vector. In certain embodiments, the viral vector may be an AAV vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8, and hybrids thereof.

[0096] Also provided are RNA transcripts encoded by the nucleic acid cassettes described herein.

[0097] Also provided are methods for reducing DRG expression of mRNA, a polypeptide encoded by mRNA, or ncRNA, relative to the expression of the polypeptide, mRNA, or ncRNA in a target tissue. In these embodiments, the methods can include constructing a nucleic acid cassette to include a DRG detargeting sequence described herein in the RNA transcript encoded therein. For example, the nucleic acid cassette can be constructed to include, in the RNA transcript encoded therein, a sequence of (i) one of SEQ ID NOS: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii). Details of the cassettes produced by this method are described herein. In some embodiments, the methods can include introducing an expression cassette described herein or RNA encoded thereby into an organism, e.g., a human subject, wherein the inclusion of any one or more of the disclosed DRG detargeting sequences reduces expression of the protein in DRG cells of the organism relative to the target tissue.

[0098] In any embodiment herein, the target cell may be a neuron, muscle cell, cardiac cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In some cases, the target cell may be a neuron, such as a cerebral cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in some embodiments, the neuron is a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron, or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0099] In any embodiment, the method may further include administering to the subject a vector (e.g., an AAV or lentiviral vector) encoding an RNA transcript, e.g., the RNA transcript is an mRNA encoding a therapeutic protein. In some embodiments, the method may include administering to the subject an RNA transcript.

[0100] Expression cassette The nucleic acid cassette may include one or more additional regulatory elements (e.g., promoters, terminators, and / or enhancers) that direct expression of the transgene in a particular cell type or a particular class of cell types. For example, a cell-type-selective regulatory element can direct gene expression in a particular cell type relative to one or more other cell types. Alternatively, or in addition, a cell-type-selective regulatory element can direct gene expression in a particular class of cells relative to one or more other classes of cells. In one embodiment, a cell-type-selective regulatory element of the invention enhances gene expression in a particular cell type or class of cells. In another embodiment, a cell-type-selective regulatory element represses gene expression in a particular cell type or class of cells. Cell-type-selective modulation of gene expression (e.g., enhancing or repressing gene expression) does not require that gene expression be affected only in the target cell type or class of cells. Rather, cell-type selective modulation of gene expression (e.g., enhancing or suppressing gene expression) requires only an increase or decrease in gene expression in the target cell type relative to one or more other cell types or classes of cells.

[0101] 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 (i) SEQ ID NO: 1, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0102] 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 (i) SEQ ID NO: 2, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0103] 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 (i) SEQ ID NO: 3, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0104] 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 (i) SEQ ID NO: 4, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0105] 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 (i) SEQ ID NO: 5, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0106] 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 (i) SEQ ID NO: 6, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0107] 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 (i) SEQ ID NO: 7, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0108] 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 (i) SEQ ID NO: 8, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0109] 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 (i) SEQ ID NO: 9, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0110] 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 (i) SEQ ID NO: 10, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0111] 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 (i) at least two distinct sequences selected from SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0112] 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 (i) at least three different sequences selected from SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0113] 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 (i) at least four different sequences selected from SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0114] 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 (i) at least five different sequences selected from SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0115] 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 (i) SEQ ID NOS: 1-10 and 43-48, in any order; (ii) a variant, functional fragment, multiple copy, or combination thereof; or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0116] 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 (i) SEQ ID NO: 43, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0117] 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 (i) SEQ ID NO: 44, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0118] 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 (i) SEQ ID NO: 45, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0119] 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 (i) SEQ ID NO: 46, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0120] 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 (i) SEQ ID NO: 47, (ii) a variant, functional fragment, multiple copy, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0121] 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 (i) SEQ ID NO: 48, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a DRG detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0122] In some embodiments, the nucleic acid cassette may comprise a CNS-selective promoter operably linked to a polynucleotide encoding a therapeutic protein and one or more DRG detargeting elements / sequences as disclosed herein. A CNS promoter is a promoter that specifically regulates gene expression in one or more cells of the central nervous system. For example, a CNS-selective promoter may specifically regulate gene expression in one or more neurons or glial cells of the CNS. In one embodiment, a CNS-selective promoter specifically regulates gene expression in one or more neurons or astrocytes. In another embodiment, a CNS-selective promoter specifically regulates gene expression in one or more astrocytes. In certain embodiments, a CNS-selective promoter enhances expression in CNS cells (e.g., neurons or glial cells such as astrocytes) compared to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, microglia, motor neurons, vascular cells, non-GABAergic neurons, or other CNS cells).

[0123] Examples of CNS-selective promoters include, but are not limited to, the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid 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, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, as well as the PaqR4 promoter. Suitable promoters are also described, for example, in International Publication No. 2018 / 187363, the sequences of which are incorporated herein by reference. Other sequences may also be used.

[0124] In some embodiments, the cassette may include a GABAergic neuron-selective promoter operably linked to a polynucleotide encoding a therapeutic protein. GABAergic cells are inhibitory neurons that produce gamma-aminobutyric acid. GABAergic cells can be identified by markers such as the expression of glutamic acid decarboxylase 2 (GAD2), GAD1, NKX2.1, DLX1, DLX5, SST, PV, and VIP. A GABAergic neuron-selective promoter is a regulatory element that specifically regulates gene expression in GABAergic neurons. For example, a GABAergic neuron-selective promoter enhances expression in GABAergic neurons compared to one or more other CNS cell types (e.g., excitatory neurons, dopaminergic neurons, astrocytes, microglia, motor neurons, vascular cells, non-GABAergic neurons, or other CNS cells).

[0125] A PV neuron-selective promoter is a promoter that specifically regulates gene expression in PV neurons, e.g., a PV neuron-selective promoter enhances expression in PV neurons relative to one or more other CNS cell types.

[0126] In certain embodiments, the neuron-selective promoter can be of human origin or include sequences of human origin. In some cases, the promoter can be of murine origin or include sequences of murine origin. In some cases, the promoter is not naturally occurring or includes sequences that are not naturally occurring. In some instances, the promoter's sequence can be 100% human. In other cases, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the promoter sequence is human. For example, a promoter can have 50% of its sequence of human origin, and the remaining 50% can be non-human (e.g., murine or fully synthetic).

[0127] In some embodiments, the therapeutic protein encoded by the mRNA is associated with a neurological disease or disorder. As described above, neurological diseases and disorders include those associated with one or more gene mutations and those with 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 disorders include Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), Dravet syndrome, early myoclonic encephalopathy (EME), epileptic lid myoclonus Jeavons syndrome, infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep (CSWS), infantile spasms (West syndrome). group), juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures isolated, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic-atonic epilepsy Douse syndrome, sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

[0128] In these embodiments, the therapeutic protein is selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, 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 expression of a gene from (i). The transcription factor encoded by the mRNA can be an engineered or naturally occurring transcription factor that regulates, eg, activates or represses, the gene of interest.

[0129] In certain embodiments, the nucleic acid constructs described herein include, in addition to a promoter, other regulatory elements, such as sequences associated with transcription initiation or termination, enhancer sequences, and efficient RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stability elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, or polyadenylation (polyA) sequences, or combinations thereof. Regulatory elements can function to regulate gene expression at the transcriptional, late-transcriptional, or translational phases of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination. In various embodiments, regulatory elements can increase gene expression selectivity in a cell type of interest, increase the rate at which RNA transcripts are produced, increase the stability of the produced RNA, and / or recruit transcription factors to the coding region that increase the rate of protein synthesis from RNA transcripts.

[0130] In certain embodiments, the cassette may further comprise a polyA sequence. Suitable polyA sequences include, for example, an artificial polyA (PA75) having a length of approximately 75 bp (see, for example, WO 2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit beta globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 Elb polyA, growth hormone polyA, or PBGD polyA. In certain embodiments, the polyA sequence is located downstream of the polynucleotide encoding the functional therapeutic protein in the nucleic acid construct described herein.

[0131] Liver detargeting In addition to the DRG detargeting embodiments described above, the present disclosure further includes a liver detargeting element. The provided liver detargeting element can be present in a nucleic acid cassette, an RNA molecule, e.g., an RNA transcript, a synthetic RNA molecule, etc., as described above for the DRG detargeting element. Additionally, methods of using the liver detargeting element to reduce transgene expression and / or activity in liver cells / tissues are provided. Such methods are similar to those detailed above for the method of using the DRG detargeting element to reduce transgene expression and / or activity in DRG cells. Accordingly, all embodiments described herein for the DRG detargeting element can be applied to the liver detargeting element described below, with the understanding that the tissue / cell detargeted by the liver detargeting element is a living tissue / liver cell, not a DRG cell.

[0132] Thus, in certain embodiments, a nucleic acid cassette is provided comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises a sequence of (i) any of SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80% identical to (i) or (ii). In these embodiments, the sequence reduces expression of the RNA transcript in liver cells. Embodiments utilizing these sequences are described in more detail below. These liver detargeting sequences can be used in any embodiment in which liver detargeting is desired.

[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 (i) SEQ ID NO: 65, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a liver-detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[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 (i) SEQ ID NO: 110, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii), including a liver-detargeting region. In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[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 (i) SEQ ID NO: 112, (ii) a variant, functional fragment, multiple copies, or combination thereof, or (iii) a liver-detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[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 (i) at least two different sequences selected from SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, multiple copy, or combination thereof; or (iii) a liver-detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[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 (i) at least three different sequences selected from SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, multiple copy, or combination thereof; or (iii) a liver-detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[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 (i) at least four different sequences selected from SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, multiple copy, or combination thereof; or (iii) a liver-detargeting region comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of (i) or (ii). In certain embodiments, the promoter is a tissue-selective or tissue-specific promoter. In certain embodiments, the promoter is a CNS-selective promoter, and the RNA transcript is an mRNA encoding a therapeutic expression product, e.g., a therapeutic protein, for a neurological disease or disorder.

[0139] Combining Arrays As shown in any of the tables below, the DRG and liver targeting sequences described above can be combined with each other or with other detargeting sequences to produce cassettes that more effectively detarget 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; in any combination, the combination can include 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, 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.

[0140] Table 1 below shows exemplary combinations of DRG detargeting elements (SEQ ID NOS: 1-10 and 43-48) that can be used in the present invention.

[0141] [Table 1]

[0142] The liver detargeting elements of SEQ ID NOs: 65, 110, and 112 can be combined with each other or with the liver detargeting elements described in International Application No. US2023065801, filed April 14, 2023 (i.e., SEQ ID NOs: 57-62, 64, and 66-71), which are incorporated herein by reference.

[0143] Table 2 below shows exemplary combinations of liver detargeting elements that may be used herein.

[0144] [Table 2]

[0145] In some embodiments, the nucleic acid cassette may comprise a therapeutic transgene encoding an RNA transcript (e.g., mRNA), wherein the RNA transcript comprises a first sequence that detargets expression in DRG cells and a second sequence that detargets expression in liver cells. In these embodiments, the first and second sequences may result in decreased expression of the RNA transcript or a polypeptide encoded thereby (e.g., if the RNA transcript is mRNA) in DRG and liver cells relative to a target tissue, e.g., neuronal cells such as cerebral cells, brain stem cells, hippocampal cells, cerebellar cells, or GABAergic cells, e.g., where the GABAergic cells are parvalbumin-expressing cells.

[0146] In this cassette, the first and second sequences are capable of expressing an RNA transcript or a polypeptide encoded thereby (e.g., an RNA transcript) in a DRG cell 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% less than the expression of the RNA transcript or polypeptide in a DRG cell from an equivalent RNA transcript but excluding the first and second sequences. is an mRNA), independently, resulting in reduced expression of the RNA transcript or the polypeptide encoded thereby in liver 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 expression of the RNA transcript or polypeptide in liver cells from an otherwise equivalent RNA transcript that does not contain the first and second sequences.

[0147] In any of these embodiments, (a) the first sequence is (i) any of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii). and (b) the second sequence is (iv) any of SEQ ID NOs: 57-62, 64-71, 110, and 112, (v) a variant, functional fragment, or combination thereof, or (vi) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (iv) or (v).

[0148] In some of these embodiments, the first sequence (i), (ii), or (iii) may provide binding sites 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) may provide binding sites for hsa-mir-22-3p, hsa-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.

[0149] In any of these embodiments, the RNA transcript may comprise a combination of sequences selected from Table 3 below, where SEQ ID NOS: 1-10 and 43-48 detarget expression in DRG cells, while SEQ ID NOS: 57-62, 64-71, 110, and 112 detarget expression in liver cells. Table 3 below shows exemplary combinations of DRG detargeting sequence elements and liver detargeting sequence elements that may be used herein.

[0150] [Table 3]

[0151] For clarity, an RNA transcript can include multiple different DRG detargeting elements combined with one or more liver detargeting elements, and / or multiple different liver detargeting elements combined with one or more DRG detargeting elements, each of which may be independently present in the RNA transcript in one or more (e.g., two, three, four, or five or more) copies. SEQ ID NO: 111 is one example of such a combination, although several others exist (as exemplified in the experiments of this disclosure).

[0152] The first and second sequences reduce transgene expression in dorsal root ganglion cells (DRG) and liver cells compared to target cells (neuronal cells, e.g., neurons), and can therefore be used in various gene therapy strategies targeting cells not present in the DRG or liver. Reducing transgene expression in DRG and liver cells compared to target cells means that the reduction in transgene expression driven by the DRG and liver detargeting sequences disclosed herein is greater in DRG cells and liver than in target cells. Thus, in certain embodiments, a reduction in transgene expression in target cells may be observed, but it is less than that observed in DRG and liver cells. This reduction in expression in DRG and liver can reduce or eliminate toxicity and / or axonal damage in subjects undergoing gene therapy targeting non-DRG cells or tissues, such as neural cells, e.g., neurons, and non-liver cells, thereby improving their safety profile.

[0153] In some embodiments, the DRG and / or liver detargeting elements disclosed herein can be used in combination with other sequences with known cell- or tissue-specific detargeting activity. For example, an expression cassette of the present disclosure can encode an RNA transcript that includes one or more detargeting sequences disclosed herein (e.g., SEQ ID NOS: 1-10, 57-62, and 64-71, either alone or in any combination) and also includes one or more sequences with known detargeting activity, such as SEQ ID NOS: 63, which has liver detargeting activity. No limitation in this regard is intended.

[0154] vector Expression vectors can be used to deliver nucleic acid molecules to target cells via transfection or transduction. Vectors can be integrative or non-integrative, which refers to the ability of a vector to integrate an expression cassette or transgene into the genome of a host cell. Examples of expression vectors include, but are not limited to, (a) non-viral vectors, such as nucleic acid vectors including linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs or PACs); episomal vectors; transposons (e.g., PiggyBacs); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0155] Expression vectors may be linear oligonucleotides or circular plasmids 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 cell membrane barriers to promote intracellular delivery of genetic material. Examples of physical methods include the use of needles, ballistic DNA, electroporation, sonoporation, photoporation, magnetofection, and hydroporation. Chemical methods generally refer to methods in which chemical carriers deliver nucleic acid molecules to cells and can include inorganic particles, lipid-based vectors, polymer-based vectors, and peptide-based vectors.

[0156] In some embodiments, expression vectors are administered to target cells using inorganic particles. Inorganic particles can refer to nanoparticles, such as nanoparticles engineered for various sizes, shapes, and / or porosity to escape the reticuloendothelial system or protect trapped molecules from degradation. Inorganic nanoparticles can be prepared from metals (e.g., iron, gold, and silver), inorganic salts, or ceramics (e.g., calcium, magnesium, or silicon phosphates or carbonates). The surfaces of these nanoparticles can be coated to facilitate DNA binding or targeted gene delivery. Magnetic nanoparticles (e.g., supermagnetic iron oxide), fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g., cylindrical fullerenes), quantum dots, and supramolecular systems can also be used.

[0157] In some embodiments, expression vectors are administered to target cells using cationic lipids (e.g., cationic liposomes). Various types of lipids have been investigated for gene delivery, such as lipid nanoemulsions (e.g., which are dispersions of one immiscible liquid in another stabilized by an emulsifier) ​​or solid lipid nanoparticles.

[0158] In some embodiments, the expression vector is administered to the target cell using a peptide-based delivery vehicle. Peptide-based delivery vehicles may have the advantage of protecting the delivered genetic material, targeting specific cell receptors, disrupting the endosomal membrane, and delivering the genetic material into the nucleus. In some embodiments, the expression vector is administered to the target cell using a polymer-based delivery vehicle. The polymer-based delivery vehicle may comprise natural proteins, peptides, and / or polysaccharides, or synthetic polymers. In one embodiment, the polymer-based delivery vehicle comprises polyethylenimine (PEI). PEI can condense DNA into positively charged particles that bind to anionic cell surface residues and are transported into the cell via endocytosis. In other embodiments, the polymer-based delivery vehicle may comprise poly-L-lysine (PLL), poly(DL-lactic acid) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, histones, protamine, dendrimers, chitosan, synthetic amino derivatives of dextran, and / or cationic acrylic polymers. In certain embodiments, the polymer-based delivery vehicle may comprise a mixture of polymers, such as PEG and PLL.

[0159] In certain embodiments, the expression vector may be a viral vector suitable for gene therapy. Favorable characteristics of viral gene therapy or gene delivery vectors include the ability to be reproducibly and stably propagated and purified to high titers; the ability to mediate targeted delivery (e.g., delivering a transgene specifically to a tissue or organ of interest without widespread vector spread to other locations); and the ability to mediate gene delivery and transgene expression without inducing adverse side effects.

[0160] Some types of viruses, such as the non-pathogenic parvoviruses called adeno-associated viruses, have been engineered for gene therapy purposes by utilizing the viral infection pathway but avoiding replication and subsequent expression of viral genes that could lead to toxicity. Such viral vectors can be obtained by deleting all or part of the coding region from the viral genome, while leaving intact sequences (e.g., terminal repeats) that may be necessary for functions such as packaging of the vector genome into viral capsids or integration of vector nucleic acid (e.g., DNA) into host chromatin.

[0161] In various embodiments, suitable viral vectors include negative-strand RNA viruses such as retroviruses (e.g., types A, B, C, and D viruses), adenoviruses, parvoviruses (e.g., adeno-associated viruses or AAV), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-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 viruses, human T-lymphotrophic virus type 1 (HTLV-1), bovine leukemia virus (BLV), lentiviruses, and spumaretroviruses. Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Viral vectors can be classified into two groups: integrating and non-integrating, according to their ability to integrate into the host genome. Oncoretroviruses and lentiviruses can integrate into host cell chromatin, while adenoviruses, adeno-associated viruses, and herpesviruses persist primarily in the cell nucleus as extrachromosomal episomes.

[0162] In certain embodiments, a suitable viral vector is a retroviral vector. Retrovirus refers to viruses of the Retroviridae family. Examples of retroviruses include oncoretroviruses such as murine leukemia virus (MLV) and lentiviruses such as human immunodeficiency virus 1 (HIV-1). The retroviral genome is a single-stranded (ss) RNA and contains various genes that can be provided in cis or trans. For example, a retroviral genome may contain cis-acting sequences such as two long terminal repeats (LTRs) along with elements for gene expression, reverse transcription, and integration into a host chromosome. Other components include a packaging signal (psi or Ψ) for specific RNA packaging into newly formed virions and a polypurine tract (PPT), which is the initiation site for plus-strand DNA synthesis during reverse transcription. In addition, a retroviral genome may contain the gag, pol, and env genes. The gag gene encodes structural proteins, the pol gene encodes an enzyme that associates with ssRNA and reverse transcribes viral RNA into DNA, and the env gene encodes the viral envelope. Generally, gag, pol, and env are provided in trans for viral replication and packaging.

[0163] In certain embodiments, the retroviral vector provided herein may be a lentiviral vector. At least five serogroups or serotypes of lentiviruses have been recognized. Viruses of different serotypes may differentially infect certain cell types and / or hosts. Lentiviruses include, for example, primate retroviruses and non-primate retroviruses. Primate retroviruses include HIV and simian immunodeficiency virus (SIV). Non-primate retroviruses include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and visna virus. Lentiviruses or lentivectors may be capable of transducing quiescent cells. Similar to oncoretroviral vectors, the design of lentivectors may be based on the separation of cis- and trans-acting sequences.

[0164] In an exemplary embodiment, the viral vector provided herein is an adeno-associated virus (AAV). AAV is a small, replication-deficient, non-enveloped animal virus that infects humans and some other primate species. AAV is not known to cause human disease and induces a mild immune response. AAV vectors can also infect both dividing and quiescent cells without integrating into the host cell genome.

[0165] The AAV genome consists of linear, single-stranded DNA approximately 4.7 kb in length. The genome consists of two open reading frames (ORFs) flanked by inverted terminal repeat (ITR) sequences approximately 145 bp in length. The ITRs consist of a 5'-terminal nucleotide sequence (5'ITR) and a 3'-terminal nucleotide sequence (3'ITR) that contain palindromic sequences. The ITRs function in cis by folding to form a T-shaped hairpin structure through complementary base pairing, which serves as a primer during the initiation of DNA replication for second-strand synthesis. The two open reading frames encode the rep and cap genes, which are involved in virion replication and packaging. In an exemplary embodiment, the AAV vectors provided herein do not contain the rep or cap genes. Such genes can be provided in trans to produce virions, as further described below.

[0166] In certain embodiments, the AAV vector may comprise a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode green fluorescent protein or an antibiotic resistance gene, such as kanamycin or ampicillin. In certain embodiments, the stuffer nucleic acid may be located outside the ITR sequence (e.g., relative to the polynucleotide encoding the therapeutic protein and the regulatory sequences located between the 5' and 3' ITR sequences).

[0167] There are various serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8. These serotypes differ in their tropism, or the type of cell they infect. AAV can contain genomes and capsids from multiple serotypes (e.g., pseudotypes). For example, an AAV can contain a serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotyping can improve transduction efficiency and alter tropism.

[0168] In some embodiments, AAV vectors or AAV viral particles, or virions, can be used to deliver constructs comprising a cell-selective regulatory element operably linked to a polynucleotide encoding a functional therapeutic protein to cells, cell types, or tissues, and can be performed either in vivo, ex vivo, or in vitro. In exemplary embodiments, such AAV vectors are replication-deficient. In some embodiments, the AAV virus is engineered or genetically modified so that it can replicate and generate virions only in the presence of helper factors.

[0169] In certain embodiments, viral vectors can be selected to produce virions with high infectivity without selectivity for a particular cell type. In certain embodiments, viral vectors can be designed to produce virions that infect many different cell types, but with transgene expression enhanced and / or optimized in the cell type of interest (e.g., PV neurons) and transgene expression 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, manipulated, or engineered using different regulatory elements that are selective for one or more cell types. In some cases, one or more regulatory elements operably linked to a polynucleotide encoding a therapeutic protein enhance selective expression of the polynucleotide in a target cell, cell type, or tissue, while one or more regulatory elements suppress transgene expression in off-target cells, cell types, or tissues, or confer significantly low, negligible, or statistically low gene expression in one or more off-target cells, cell types, or tissues.

[0170] In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred.

[0171] In an exemplary embodiment, the present application provides an expression vector designed for delivery by AAV. The AAV can be any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8, or a chimeric, hybrid, or variant AAV. The AAV can also be a self-complementary AAV (scAAV), where the coding region is designed to form an intramolecular double-stranded DNA template. Upon infection with such a vector, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. The design of scAAV vectors has been described in various publications, including McCarty et al Gene Therapy 2001 8:1248-54.

[0172] In certain embodiments, an expression vector designed for delivery by AAV comprises a 5' ITR and a 3' ITR. In certain embodiments, an expression vector designed for delivery by AAV comprises a 5' ITR, a promoter, the above construct, and a 3' ITR. In certain embodiments, an expression vector designed for delivery by AAV comprises a 5' ITR, an enhancer, a promoter, the above construct, and a 3' ITR.

[0173] host cell In another aspect, the present invention relates to a host cell comprising the nucleic acid cassette described above. The host cell may be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In an exemplary embodiment, the host cell refers to any cell line that is susceptible to infection by the virus of interest and is suitable for in vitro culture.

[0174] In certain embodiments, the host cells provided herein can be used for ex vivo gene therapy purposes. In such embodiments, the cells are transfected with the above-described nucleic acid molecules or expression cassettes and then transplanted into a patient or subject. The transplanted cells can be of autologous, allogeneic, or xenogeneic origin. For clinical use, cell isolation will generally be performed under Good Manufacturing Practices (GMP) conditions. Prior to transplantation, cell quality and the absence of microorganisms or other contaminants are typically checked, and preconditioning, such as with radiation and / or immunosuppressive treatment, may be performed. Additionally, the host cells can be transplanted together with growth factors to stimulate cell proliferation and / or differentiation.

[0175] In certain embodiments, host cells can be used for ex vivo gene therapy to the CNS. Preferably, the cells are eukaryotic cells, such as mammalian cells, including but not limited to, cells from 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. Those skilled in the art will select more appropriate cells according to the patient or subject to be transplanted.

[0176] In certain embodiments, the host cells provided herein may be cells with self-renewal and pluripotency properties, such as stem cells or induced pluripotent stem cells. The stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells (MSCs) can differentiate into at least one of osteoblasts, chondrocytes, adipocytes, or myocytes and can be isolated from any type of tissue. Generally, MSCs are isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. Methods for obtaining them are well known to those skilled in the art. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of multipotent stem cell that can be generated directly from adult cells. Yamanaka et al. induced iPS cells by introducing Oct3 / 4, Sox2, Klf4, and c-Myc genes into mouse and human fibroblasts and expressing the genes in the cells (WO 2007 / 069666). Subsequently, Thomson et al. generated human iPS cells using Nanog and Lin28 instead of Klf4 and c-Myc (WO 2008 / 118820).

[0177] In an exemplary embodiment, the host cells provided herein are packaging cells. The cells can be adherent or suspension cells. The packaging cells and the helper vector or virus, or DNA construct, together provide in trans all missing functions required for complete replication and packaging of the viral vector.

[0178] Preferably, the packaging cells are eukaryotic cells, such as mammalian cells, including monkey, human, canine, and rodent cells. Examples of human cells include PER.C6 cells (WO 01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus lung cells (ATCC CL-160). Examples of non-human primate cells include Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). Examples of canine cells include MDCK cells (ATCC CCL-34). Examples of rodent cells include hamster cells such as BHK21-F, HKCC, or CHO cells.

[0179] As an alternative to mammalian sources, cell lines for use in the present invention may be derived from avian sources such as chicken, duck, goose, quail, or pheasant. Examples of avian cell lines include avian embryonic stem cells (WO 01 / 85938 and WO 03 / 076601), immortalized duck retinal cells (WO 2005 / 042728), and avian embryonic stem cell-derived cells, including chicken cells (WO 2006 / 108846) or duck cells, such as the EB66 cell line (WO 2008 / 129058 and WO 2008 / 142124).

[0180] In another embodiment, the host cells are insect cells such as SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB-Sf21), MG1 cells (BTI-TN-MG1), or High Five™ cells (BTI-TN-5B1-4).

[0181] In certain embodiments, the host cells provided herein comprise a nucleic acid construct (e.g., a plasmid) carrying a recombinant AAV vector / genome containing the cassettes described above, and may further comprise one or more additional nucleic acid constructs, such as, for example, (i) a nucleic acid construct encoding the rep and cap genes but not carrying ITR sequences (e.g., an AAV helper plasmid), and / or (ii) a nucleic acid construct (e.g., a plasmid) that provides adenoviral functions necessary for AAV replication. In exemplary embodiments, the host cells provided herein comprise i) the nucleic acid construct or expression vector described above, ii) a nucleic acid construct encoding the AAV rep and cap genes that does not carry ITR sequences, and iii) a nucleic acid construct comprising adenoviral helper genes (described further below).

[0182] In certain embodiments, the rep gene, cap gene, and adenovirus helper genes can be combined on a single plasmid (Blouin V et al. J Gene Med. 2004; 6(suppl): S223-S228, Grimm D. et al. Hum. Gene Ther. 2003; 7: 839-850). Thus, in another exemplary embodiment, the host cell provided herein comprises i) a nucleic acid molecule or expression cassette, and ii) a plasmid encoding the AAV rep and cap genes, which does not carry ITR sequences, and further comprises adenovirus helper genes. Alternative methods are known. For example, the rep, cap, and adenovirus helper genes need not be present on the same plasmid, but can be provided on different plasmids, or the rep and cap genes can be provided on a different plasmid from the adenovirus helper genes.

[0183] In certain embodiments, suitable hosts for large-scale production of AAV vectors are insect cells that can be infected with a combination of recombinant baculoviruses (Urabe et al. Hum. Gene Ther. 2002;13:1935-1943). For example, SF9 cells can be co-infected with three baculovirus vectors that express AAV rep, AAV cap, and the AAV vector to be packaged, respectively. The recombinant baculovirus vectors will provide the viral helper gene functions necessary for viral replication and / or packaging.

[0184] Further guidance on the construction and production of virions for gene therapy according to the present invention can be found in: Viral Vectors for Gene Therapy, Methods and Protocols. Series: Methods in Molecular Biology, Vol. 737. Merten and Al-Rubeai (Eds.); 2011 Humana Press (Springer); Gene Therapy. M. Giacca. 2010 Springer-Verlag; Heilbronn R. and Weger S. Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics. In: Drug Delivery, Handbook of Experimental Pharmacology 197; M. Schafer-Korting (Ed.). 2010 Springer-Verlag; pp. 143-170; Adeno-Associated Virus: Methods and Protocols. R. O. Snyder and P. Moullier (Eds). 2011 Humana Press (Springer); Bunning H. et al.Recent developments in adeno-associated virus technology.J.Gene Med.2008;10:717-733; and Adenovirus: Methods and Protocols. M. Chilion and A. Bosch (Eds.); Third. Edition. 2014 Humana Press (Springer).

[0185] Virions and methods for producing virions In certain embodiments, the present application provides a viral particle comprising a viral vector. The terms "viral particle" and "virion" are used interchangeably herein and refer to an infectious and typically replication-deficient viral particle comprising a viral genome (e.g., a viral expression vector) packaged within a capsid, and in some cases, for example, in the case of retroviruses, a lipid envelope surrounding the capsid. "Capsid" refers to the structure in which the viral genome is packaged. A capsid consists of several oligomeric structural subunits made of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2, and VP3. In one embodiment, the virion provided herein is a recombinant AAV virion or rAAV virion obtained by packaging an AAV vector in a protein shell.

[0186] In certain embodiments, the recombinant AAV virion provided herein can be prepared by encapsidating the AAV genome from a specific AAV serotype in a viral particle formed by the native Cap protein corresponding to the AAV of the same specific serotype.In other embodiments, the AAV viral particle provided herein comprises a viral vector that contains the ITR of a given AAV serotype packaged in proteins from different serotypes.See, for example, Bunning H et al.J Gene Med 2008;10:717-733. For example, viral vectors with ITRs from a given AAV serotype can be expressed as: a) viral particles composed of capsid proteins from the same or different AAV serotypes (e.g., AAV2 ITRs and AAV9 capsid proteins, AAV2 ITRs and AAV8 capsid proteins, etc.); b) mosaic viral particles composed of a mixture of capsid proteins from different AAV serotypes or variants (e.g., AAV2 ITRs with AAV1 and AAV9 capsid proteins); c) chimeric viral particles composed of capsid proteins truncated by domain swapping between different AAV serotypes or variants (e.g., AAV2 ITRs with AAV8 capsid proteins with AAV9 domains); or d) targeted viral particles engineered to display selective binding domains that enable stringent interaction with target cell-specific receptors (e.g., AAV5 capsid proteins with AAV9 capsid proteins genetically truncated by insertion of peptide ligands). It may be packaged into the ITRs or into non-genetically engineered AAV9 capsid proteins by coupling peptide ligands to the capsid surface.

[0187] Those skilled in the art will understand that the AAV virions provided herein can contain capsid proteins of any AAV serotype. In one embodiment, the viral particle contains capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9, which are more suitable for delivery to the CNS (M. Hocquemiller et al., Hum Gene Ther 27(7):478-496(2016)). In a specific embodiment, the viral particle contains a nucleic acid construct of the present invention, wherein the 5TTR and 3TTR sequences of the nucleic acid construct are those of the AAV2 serotype and the capsid proteins are those of the AAV9 serotype.

[0188] Many methods are known for producing rAAV viral particles, including transfection, stable cell line production, and adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J.E. et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. rAAV production cultures for producing rAAV viral particles all require 1) suitable host cells, including, for example, a human-derived cell line such as HeLa, A549, or 293 cells, or an insect-derived cell line such as SF-9 in the case of baculovirus production systems; 2) suitable helper virus functions provided by wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene flanked by AAV ITR sequences; and 5) suitable media and media components to support rAAV production.

[0189] In various embodiments, the host cells described herein contain three components: (1) rep and cap genes, (2) genes providing helper functions, and (3) a transgene flanked by ITRs. The AAV rep gene, AAV cap gene, and genes providing helper functions can be introduced into a cell by incorporating the genes into a vector, e.g., a plasmid, and introducing the vector into a host cell. The rep, cap, and helper function genes can be incorporated into the same or different plasmids. In a preferred embodiment, the AAV rep and cap genes may be incorporated into one plasmid, and the genes providing helper functions are incorporated into another plasmid. The various plasmids (e.g., containing the AAV rep and cap genes, helper functions, or transgenes) for generating a host cell for virion production can be introduced into a cell by using any suitable method known in the art. Examples of transfection methods include, but are not limited to, co-precipitation with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and biolistic transfection. In certain embodiments, plasmids providing the rep and cap genes, helper functions, and transgenes can be introduced into cells simultaneously. In another embodiment, the rep and cap genes and plasmids providing helper functions can be introduced into cells before or after introduction of the plasmid containing the transgene. In an exemplary embodiment, cells are transfected with three plasmids simultaneously (e.g., a triple transfection method): (1) a plasmid containing the transgene, (2) a plasmid containing the AAV rep and cap genes, and (3) a plasmid containing genes providing helper functions. Exemplary host cells can be 293, A549, or HeLa cells.

[0190] In other embodiments, one or more of (1) the AAV rep and cap genes, (2) genes providing helper functions, and (3) a transgene (e.g., a PV-selective regulatory element operably linked to a polynucleotide encoding a therapeutic protein disclosed herein) may be carried by the packaging cell either episomally and / or integrated into the genome of the packaging cell. In one embodiment, the host cell may be a packaging cell in which the AAV rep and cap genes and helper functions are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene. In another embodiment, the host cell is a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene and a plasmid containing the helper functions. In another embodiment, the host cell may be a packaging cell in which the helper functions are stably maintained in the host cell, and the host cell is transiently transfected with a plasmid containing the transgene and a plasmid containing the rep and cap genes. In another embodiment, the host cell can be a producer cell line stably transfected with the rep and cap genes, helper functions, and transgene sequences. Exemplary packaging and producer cells can be derived from 293, A549, or HeLa cells.

[0191] In another embodiment, the producer cell line is an insect cell line (typically Sf9 cells) infected with a baculovirus expression vector that provides the Rep and Cap proteins. This system does not require adenovirus helper genes (Ayuso E, et al., Curr. Gene Ther. 2010, 10:423-436).

[0192] As used herein, the term "cap protein" refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3). Examples of functional activities of a cap protein include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote AAV DNA packaging into capsids (i.e., encapsidation), bind to a cellular receptor, and promote virion entry into a host cell. In principle, any Cap protein can be used in the context of the present invention.

[0193] Cap proteins have been reported to affect host tropism, cell, tissue, or organ specificity, receptor usage, infection efficiency, and immunogenicity of AAV viruses. Therefore, AAV caps for use in rAAVs can be selected, for example, taking into consideration the subject's species (e.g., human or non-human), the subject's immunological status, the subject's suitability for long-term or short-term treatment, or a particular therapeutic application (e.g., treatment of a particular disease or disorder, or delivery to a particular cell, tissue, or organ). In certain embodiments, the cap protein is derived from an AAV from the group consisting of AAV1, AAV2, AAV5, AAV8, and AAV9 serotypes. In an exemplary embodiment, the cap protein is derived from AAV9.

[0194] In some embodiments, an AAV Cap for use in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV caps or its encoding nucleic acid, hi some embodiments, the AAV cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV caps.

[0195] In some embodiments, the AAV cap is chimeric and comprises domains from two, three, four, or more of the aforementioned AAV caps. In some embodiments, the AAV cap is a mosaic of VP1, VP2, and VP3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises two or more of the aforementioned caps.

[0196] In some embodiments, AAV caps for use in rAAV virions are engineered to contain heterologous sequences or other modifications. For example, peptide or protein sequences that confer selective targeting or immune evasion can be engineered into the cap protein. Alternatively, or in addition, the cap can be chemically modified so that the surface of the rAAV is pegylated (i.e., PEGylated), which can promote immune evasion. The cap protein can also be mutagenized (e.g., to eliminate its native receptor binding or to mask immunogenic epitopes).

[0197] The term "rep protein," as used herein, refers to a polypeptide having at least one functional activity of a native AAV rep protein (e.g., rep40, 52, 68, 78). Examples of functional activities of a rep protein include any activity associated with the physiological function of a protein, including promoting DNA replication by recognizing, binding to, and nicking the AAV DNA replication origin, and DNA helicase activity. Additional functions include regulating transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into a host chromosome. In certain embodiments, the AAV rep gene may be from an AAV serotype selected from the group consisting of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrhlO, more preferably AAV1, AAV2, AAV5, AAV8, and AAV9.

[0198] In some embodiments, AAV rep proteins for use in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV rep proteins or their encoding nucleic acids, hi some embodiments, the AAV rep is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV rep proteins.

[0199] The phrases "helper functions" or "helper genes," as used herein, refer to viral proteins on which AAV depends for replication. Helper functions include proteins required for AAV replication, including, but not limited to, proteins involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Helper functions include, but are not limited to, adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, and UL29, and herpesvirus polymerase. In a preferred embodiment, the proteins on which AAV depends for replication are derived from adenovirus.

[0200] In some embodiments, viral proteins on which AAVs depend for replication for use in the methods of the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned viral proteins or their encoding nucleic acids, hi some embodiments, the viral protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned viral proteins.

[0201] Methods for assaying the function of the cap protein, the rep protein, and the viral proteins on which AAV depends for replication are well known in the art.

[0202] Host cells for expressing a transgene of interest can be grown under conditions suitable for the assembly of AAV virions. In certain embodiments, the host cells are grown for a suitable period of time to promote the assembly of AAV virions and their release into the 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 sooner, depending on the culture conditions and the particular host cells used), the level of production generally decreases significantly. Generally, the culture time is measured from the time of virus production. For example, in the case of AAV, virus production generally begins upon supplying helper virus functions in a suitable host cell as described herein. Generally, cells are harvested about 48 to about 100 hours after helper virus infection (or after virus production has begun), preferably about 48 to about 96 hours, preferably about 72 to about 96 hours, preferably about 68 to about 72 hours.

[0203] rAAV production cultures can be grown under a variety of conditions (e.g., over a wide temperature range, for various lengths of time) suitable for the particular host cells being utilized. rAAV production cultures include attachment-dependent cultures, which can be cultured in suitable attachment-dependent vessels such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including spinner flasks, stirred-tank bioreactors, and disposable systems such as the Wave bag system.

[0204] Suitable media known in the art can be used for the production of rAAV virions. These media include, but are not limited to, media manufactured by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM) and Dulbecco's Modified Eagle Medium (DMEM), each of which is incorporated herein by reference in its entirety. In certain embodiments, rAAV production culture media can be supplemented with serum or serum-derived recombinant proteins at levels of 0.5% to 20% (v / v or w / v). Alternatively, rAAV vectors can be produced in serum-free conditions, which may also be referred to as animal-derived product-free media.

[0205] After culturing the host cells to produce AAV virions, the resulting virions can then be harvested and purified. In certain embodiments, AAV virions can be obtained from (1) the host cells of a production culture by lysis of the host cells, and / or (2) from the culture medium of the cells after a period of time, preferably 72 hours, after transfection. rAAV virions can be harvested from spent medium from a production culture, provided that the cells are cultured under conditions that cause the release of rAAV virions from 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.

[0206] After recovery, the rAAV virions can be purified. As used herein, the term "purified" includes preparations of rAAV virions that lack at least some of the other components that may be present in the rAAV virions naturally or when initially prepared. Thus, for example, purified rAAV virions can be prepared using isolation techniques to enrich them from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in various ways, such as by the percentage of DNase-resistant particles (DRPs) or genome copies (gc) present in solution, or by infectivity, or it can be measured with respect to secondary interfering substances that may be present in the source mixture, such as contaminants including production culture contaminants or in-process contaminants, including helper viruses, media components, and the like.

[0207] In certain embodiments, rAAV production culture harvests may be clarified to remove host cell debris, hi some embodiments, production culture harvests may be clarified using a variety of standard techniques, such as centrifugation or filtration through filters with a pore size of 0.2 pm or greater (e.g., cellulose acetate filters or a series of depth filters).

[0208] In certain embodiments, the rAAV production culture harvest is further treated with Benzonase™ to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase™ digestion is carried out under standard conditions, e.g., a final concentration of 1-2.5 units / ml of Benzonase™, at temperatures ranging from room temperature to 37°C, for 30 minutes to several hours.

[0209] In certain embodiments, rAAV virions can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. Methods for purifying rAAV particles can be found, for example, in Xiao et al. (1998) Journal of Virology 72:2224-2232, U.S. Patent Nos. 6,989,264 and 8,137,948, and WO 2010 / 148143.

[0210] In certain embodiments, purified AAV virions can be dialyzed against PBS, filtered, and stored at −80° C. The titer of the viral genome can be determined by quantitative PCR using linearized plasmid DNA as a standard curve (see, e.g., Lock M, et al., Hum. Gene Ther. 2010;21:1273-1285).

[0211] Pharmaceutical Composition In certain embodiments, the present application provides a composition comprising a nucleic acid cassette, e.g., an expression cassette, e.g., an rAAV comprising the above-described expression cassette, or RNA encoded thereby, e.g., mRNA or ncRNA, and a pharmaceutically acceptable carrier. (Note that the RNA may be produced synthetically and therefore not necessarily via transcription of the nucleic acid cassette described herein.) In some embodiments, virions containing the cassette and a pharmaceutically acceptable carrier are provided. In exemplary embodiments, such compositions are suitable for gene therapy applications. Pharmaceutical compositions are preferably sterile and stable under the conditions of manufacture and storage. Sterile solutions can be achieved, for example, by filtration through a sterile filtration membrane.

[0212] Acceptable carriers and excipients in pharmaceutical compositions are preferably non-toxic to recipients at the dosages and concentrations used. Acceptable carriers and excipients include buffers such as phosphate, citric acid, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol. The pharmaceutical compositions of the present disclosure can be administered parenterally in the form of an injectable formulation. Injectable pharmaceutical compositions can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water and physiological saline.

[0213] The pharmaceutical compositions of the present disclosure can be formulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules. The pharmaceutical compositions of the present disclosure can also be formulated with other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Pharmaceutical compositions for gene therapy can be in an acceptable diluent or can comprise a slow-release matrix in which the gene delivery vehicle is embedded.

[0214] The pharmaceutical compositions provided herein may be formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration. Pharmaceutical compositions may also be formulated for or administered via nasal, spray, oral, aerosol, rectal, or vaginal administration. In one embodiment, the pharmaceutical compositions provided herein are administered to the CNS or cerebrospinal fluid (CSF), for example, by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. The tissue target may be specific, e.g., the CNS, or it may be a combination of several tissues, e.g., muscle and CNS tissue. Exemplary tissues or other targets may include the liver, skeletal muscle, cardiac muscle, fat deposits, kidney, lung, vascular endothelium, epithelium, hematopoietic cells, cancer cells, CNS, and / or CSF. In a preferred embodiment, the pharmaceutical compositions provided herein are administered by CNS or CSF injection, for example, intraparenchymal, intrathecal, intracisternal, or intraventricular injection, any one or more of which methods can be used to administer the pharmaceutical compositions of the present disclosure.

[0215] In certain embodiments, the pharmaceutical compositions provided herein comprise an “effective amount” or a “therapeutically effective amount.” As used herein, such an amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.

[0216] The dosage of the pharmaceutical composition of the present disclosure depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics (e.g., age, weight, overall health). The dosage can be adjusted to provide an optimal therapeutic response. Typically, the dosage will be an amount that effectively treats the disease without inducing significant toxicity. In certain embodiments, the pharmaceutical composition may be formulated into a unit dose as needed.

[0217] The pharmaceutical composition of the present disclosure can be administered to a subject in need thereof when medically necessary. In an exemplary embodiment, a single administration is sufficient. In one embodiment, the pharmaceutical composition is suitable for use in a human subject and is administered by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. In one embodiment, the pharmaceutical composition is delivered via a peripheral vein by bolus injection. In another embodiment, the pharmaceutical composition is delivered via a peripheral vein by infusion.

[0218] In another aspect, the present application further provides kits comprising the nucleic acid molecules, vectors, host cells, virions, or pharmaceutical compositions described herein in one or more containers. The kits may include instructions or packaging materials describing how to administer the nucleic acid molecules, vectors, host cells, or virions contained within the kit to a patient. The containers of the kits may be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kits may include one or more ampoules or syringes containing the nucleic acid molecules, vectors, host cells, virions, or pharmaceutical compositions in a suitable liquid or solution form.

[0219] Treatment methods The nucleic acid cassettes, expression cassettes, expression vectors, viral vectors, viral particles, or pharmaceutical compositions of the present invention can be used to treat various disorders, such as neurological disorders. In some embodiments, the chemical compounds, proteins, or nucleic acid molecules of the present invention can be used to treat or ameliorate one or more symptoms associated with a genetic mutation or an underexpressed or non-expressed gene in a subject. In certain embodiments, the treatment can be treating a subject via gene therapy, which can be administered directly to a subject in need thereof (e.g., directly to the CNS) or systemically via injection and / or infusion. The therapy can be formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration, or via nasal, spray, oral, aerosol, rectal, or vaginal administration, e.g., by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. The tissue target can be specific, e.g., the CNS, or can be a combination of several tissues.

[0220] In any embodiment herein, the target cell may be a neuron, muscle cell, cardiac cell, skin cell, immune cell, hematopoietic cell, cancer cell, pancreatic cell, or kidney cell. In some cases, the target cell may be a neuron, such as a cerebral cell, brain stem cell, hippocampal cell, or cerebellar cell. For example, in some embodiments, the neuron is a GABAergic cell, such as a parvalbumin-expressing cell. In some cases, the target cell may be a CNS cell, such as an excitatory neuron, a dopaminergic neuron, a glial cell, an ependymal cell, an oligodendrocyte, an astrocyte, a microglia, a motor neuron, a vascular cell, a GABAergic neuron, or a non-GABAergic neuron (e.g., a cell that does not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP), a non-PV neuron (e.g., a GABAergic neuron that does not express parvalbumin), or another CNS cell (e.g., a CNS cell type that does not express PV, GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP).

[0221] In any embodiment, the therapy may be used to increase the production or expression of a target protein in cells such as GABA neurons or parvalbumin neurons.

[0222] In certain embodiments, the treatments provided herein do not result in adverse reactions in a subject. Treatment with the nucleic acid molecules, expression vectors, pharmaceutical compositions, or virions described herein may cause fewer or less severe adverse reactions in a subject than treatment with a similar gene therapy comprising the same transgene linked to a non-parvalbumin neuron-selective regulatory element.

[0223] Sequence Listing Table 4 below provides specific sequences that may be referenced in other parts of this disclosure.

[0224] SEQ ID NOs: 1-10 and 43-48 are DRG detargeting sequences that may be present in RNA transcripts encoded by the nucleic acid cassettes of the present disclosure.

[0225] SEQ ID NOs: 57-62, 64-71, 110, and 112 are liver detargeting sequences that may be present in RNA transcripts encoded by the nucleic acid cassettes of the present disclosure.

[0226] Table 4 also provides DNA versions of the RNA sequences (which may be present in the cassette itself), as well as the sequences of specific miRNAs and pre-miRNAs that may bind to these RNA sequences.

[0227] Additional sequences are also listed in Table 4, including control sequences and sequences of several exemplary combinations of sequences that detarget expression in both DRG and liver cells.

[0228] [Table 4-1]

[0229] [Table 4-2]

[0230] [Table 4-3]

[0231] [Table 4-4]

[0232] [Table 4-5]

[0233] [Table 4-6]

[0234] [Table 4-7]

[0235] [Table 4-8]

[0236] [Table 4-9]

[0237] Embodiment Embodiments of the present disclosure include nucleic acid cassettes encoding transgenes comprising one or more DRG detargeting elements, one or more liver detargeting elements, or both, RNA transcripts derived therefrom, and isolated and / or synthetic RNA molecules comprising one or more DRG detargeting elements, one or more liver detargeting elements, or both. Embodiments further include methods of making and using such nucleic acid cassettes and RNA molecules, and methods of using them as therapeutic agents. The following embodiments are intended to further illustrate certain aspects of the present disclosure, but are not intended to limit its scope.

[0238] Embodiment 1. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the sequence of: (i) any of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0239] Embodiment 2. The nucleic acid cassette of embodiment 1, wherein the RNA transcript comprises a sequence of at least 15 contiguous nucleotides of any of SEQ ID NOs: 1-10 and 43-48, which reduces expression in dorsal root ganglion (DRG) cells.

[0240] Embodiment 3. The nucleic acid cassette of embodiment 1 or 2, wherein the RNA transcript further comprises a second sequence of (i), (ii), or (iii).

[0241] Embodiment 4. The nucleic acid cassette of embodiment 3, wherein the RNA transcript further comprises a third sequence of (i), (ii), or (iii).

[0242] Embodiment 5. The nucleic acid cassette of embodiment 4, wherein the RNA transcript further comprises a fourth sequence of (i), (ii), or (iii).

[0243] Embodiment 6. The nucleic acid cassette of any one of embodiments 1 to 5, wherein the RNA transcript comprises two or more copies of the sequence of (i), (ii), or (iii).

[0244] Embodiment 7. The nucleic acid cassette of embodiment 6, wherein the RNA transcript comprises three or more copies of the sequence of (i), (ii), or (iii).

[0245] Embodiment 8. The nucleic acid cassette of embodiment 7, wherein the RNA transcript comprises four or more copies of the sequence of (i), (ii), or (iii).

[0246] Embodiment 9. The nucleic acid cassette of embodiment 8, wherein the RNA transcript comprises five or more copies of the sequence of (i), (ii), or (iii).

[0247] Embodiment 6. A nucleic acid cassette described in any one of embodiments 1 to 5, wherein the sequence of (i), (ii), or (iii) provides binding sites 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.

[0248] Embodiment 11. The nucleic acid cassette of any one of embodiments 1 to 10, wherein the RNA transcript is an mRNA, and optionally, the sequence of (i), (ii), or (iii) is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0249] Embodiment 12. The nucleic acid cassette of embodiment 11, wherein the sequence of (i), (ii), or (iii) is located in the 3'UTR region of the mRNA.

[0250] Embodiment 13. The nucleic acid cassette of embodiment 11, wherein the sequence of (i), (ii), or (iii) is located in the 5'UTR region of the mRNA.

[0251] Embodiment 14. The nucleic acid cassette of embodiment 11, wherein the sequence of (i), (ii), or (iii) is located in an intron of the mRNA.

[0252] Embodiment 15. The nucleic acid cassette of any one of embodiments 1 to 14, wherein the nucleic acid cassette does not occur in nature.

[0253] Embodiment 16. The nucleic acid cassette of any one of embodiments 1 to 15, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0254] Embodiment 17. The nucleic acid cassette of embodiment 16, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0255] Embodiment 18. The nucleic acid cassette of any one of embodiments 1 to 17, wherein the nucleic acid cassette comprises an enhancer.

[0256] Embodiment 19. The nucleic acid cassette of any one of embodiments 1 to 18, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder, and optionally, the RNA transcript is an mRNA encoding a therapeutic protein associated with a neurological disease or disorder.

[0257] Embodiment 20. The neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome). 20. The nucleic acid cassette of embodiment 19, wherein the nucleic acid cassette is selected from the group consisting of juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0258] Embodiment 21. The therapeutic protein encoded by the mRNA is selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, PCDH19, 21. The nucleic acid cassette of embodiment 19 or 20, wherein the nucleic acid cassette is selected from: (i) a protein encoded by a gene selected from 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 expression of a gene from (i).

[0259] Embodiment 22. (a) the RNA transcript comprises a sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a CNS-selective promoter; (c) The nucleic acid cassette according to any one of embodiments 1 to 21, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0260] Embodiment 23. (a) the RNA transcript comprises a sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally (c) Therapeutic RNA transcripts are: 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, 23. The nucleic acid cassette of any one of embodiments 1 to 22, which is a therapeutic protein encoded by a gene selected from PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) an mRNA encoding a transcription factor that regulates expression of a gene from (i).

[0261] Embodiment 24. The nucleic acid cassette of any one of embodiments 1 to 23, wherein the sequence of (i), (ii), or (iii) results in decreased expression of the RNA transcript in a DRG cell compared to expression of the RNA transcript in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) results in decreased expression of a polypeptide encoded by the mRNA in a DRG cell compared to expression of the polypeptide in a DRG cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0262] Embodiment 25. The nucleic acid cassette of embodiment 24, wherein the sequence of (i), (ii), or (iii) results in a reduction in expression of the RNA transcript and / or polypeptide encoded by the mRNA in a DRG cell at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to the expression of the RNA transcript or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

[0263] Embodiment 26. The nucleic acid cassette of embodiment 24 or 25, wherein the sequence of (i), (ii), or (iii) results in reduced expression of the RNA transcript and / or polypeptide encoded by the mRNA in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript and / or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

[0264] Embodiment 27. The nucleic acid cassette of any one of embodiments 1 to 26, wherein the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the RNA transcript in the target cell compared to the expression of the RNA transcript in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the polypeptide encoded by the mRNA in the target cell compared to the expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0265] Embodiment 28. The nucleic acid cassette of embodiment 27, wherein the sequence of (i), (ii), or (iii) does not reduce expression of the RNA transcript and / or the polypeptide encoded thereby (if the RNA transcript is mRNA) in a target cell compared to expression of the polypeptide in the target cell from an equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

[0266] Embodiment 29. The nucleic acid cassette of embodiment 27, wherein the sequence of (i), (ii) or (iii) results in expression of an RNA transcript and / or a polypeptide encoded thereby in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the polypeptide in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

[0267] Embodiment 30. The nucleic acid cassette of any one of embodiments 27 to 29, wherein the target cell is a neuronal cell.

[0268] Embodiment 31. The nucleic acid cassette of embodiment 30, wherein the neuronal cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

[0269] Embodiment 32. The nucleic acid cassette of embodiment 31, wherein the neuronal cell is a GABAergic cell.

[0270] Embodiment 33 The nucleic acid cassette of embodiment 32, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0271] Embodiment 34. A nucleic acid cassette according to any one of embodiments 1 to 33, wherein the nucleic acid cassette is a linear construct or a vector.

[0272] Embodiment 35. The nucleic acid cassette of embodiment 34, wherein the vector is a plasmid.

[0273] Embodiment 36. The nucleic acid cassette of embodiment 34, wherein the vector is a viral vector.

[0274] Embodiment 37. The nucleic acid cassette of embodiment 36, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0275] Embodiment 38. The nucleic acid cassette of embodiment 37, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0276] Embodiment 39. The nucleic acid cassette of embodiment 37 or 38, wherein the AAV is scAAV.

[0277] Embodiment 40. The nucleic acid cassette of embodiment 36, wherein the viral vector is a lentiviral vector.

[0278] Embodiment 41. RNA having a sequence encoded by the nucleic acid cassette of any one of embodiments 1 to 40.

[0279] Embodiment 42. A nucleic acid cassette comprising a transgene encoding an RNA transcript, wherein the RNA transcript is a therapeutic RNA transcript, e.g., an mRNA encoding a therapeutic protein, and comprises a binding site for a miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p, or a complement thereof.

[0280] Embodiment 43. The nucleic acid cassette of embodiment 42, 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 complements.

[0281] Embodiment 44. The nucleic acid cassette of embodiment 42, 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 complements.

[0282] Embodiment 45. The nucleic acid cassette of embodiment 42, comprising two binding sites for a miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p, or its complement.

[0283] Embodiment 46. The nucleic acid cassette of embodiment 42, comprising three binding sites for a miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p, or its complement.

[0284] Embodiment 47. The nucleic acid cassette of embodiment 42, comprising four binding sites for a miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p, or its complement.

[0285] Embodiment 48. The nucleic acid cassette of embodiment 42, comprising more than four binding sites for a miRNA selected from mir-196b-5p, mir-10b-5p, mir-24-2-5p, hsa-mir-183-3p, mir-196a-5p, and mir-494-3p, or a complement thereof.

[0286] Embodiment 49. The nucleic acid cassette according to any one of embodiments 42 to 48, wherein the miRNA is mir-196b-5p.

[0287] Embodiment 50. The nucleic acid cassette of any one of embodiments 42 to 48, wherein the miRNA is mir-10b-5p.

[0288] Embodiment 51. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the miRNA is mir-24-2-5p.

[0289] Embodiment 52. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the miRNA is hsa-mir-183-3p.

[0290] Embodiment 53. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the miRNA is mir-196a-5p.

[0291] Embodiment 54. The nucleic acid cassette according to any one of embodiments 42 to 48, wherein the miRNA is mir-494-3p.

[0292] Embodiment 55. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the RNA transcript comprises multiple copies of the same miRNA binding site.

[0293] Embodiment 56. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the RNA transcript comprises a plurality of different miRNA binding sites.

[0294] Embodiment 57. A nucleic acid cassette according to any one of embodiments 42 to 48, wherein the RNA transcript comprises multiple copies of the same miRNA binding site and multiple different miRNA binding sites.

[0295] Embodiment 58. A nucleic acid cassette according to any one of embodiments 42 to 57, wherein the RNA transcript further comprises a sequence of at least 10 consecutive nucleotides of any of SEQ ID NOs: 1 to 10 and 43 to 48, which reduces expression in DRG cells.

[0296] Embodiment 59. A nucleic acid cassette according to any one of embodiments 42 to 57, wherein the RNA transcript further comprises at least two sequences of at least 20 consecutive nucleotides of any of SEQ ID NOs: 1 to 10 and 43 to 48, which sequences reduce expression in DRG cells.

[0297] Embodiment 60. The nucleic acid cassette of any one of embodiments 42 to 59, wherein the RNA transcript is an mRNA and the miRNA binding site is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0298] Embodiment 61. The nucleic acid cassette of embodiment 60, wherein the miRNA binding site is located in the 3'UTR region of the mRNA.

[0299] Embodiment 62. The nucleic acid cassette of embodiment 60, wherein the miRNA binding site is located in the 5'UTR region of the mRNA.

[0300] Embodiment 63. The nucleic acid cassette of embodiment 60, wherein the miRNA binding site is located in an intron of the mRNA.

[0301] Embodiment 64. The nucleic acid cassette of any one of embodiments 42 to 63, wherein the nucleic acid cassette does not occur in nature.

[0302] Embodiment 65. The nucleic acid cassette of any one of embodiments 42 to 64, wherein the nucleic acid cassette comprises a promoter.

[0303] Embodiment 66. The nucleic acid cassette of any one of embodiments 42 to 65, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0304] Embodiment 67. The nucleic acid cassette of embodiment 66, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0305] Embodiment 68. A nucleic acid cassette according to any one of embodiments 42 to 67, wherein the nucleic acid cassette comprises an enhancer.

[0306] Embodiment 69. The nucleic acid cassette of any one of embodiments 42 to 68, wherein the therapeutic RNA transcript is for treating a neurological disease or disorder, and optionally, the therapeutic RNA transcript is an mRNA encoding a therapeutic protein associated with a neurological disease or disorder.

[0307] Embodiment 70. The therapeutic protein is selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, 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 expression of a gene from (i).

[0308] Embodiment 71. (a) the RNA transcript is (i) any one of SEQ ID NOs: 1 to 10 and 43 to 48; (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a CNS-selective promoter; (c) The nucleic acid cassette of any one of embodiments 42 to 70, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0309] Embodiment 72. (a) the RNA transcript comprises a sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally (c) RNA transcripts were identified for: 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, and POL 72. The nucleic acid cassette of embodiment 71, which is a therapeutic protein encoded by a gene selected from G1, 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) an mRNA encoding a transcription factor that regulates expression of a gene from (i).

[0310] Embodiment 73. A nucleic acid cassette described in any one of embodiments 42 to 72, wherein the miRNA binding site results in reduced expression of the RNA transcript and / or the polypeptide encoded thereby (if the RNA transcript is mRNA) in a DRG cell compared to expression of the polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not contain the miRNA binding site.

[0311] Embodiment 74. A nucleic acid cassette described in any one of embodiments 42 to 73, wherein the miRNA binding site results in a reduction in expression of the RNA transcript and / or the polypeptide encoded thereby in DRG cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to the expression of the polypeptide in DRG cells from an equivalent RNA transcript that does not contain the miRNA binding site.

[0312] Embodiment 75. The nucleic acid cassette of any one of embodiments 42 to 74, wherein the miRNA binding site results in reduced expression of a polypeptide encoded by the RNA transcript in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the miRNA binding site.

[0313] Embodiment 76. The nucleic acid cassette of any one of embodiments 42 to 75, wherein the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the RNA transcript in the target cell compared to the expression of the RNA transcript in the target cell from an equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the polypeptide encoded by the mRNA in the target cell compared to the expression of the polypeptide in the target cell from an equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0314] Embodiment 77. The nucleic acid cassette of embodiment 76, wherein the sequence of (i), (ii), or (iii) does not reduce expression of an RNA transcript in a target cell compared to expression of the RNA transcript in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) does not reduce expression of a polypeptide encoded by the mRNA in the target cell compared to expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0315] Embodiment 78. The sequence of (i), (ii), or (iii) results in expression of an RNA transcript in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii), and wherein the RNA transcript is an mRNA, (i) 77. The nucleic acid cassette of embodiment 76, wherein the sequence of (i), (ii), or (iii) results in expression of a polypeptide encoded by the mRNA in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the polypeptide in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0316] Embodiment 79. The nucleic acid cassette of any one of embodiments 42 to 78, wherein the target cell is a neuronal cell.

[0317] Embodiment 80. The nucleic acid cassette of embodiment 79, wherein the neuronal cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0318] Embodiment 81. The nucleic acid cassette of embodiment 80, wherein the neuronal cell is a GABAergic cell.

[0319] Embodiment 82 The nucleic acid cassette of embodiment 81, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0320] Embodiment 83. The nucleic acid cassette of any one of embodiments 42 to 82, wherein the nucleic acid cassette is a linear construct or a vector.

[0321] Embodiment 84. The nucleic acid cassette of embodiment 83, wherein the vector is a plasmid.

[0322] Embodiment 85. The nucleic acid cassette of embodiment 83, wherein the vector is a viral vector.

[0323] Embodiment 86. The nucleic acid cassette of embodiment 85, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0324] Embodiment 87. The nucleic acid cassette of embodiment 86, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0325] Embodiment 88. The nucleic acid cassette of embodiment 86 or 87, wherein the AAV is a scAAV.

[0326] Embodiment 89. The nucleic acid cassette of embodiment 85, wherein the viral vector is a lentiviral vector.

[0327] Embodiment 90. RNA encoded by the nucleic acid cassette of any one of embodiments 42 to 89.

[0328] Embodiment 91. The nucleic acid cassette of any one of embodiments 42 to 89, wherein the RNA transcript is an mRNA encoding a polypeptide.

[0329] Embodiment 92. The nucleic acid cassette of embodiment 91, wherein the polypeptide is a therapeutic protein.

[0330] Embodiment 93. RNA having a sequence encoded by the nucleic acid cassette of any one of embodiments 42 to 92.

[0331] Embodiment 94. A method of reducing 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 an mRNA) while maintaining expression of the therapeutic RNA transcript or therapeutic protein in a target tissue, comprising comprising comprising in the therapeutic RNA transcript a sequence of: (i) any of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0332] Embodiment 95. The method of embodiment 94, wherein the therapeutic RNA transcript further comprises a second sequence of (i), (ii), or (iii).

[0333] Embodiment 96 The method of embodiment 94, wherein the therapeutic RNA transcript further comprises a third sequence of (i), (ii), or (iii).

[0334] Embodiment 97. The method of embodiment 94, wherein the therapeutic RNA transcript further comprises a fourth sequence of (i), (ii), or (iii).

[0335] Embodiment 98. The method of embodiment 94, wherein the therapeutic RNA transcript comprises five or more sequences of (i), (ii), or (iii).

[0336] Embodiment 99. The method of any one of embodiments 94 to 98, wherein the therapeutic RNA transcript comprises two or more copies of the sequence of (i), (ii), or (iii).

[0337] Embodiment 100. The method of any one of embodiments 94-99, wherein the therapeutic RNA transcript comprises three or more copies of the sequence of (i), (ii), or (iii).

[0338] Embodiment 101. The method of any one of embodiments 94 to 100, wherein the therapeutic RNA transcript comprises four or more copies of the sequence of (i), (ii), or (iii).

[0339] Embodiment 102. The method of any one of embodiments 94 to 101, wherein the therapeutic RNA transcript comprises five or more copies of the sequence of (i), (ii), or (iii).

[0340] Embodiment 103. The method of any one of embodiments 94-102, wherein the therapeutic RNA transcript comprises at least 10 contiguous nucleotides of any of SEQ ID NOs: 1-10 and 43-48, which have reduced expression in DRG cells.

[0341] Embodiment 104. The method of any one of embodiments 94 to 103, wherein the therapeutic RNA transcript is an mRNA and the sequence of (i), (ii), or (iii) is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0342] Embodiment 105. The method of embodiment 104, wherein the sequence of (i), (ii) or (iii) is located in the 3'UTR region of the mRNA.

[0343] Embodiment 106. The method of embodiment 104, wherein the sequence of (i), (ii) or (iii) is located in the 5'UTR region of the mRNA.

[0344] Embodiment 107. The method of embodiment 104, wherein the sequence of (i), (ii) or (iii) is located in an intron of the mRNA.

[0345] Embodiment 108. The method of any one of embodiments 94 to 107, wherein the method comprises administering to the subject a nucleic acid cassette encoding a therapeutic RNA transcript.

[0346] Embodiment 109. The method of any one of embodiments 94 to 108, wherein the administering is systemic administration.

[0347] Embodiment 110. The method of any one of embodiments 94 to 108, wherein the administering is topical administration.

[0348] Embodiment 111. The method of embodiment 110, wherein the nucleic acid is administered locally to brain or CNS tissue.

[0349] Embodiment 112. The method of embodiment 110 or 111, wherein administering is by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0350] Embodiment 113. The method of any one of embodiments 94 to 112, wherein the therapeutic RNA transcript is for treating a neurological disease or disorder.

[0351] Embodiment 114. The RNA transcript is selected from the group consisting of: (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG 114. The method of embodiment 113, wherein the mRNA encodes a therapeutic protein selected from (i), (ii) a protein encoded by a gene selected from: 1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (iii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates expression of a gene from (i).

[0352] Embodiment 115. The method of any one of embodiments 108 to 114, wherein the subject has a neurological disease or disorder.

[0353] Embodiment 116. The subject is a patient with Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome), young adulthood. 116. The method of embodiment 115, wherein the patient has chronic myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0354] Embodiment 117. The method of any one of embodiments 108 to 116, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0355] Embodiment 118. The method of embodiment 117, wherein the CNS-selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0356] Embodiment 119. (a) the therapeutic RNA transcript comprises the sequence of (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a CNS-selective promoter; (c) The method of any one of embodiments 108 to 118, wherein the therapeutic RNA transcript is for treating a neurological disease or disorder.

[0357] Embodiment 120. (a) the therapeutic RNA transcript comprises the sequence of (i) any of SEQ ID NOS: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally (c) The therapeutic RNA transcript is an mRNA, and the mRNA is selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, PCDH19, P 120. The method of embodiment 119, wherein the therapeutic protein is encoded by a gene selected from LCB1, 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 expression of a gene from (i).

[0358] Embodiment 121. The method of any one of embodiments 94 to 120, wherein the sequence of (i), (ii), or (iii) results in a reduction in expression of the RNA transcript in DRG cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to the expression of the RNA transcript in DRG cells from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) results in a reduction in expression of the protein encoded by the mRNA in DRG cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to the expression of the protein in DRG cells from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0359] Embodiment 122. The sequence of (i), (ii), or (iii) results in reduced expression of an RNA transcript in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), wherein the RNA transcript is an mRNA, 122. The method of any one of embodiments 94-121, wherein the sequence of (ii) or (iii) results in reduced expression of the protein encoded by the mRNA 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 expression of the protein in DRG cells from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0360] Embodiment 123. The method of any one of embodiments 94 to 122, wherein the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the RNA transcript in the target cell compared to the expression of the RNA transcript in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), and, if the RNA transcript is an mRNA, the sequence of (i), (ii), or (iii) does not result in a significantly reduced expression of the protein encoded by the mRNA in the target cell compared to the expression of the protein in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0361] Embodiment 124. The method of embodiment 123, wherein the sequence of (i), (ii), or (iii) does not reduce expression of the RNA transcript and / or the protein encoded thereby (i.e., where the RNA transcript is mRNA) in the target cell compared to expression of the RNA transcript and / or protein in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0362] Embodiment 125. The sequence of (i), (ii), or (iii) results in expression of an RNA transcript in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii), wherein the RNA transcript is an mRNA, 125. The method of any one of embodiments 94-124, wherein the sequence of (ii) or (iii) results in expression of the protein encoded by the mRNA in the target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the protein in the target cell from an otherwise equivalent mRNA that does not comprise the sequence of (i), (ii), or (iii).

[0363] Embodiment 126. The method of embodiment 124 or 125, wherein the target cell is a neuronal cell.

[0364] Embodiment 127. The method of embodiment 126, wherein the neural cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

[0365] Embodiment 128. The method of embodiment 127, wherein the neuronal cells are GABAergic cells.

[0366] Embodiment 129 The method of embodiment 128, wherein the GABAergic cells are parvalbumin-expressing cells.

[0367] Embodiment 130. The method of any one of embodiments 94 to 129, wherein the RNA transcript is expressed from a nucleic acid cassette.

[0368] Embodiment 131. The method of embodiment 130, wherein the nucleic acid cassette is a linear construct.

[0369] Embodiment 132. The method of embodiment 130, wherein the nucleic acid cassette is a vector.

[0370] Embodiment 133. The method of embodiment 132, wherein the vector is a plasmid.

[0371] Embodiment 134. The method of embodiment 132, wherein the vector is a viral vector.

[0372] Embodiment 135. The method of embodiment 134, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0373] Embodiment 136. The method of embodiment 135, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0374] Embodiment 137. The method of embodiment 135 or 136, wherein the AAV is scAAV.

[0375] Embodiment 138. The method of embodiment 134, wherein the viral vector is a lentiviral vector.

[0376] Embodiment 139. The method of any one of embodiments 132 to 138, wherein the method comprises administering a vector to a subject.

[0377] Embodiment 140. The method of any one of embodiments 132 to 138, further comprising administering a vector to a subject.

[0378] Embodiment 141. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the sequence of (i) any of SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0379] Embodiment 142. The nucleic acid cassette of embodiment 141, wherein the sequence reduces expression of an RNA transcript in liver cells.

[0380] Embodiment 143. The nucleic acid cassette of embodiment 141 or 142, wherein the RNA transcript further comprises a second sequence of (i), (ii), or (iii).

[0381] Embodiment 144. The nucleic acid cassette of embodiment 143, wherein the RNA transcript further comprises a third sequence of (i), (ii), or (iii).

[0382] Embodiment 145. The nucleic acid cassette of embodiment 144, wherein the RNA transcript further comprises a fourth sequence of (i), (ii), or (iii).

[0383] Embodiment 146. The nucleic acid cassette of any one of embodiments 141 to 145, wherein the RNA transcript comprises two or more copies of the sequence of (i), (ii), or (iii).

[0384] Embodiment 147. The nucleic acid cassette of embodiment 146, wherein the RNA transcript comprises three or more copies of the sequence of (i), (ii), or (iii).

[0385] Embodiment 148. The nucleic acid cassette of embodiment 147, wherein the RNA transcript comprises four or more copies of the sequence of (i), (ii), or (iii).

[0386] Embodiment 149. The nucleic acid cassette of embodiment 148, wherein the RNA transcript comprises five or more copies of the sequence of (i), (ii), or (iii).

[0387] Embodiment 150. The nucleic acid cassette of any one of embodiments 141 to 149, wherein the RNA transcript is an mRNA and the sequence is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0388] Embodiment 151. The nucleic acid cassette of embodiment 150, wherein the sequence is located in the 3'UTR region of the mRNA.

[0389] Embodiment 152. The nucleic acid cassette of embodiment 150, wherein the sequence is located in the 5'UTR region of the mRNA.

[0390] Embodiment 153. The nucleic acid cassette of embodiment 150, wherein the sequence is located in an intron of the mRNA.

[0391] Embodiment 154. The nucleic acid cassette of any one of embodiments 141 to 153, wherein the nucleic acid cassette does not occur in nature.

[0392] Embodiment 155. The nucleic acid cassette of any one of embodiments 141 to 154, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0393] Embodiment 156. The nucleic acid cassette of embodiment 155, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0394] Embodiment 157. The nucleic acid cassette of any one of embodiments 141 to 156, wherein the nucleic acid cassette comprises an enhancer.

[0395] Embodiment 158. The nucleic acid cassette of any one of embodiments 141 to 157, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0396] Embodiment 159. The neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome). , juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0397] Embodiment 160. The therapeutic RNA transcript is an mRNA encoding a therapeutic protein, the therapeutic protein being selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL 2, 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 expression of a gene from (i).

[0398] Embodiment 161. (a) the RNA transcript comprises the sequence, (b) the nucleic acid cassette comprises a CNS-selective promoter; (c) The nucleic acid cassette of any one of embodiments 141 to 160, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0399] Embodiment 162. (a) the RNA transcript comprises the sequence, (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally (c) The RNA transcript is an mRNA, and the mRNA is selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, and GAB RAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, Myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, 162. The nucleic acid cassette of any one of embodiments 141 to 161, encoding a therapeutic protein encoded by a gene selected from 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 expression of a gene from (i).

[0400] Embodiment 163. The nucleic acid cassette of any one of embodiments 141 to 162, wherein the sequence results in decreased expression of the RNA transcript and / or the polypeptide encoded thereby (i.e., where the RNA transcript is mRNA) in a liver cell compared to expression of the RNA transcript and / or the polypeptide in a liver cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0401] Embodiment 164. The nucleic acid cassette of embodiment 163, wherein the sequence results in a reduction in expression of the RNA transcript and / or the polypeptide encoded thereby in a liver cell at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to the expression of the RNA transcript and / or polypeptide in a liver cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0402] Embodiment 165. The nucleic acid cassette of embodiment 163 or 164, wherein the sequence results in reduced expression of the RNA transcript and / or the polypeptide encoded thereby in liver 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 expression of the RNA transcript and / or polypeptide in liver cells from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0403] Embodiment 166. The nucleic acid cassette of any one of embodiments 141 to 165, wherein the sequence does not result in a significant decrease in expression of the RNA transcript and / or the polypeptide encoded thereby in a target cell compared to expression of the RNA transcript and / or the polypeptide in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0404] Embodiment 167. The nucleic acid cassette of embodiment 166, wherein the sequence does not reduce expression of the RNA transcript and / or the polypeptide encoded thereby in a target cell compared to expression of the RNA transcript and / or the polypeptide in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0405] Embodiment 168. The nucleic acid cassette of embodiment 167, wherein the sequence results in expression of an RNA transcript and / or a polypeptide encoded thereby in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript and / or polypeptide in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0406] Embodiment 169. A nucleic acid cassette according to any one of embodiments 166 to 168, wherein the target cell is a neuronal cell.

[0407] Embodiment 170. The nucleic acid cassette of embodiment 169, wherein the neuronal cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0408] Embodiment 171. The nucleic acid cassette of embodiment 169, wherein the neuronal cell is a GABAergic cell.

[0409] Embodiment 172. The nucleic acid cassette of embodiment 170, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0410] Embodiment 173. The nucleic acid cassette of any one of embodiments 141 to 172, wherein the nucleic acid cassette is a linear construct or a vector.

[0411] Embodiment 174. The nucleic acid cassette of embodiment 173, wherein the vector is a plasmid.

[0412] Embodiment 175. The nucleic acid cassette of embodiment 173, wherein the vector is a viral vector.

[0413] Embodiment 176. The nucleic acid cassette of embodiment 175, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0414] Embodiment 177. The nucleic acid cassette of embodiment 176, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0415] Embodiment 178. The nucleic acid cassette of embodiment 176 or 177, wherein the AAV is a scAAV.

[0416] Embodiment 179. The nucleic acid cassette of embodiment 173, wherein the viral vector is a lentiviral vector.

[0417] Embodiment 180. An RNA having a sequence encoded by the nucleic acid cassette of any one of embodiments 141 to 180.

[0418] Embodiment 181. A method of reducing hepatic expression of a therapeutic RNA transcript and / or protein encoded thereby (i.e., where the RNA transcript is mRNA) while maintaining expression of the RNA transcript and / or protein in a target tissue, comprising comprising a sequence of: (i) any of SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii).

[0419] Embodiment 182. The method of embodiment 181, wherein the RNA transcript is an mRNA and the sequence is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0420] Embodiment 183. The method of embodiment 182, wherein the sequence is located in the 3'UTR region of the mRNA.

[0421] Embodiment 184. The method of embodiment 182, wherein the sequence is located in the 5'UTR region of the mRNA.

[0422] Embodiment 185. The method of embodiment 182, wherein the sequence is located in an intron of the mRNA.

[0423] Embodiment 186. The method of any one of embodiments 181 to 185, wherein the method comprises administering to the subject a nucleic acid cassette encoding an RNA transcript.

[0424] Embodiment 187. The method of embodiment 186, wherein the administering is systemic administration.

[0425] Embodiment 188. The method of embodiment 186, wherein the administering is topical administration.

[0426] Embodiment 189. The method of embodiment 188, wherein the nucleic acid is administered locally to brain or CNS tissue.

[0427] Embodiment 190. The method of embodiment 188 or 189, wherein administering is by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0428] Embodiment 191. The method of any one of embodiments 181 to 190, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0429] Embodiment 192. The therapeutic RNA transcript is an mRNA encoding a therapeutic protein, the therapeutic protein being selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, 192. The method of embodiment 191, wherein the protein is selected from: (i) a protein encoded by a gene selected from 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 expression of a gene from (i).

[0430] Embodiment 193. The method of any one of embodiments 186 to 193, wherein the subject has a neurological disease or disorder.

[0431] Embodiment 194. The subject is a patient with Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome), young adulthood. 187. The method of embodiment 186, wherein the patient has chronic myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0432] Embodiment 195. The method of any one of embodiments 181 to 194, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0433] Embodiment 196. The method of embodiment 195, wherein the CNS-selective promoter is selected from the group consisting of Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0434] Embodiment 197. (a) the RNA transcript comprises the sequence, (b) the nucleic acid cassette comprises a CNS-selective promoter; (c) The method of any one of embodiments 181 to 196, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0435] Embodiment 198. (a) the RNA transcript comprises the sequence, (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally (c) The RNA transcript is an mRNA, and the mRNA is selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, and GAB RAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A 198. The method of any one of embodiments 181-197, wherein the therapeutic protein is encoded by a gene selected from: (i), 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 expression of a gene from (i).

[0436] Embodiment 199. The method of any one of embodiments 181 to 198, wherein inclusion of the sequence results in a decrease in expression of the RNA transcript and / or the protein encoded thereby (i.e., where the RNA transcript is mRNA) in liver cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the RNA transcript and / or protein in liver cells from an otherwise equivalent RNA transcript that does not include the sequence.

[0437] Embodiment 200. The method of any one of embodiments 181 to 199, wherein inclusion of the sequence results in reduced expression of the RNA transcript and / or the protein encoded thereby in liver 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 expression of the RNA transcript and / or protein in liver cells from an otherwise equivalent RNA transcript that does not include the sequence.

[0438] Embodiment 201. The method of any one of embodiments 181 to 200, wherein the sequence does not result in a significant decrease in expression of the RNA transcript and / or the protein encoded thereby in the target cell compared to expression of the RNA transcript and / or protein in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0439] Embodiment 202. The method of embodiment 201, wherein the sequence does not reduce expression of the RNA transcript and / or the protein encoded thereby in the target cell compared to expression of the RNA transcript and / or protein in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0440] Embodiment 203. The method of any one of embodiments 181 to 202, wherein the sequence results in expression of the RNA transcript and / or the protein encoded thereby in the target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript and / or protein in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

[0441] Embodiment 204. The method of any one of embodiments 201 to 203, wherein the target cell is a neuronal cell.

[0442] Embodiment 205. The method of embodiment 204, wherein the neural cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

[0443] Embodiment 206. The method of embodiment 205, wherein the neuronal cells are GABAergic cells.

[0444] Embodiment 207 The method of embodiment 206, wherein the GABAergic cells are parvalbumin-expressing cells.

[0445] Embodiment 208. The method of any one of embodiments 181 to 207, wherein the RNA transcript is expressed from a nucleic acid cassette.

[0446] Embodiment 209. The method of embodiment 208, wherein the nucleic acid cassette is a linear construct.

[0447] Embodiment 210. The method of embodiment 208 or 209, wherein the nucleic acid cassette is a vector.

[0448] Embodiment 211. The method of embodiment 210, wherein the vector is a plasmid.

[0449] Embodiment 212. The method of embodiment 210, wherein the vector is a viral vector.

[0450] Embodiment 213. The method of embodiment 212, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0451] Embodiment 214. The method of embodiment 213, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0452] Embodiment 215. The method of embodiment 213 or 214, wherein the AAV is scAAV.

[0453] Embodiment 216. The method of embodiment 212, wherein the viral vector is a lentiviral vector.

[0454] Embodiment 217. The method of any one of embodiments 210 to 216, wherein the method comprises administering a vector to a subject.

[0455] Embodiment 218. The method of any one of embodiments 181 to 207, wherein the method comprises administering an RNA transcript to the subject.

[0456] Embodiment 219. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript comprising a first sequence that detargets expression in dorsal root ganglion (DRG) cells and a second sequence that detargets expression in liver cells.

[0457] Embodiment 220. The nucleic acid cassette of embodiment 219, wherein the first and second sequences result in reduced expression of the RNA transcript or the polypeptide encoded thereby (i.e., if the RNA transcript is mRNA) in DRG and liver cells compared to the target tissue.

[0458] Embodiment 221. The first and second sequences are: a reduction in expression of the RNA transcript or polypeptide encoded thereby (i.e., where the RNA transcript is mRNA) in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not contain the first and second sequences, independently 221. The nucleic acid cassette of embodiment 219 or 220, which results in reduced expression of the RNA transcript or the polypeptide encoded thereby in liver 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 expression of the RNA transcript or polypeptide in liver cells from an equivalent RNA transcript but that does not comprise the first and second sequences.

[0459] Embodiment 222. (a) the first sequence is (i) any of SEQ ID NOs: 1-10 and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (i) or (ii); 222. The nucleic acid cassette of any one of embodiments 219 to 221, wherein (b) the second sequence is (iv) any of SEQ ID NOs: 57 to 62, 64 to 71, 110, and 112, (v) a variant, functional fragment, or combination thereof, or (vi) a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to (iv) or (v).

[0460] Embodiment 222A: A nucleic acid cassette described in any one of embodiments 219 to 222, wherein the first sequence (i), (ii), or (iii) provides binding sites 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 binding sites 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.

[0461] Embodiment 223. the first sequence comprises at least 15 consecutive nucleotides of any of SEQ ID NOs: 1-10 and 43-48, which reduce expression in dorsal root ganglion (DRG) cells; The nucleic acid cassette of any one of embodiments 219 to 222A, wherein the second sequence comprises at least 15 consecutive nucleotides of any of SEQ ID NOs: 57 to 62, 64 to 71, 110, and 112, which reduces expression in liver cells.

[0462] Embodiment 224. A nucleic acid cassette according to any one of embodiments 219 to 223, wherein the RNA transcript comprises at least two copies of any of the sequences (i) to (vi).

[0463] Embodiment 225. The nucleic acid cassette of embodiment 224, wherein the RNA transcript comprises at least three or at least four copies of any of the sequences (i) to (vi).

[0464] Embodiment 226. The nucleic acid of any one of embodiments 219 to 225, wherein the RNA transcript comprises a combination of sequences selected from Table 3.

[0465] Embodiment 227. The nucleic acid cassette of any one of embodiments 219 to 227, wherein the RNA transcript is an mRNA and the first and second sequences are independently located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

[0466] Embodiment 228. The nucleic acid cassette of embodiment 227, wherein the first and second sequences are located in the 3'UTR region of the mRNA.

[0467] Embodiment 229. The nucleic acid cassette of embodiment 227, wherein the first and second sequences are located in the 5'UTR region of the mRNA.

[0468] Embodiment 230. The nucleic acid cassette of embodiment 227, wherein the first and second sequences are located in an intron of the mRNA.

[0469] Embodiment 231. The nucleic acid cassette of any one of embodiments 219 to 230, wherein the nucleic acid cassette does not occur in nature.

[0470] Embodiment 232. The nucleic acid cassette of any one of embodiments 219 to 231, wherein the nucleic acid cassette comprises a CNS-selective promoter.

[0471] Embodiment 233. The nucleic acid cassette of embodiment 232, wherein the CNS-selective promoter is selected from the group consisting of the Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, synapsin I promoter, 67 kDa glutamic acid decarboxylase (GAD67) promoter, homeobox D1x5 / 6 promoter, glutamate receptor 1 (GluR1) promoter, preprotachykinin 1 (Tac1) promoter, neuron-specific enolase (NSE) promoter, dopaminergic receptor 1 (Drd1a) promoter, MAP1B promoter, Tα1 α-tubulin promoter, decarboxylase promoter, dopamine β-hydroxylase promoter, NCAM promoter, HES-5 promoter, α-internexin promoter, peripherin promoter, and GAP-43 promoter, and PaqR4 promoter.

[0472] Embodiment 234. The nucleic acid cassette of any one of embodiments 219 to 233, wherein the nucleic acid cassette comprises an enhancer.

[0473] Embodiment 235. The nucleic acid cassette of any one of embodiments 219 to 234, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0474] Embodiment 236. The neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome). , juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0475] Embodiment 237. The RNA transcript is an mRNA encoding a therapeutic protein, the therapeutic protein being selected from the group consisting of (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin1 / EFHC1, NPRL2, 237. The nucleic acid cassette of embodiment 235 or 236, wherein the nucleic acid cassette is selected from: (i) a protein encoded by a gene selected from PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (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 expression of a gene from (i).

[0476] Embodiment 238. the nucleic acid cassette comprises a CNS-selective promoter; 238. The nucleic acid cassette of any one of embodiments 219 to 237, wherein the RNA transcript is a therapeutic RNA transcript for treating a neurological disease or disorder.

[0477] Embodiment 239. the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and a GAP-43 promoter, and a PaqR4 promoter; and optionally The RNA transcripts are mRNAs, and the mRNAs are: ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GAB RAI, GABRB3, GAB RD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL 2, PCDH19, PLCB1, PNKP, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, 239. The nucleic acid cassette of any one of embodiments 219 to 238, encoding a therapeutic protein encoded by a gene selected from 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 expression of a gene from (i).

[0478] Embodiment 240. The nucleic acid cassette of any one of embodiments 219 to 239, wherein the first and second sequences result in decreased expression of the RNA transcript and / or the polypeptide encoded thereby (i.e., where the RNA transcript is mRNA) in DRG and liver cells compared to expression of the RNA transcript and / or the polypeptide in DRG and liver cells from an otherwise equivalent RNA transcript not comprising the first and second sequences.

[0479] Embodiment 241. The nucleic acid cassette of embodiment 240, wherein the first and second sequences result in a reduction in expression of the RNA transcript and / or the polypeptide encoded thereby in DRG and liver cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold compared to expression of the RNA transcript and / or polypeptide in DRG and liver cells from an otherwise equivalent RNA transcript that does not comprise the first and second sequences.

[0480] Embodiment 242. The nucleic acid cassette of embodiment 240 or 241, wherein the first and second sequences result in reduced expression of the RNA transcript and / or the polypeptide encoded thereby in DRG and liver 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 expression of the RNA transcript and / or polypeptide in DRG and liver cells from an otherwise equivalent RNA transcript not comprising the first and second sequences.

[0481] Embodiment 243. The nucleic acid cassette of any one of embodiments 219 to 242, wherein the first and second sequences do not result in a significantly reduced expression of the RNA transcript and / or the polypeptide encoded thereby in a target cell compared to the expression of the RNA transcript and / or the polypeptide in the target cell from an equivalent RNA transcript but excluding the first and second sequences.

[0482] Embodiment 244. The nucleic acid cassette of embodiment 243, wherein the first and second sequences do not reduce expression of the RNA transcript and / or the polypeptide encoded thereby in a target cell compared to expression of the RNA transcript and / or the polypeptide in the target cell from an equivalent RNA transcript but excluding the first and second sequences.

[0483] Embodiment 245. The nucleic acid cassette of any one of embodiments 219 to 244, wherein the first and second sequences result in expression of an RNA transcript and / or a polypeptide encoded thereby 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 of the RNA transcript and / or polypeptide in the target cell from an otherwise equivalent RNA transcript not comprising the first and second sequences.

[0484] Embodiment 246. The nucleic acid cassette of any one of embodiments 243 to 245, wherein the target cell is a neuronal cell.

[0485] Embodiment 247. The nucleic acid cassette of embodiment 246, wherein the neuronal cell is a cerebral cell, a brain stem cell, a hippocampal cell, or a cerebellar cell.

[0486] Embodiment 248. The nucleic acid cassette of embodiment 247, wherein the neuronal cell is a GABAergic cell.

[0487] Embodiment 249. The nucleic acid cassette of embodiment 248, wherein the GABAergic cell is a parvalbumin-expressing cell.

[0488] Embodiment 250. The nucleic acid cassette of any one of embodiments 219 to 249, wherein the nucleic acid cassette is a linear construct or a vector.

[0489] Embodiment 251. The nucleic acid cassette of embodiment 250, wherein the vector is a plasmid.

[0490] Embodiment 252. The nucleic acid cassette of embodiment 250, wherein the vector is a viral vector.

[0491] Embodiment 253. The nucleic acid cassette of embodiment 252, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0492] Embodiment 254. The nucleic acid cassette of embodiment 253, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-DJ.

[0493] Embodiment 255. The nucleic acid cassette of embodiment 253 or 254, wherein the AAV is a scAAV.

[0494] Embodiment 256. The nucleic acid cassette of embodiment 252, wherein the viral vector is a lentiviral vector.

[0495] Embodiment 257. An RNA transcript having a sequence encoded by the nucleic acid cassette of any one of embodiments 219 to 256.

[0496] Embodiment 258. A method for reducing dorsal root ganglion (DRG) and liver expression of a therapeutic RNA transcript and / or protein encoded thereby (i.e., where the RNA transcript is mRNA) while maintaining expression of the RNA transcript and / or protein in target tissues, the method comprising adding a first and a second sequence to the RNA transcript, wherein the first sequence detargets expression in DRG cells and the second sequence detargets expression in liver cells.

[0497] Embodiment 259. The first and second sequences are: a reduction in expression of the RNA transcript and / or polypeptide encoded thereby in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not contain the first and second sequences, independently; 221. The method of embodiment 219 or 220, which results in reduced expression of the RNA transcript and / or the polypeptide encoded thereby in liver cells to a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript or polypeptide in liver cells from an otherwise equivalent RNA transcript that does not comprise the first and second sequences.

[0498] Embodiment 260. The method of embodiment 258 or 258, wherein the RNA transcript is encoded by a nucleic acid cassette according to any one of embodiments 219 to 256.

[0499] Embodiment 261. A method for expressing a therapeutic protein, comprising: A method comprising administering to a subject the nucleic acid cassette described in any one of embodiments 219 to 256.

[0500] Embodiment 262. The method of embodiment 261, wherein the administering is systemic administration.

[0501] Embodiment 263. The method of embodiment 261, wherein the administering is topical administration.

[0502] Embodiment 264. The method of embodiment 263, wherein the nucleic acid cassette is administered locally to brain or CNS tissue.

[0503] Embodiment 265. The method of embodiment 263 or 264, wherein administering is by intraparenchymal, intrathecal, intracisternal, intraventricular, or intracranial administration.

[0504] Embodiment 266. The method of any one of embodiments 261 to 265, wherein the subject has a neurological disease or disorder.

[0505] Embodiment 267. The subject is a patient with Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effect of Parkinson's medication), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with centrotemporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and wave (CSWS), infantile spasms (West syndrome), young adulthood. 267. The method of embodiment 266, wherein the patient has chronic myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

[0506] Embodiment 268. The method of any one of embodiments 261 to 267, wherein the RNA transcript and / or the protein encoded thereby (i.e., where the RNA transcript is mRNA) is expressed in DRG and liver cells at a level that is at least 1.5-fold, at least 2-fold, at least 5-fold, or at least 10-fold greater than the expression of the RNA transcript and / or protein in DRG and liver cells from an otherwise equivalent RNA transcript that does not comprise the first and second sequences.

[0507] Embodiment 269. The method of any one of embodiments 261 to 268, wherein the RNA transcript and / or the protein encoded thereby is expressed in DRG and liver 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 of the RNA transcript and / or protein in DRG and liver cells from an otherwise equivalent RNA transcript that does not comprise the first and second sequences.

[0508] Embodiment 270. The method of any one of embodiments 261 to 269, wherein expression of the RNA transcript and / or the protein encoded thereby is not reduced in the target cell compared to expression of the RNA transcript and / or protein in the target cell from an equivalent RNA transcript that does not comprise the first and second sequences.

[0509] Embodiment 271. The method of any one of embodiments 261 to 270, wherein the RNA transcript and / or the protein encoded thereby is not reduced in the target cell compared to expression of the RNA transcript and / or protein in the target cell from an equivalent RNA transcript that does not comprise the first and second sequences.

[0510] Embodiment 272. The method of any one of embodiments 261 to 271, wherein the RNA transcript and / or the protein encoded thereby (i.e., where the RNA transcript is mRNA) is expressed in the target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript or protein in the target cell from an otherwise equivalent RNA transcript that does not comprise the first and second sequences.

[0511] Embodiment 273. The method of any one of embodiments 270 to 272, wherein the target cell is a neuronal cell.

[0512] Embodiment 274. The method of embodiment 273, wherein the neural cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

[0513] Embodiment 275. The method of embodiment 274, wherein the neuronal cells are GABAergic cells.

[0514] Embodiment 276 The method of embodiment 275, wherein the GABAergic cells are parvalbumin-expressing cells. [Example]

[0515] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended by the inventors to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular(ly); ip, intraperitoneal(ly...

Claims

1. 1. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises the sequence of (i) SEQ ID NO: 2 and any of SEQ ID NOs: 1, 3-10, and 43-48; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80% identical to (i) or (ii).

2. The nucleic acid cassette of claim 1, wherein the RNA transcript comprises a sequence of at least 15 consecutive nucleotides of any of SEQ ID NOs: 1-10 and 43-48, which reduces expression in dorsal root ganglion (DRG) cells.

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

4. 4. The nucleic acid cassette of claim 1, wherein the RNA transcript comprises two or more, three or more, four or more, or five or more copies of the sequence of (i), (ii), or (iii).

5. 5. The nucleic acid cassette of claim 1, wherein the sequence of (i), (ii), or (iii) provides binding sites 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. 6. The nucleic acid cassette of claim 1, wherein the RNA transcript is an mRNA, and the sequence of (i), (ii), or (iii) is located in one or more of the 3' UTR region of the mRNA, the 5' UTR of the mRNA, or an intron of the mRNA.

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. 8. The nucleic acid cassette of claim 7, 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 glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter.

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

10. The neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal lobe spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absence, epileptic encephalopathy of sleep with continuous spike and slow waves (CSWS), infantile spasms (West syndrome), The nucleic acid cassette of claim 9, which is selected from the group consisting of juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

11. The RNA transcript is an mRNA, and the mRNA is selected from the group consisting of: (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FO LR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KC NT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNK 11. The nucleic acid cassette of claim 9 or 10, encoding a therapeutic protein selected from: (i) a protein encoded by a gene selected from P, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates expression of a gene from (i).

12. (a) the RNA transcript is mRNA, and the mRNA comprises the sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii); (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter; (c) the mRNA is ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHR NA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GABRA1, GAB RB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV3.2 , KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PRR 12. The nucleic acid cassette of any one of claims 1 to 11, encoding a therapeutic protein encoded by a gene selected from T2, 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 expression of a gene from (i).

13. 13. The nucleic acid cassette of any one of claims 1 to 12, wherein the sequence of (i), (ii), or (iii) results in decreased expression of the RNA transcript and / or the polypeptide encoded by the RNA transcript in a DRG cell compared to expression of the RNA transcript and / or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

14. 14. The nucleic acid cassette of any one of claims 1 to 13, wherein the sequence of (i), (ii), or (iii) results in reduced expression of the RNA transcript and / or polypeptide encoded by the RNA transcript in a DRG cell to a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript and / or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

15. 15. The nucleic acid cassette of any one of claims 1 to 14, wherein the sequence of (i), (ii), or (iii) does not result in decreased expression of the RNA transcript and / or the polypeptide encoded by the RNA transcript in a target cell compared to expression of the RNA transcript and / or polypeptide in a target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

16. 16. The nucleic acid cassette of any one of claims 1 to 15, wherein the sequence of (i), (ii), or (iii) results in expression of the RNA transcript and / or the polypeptide encoded by the RNA transcript 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 of the RNA transcript and / or polypeptide in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence of (i), (ii), or (iii).

17. The nucleic acid cassette of claim 15 or 16, wherein the target cell is a neuronal cell.

18. The nucleic acid cassette of claim 17, wherein the neuronal cell is a cerebral cell, a brain 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. 20. The nucleic acid cassette of claim 19, wherein the viral vector is an adeno-associated viral (AAV) vector.

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

22. 1. A method of reducing dorsal root ganglion (DRG) expression of an RNA transcript and / or a therapeutic protein encoded thereby, comprising comprising in said RNA transcript a sequence of (i) any of SEQ ID NOs: 1-10 and 43-48, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

23. The sequence is 23. The method of claim 22, wherein the method results in reduced expression of the RNA transcript and / or polypeptide encoded thereby in a DRG cell at a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript and / or polypeptide in a DRG cell from an otherwise equivalent RNA transcript that does not contain the sequence.

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

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

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

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

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

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

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

31. The subject is selected from the group consisting of Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal lobe spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and slow waves (CSWS), infantile spasms (West syndrome), juvenile 31. The method of claim 30, wherein the patient has myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

32. 32. The method of any one of claims 25-31, wherein the therapeutic RNA transcript is an mRNA and 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 expression of protein in DRG cells from an otherwise equivalent mRNA that does not comprise the sequence.

33. 33. The method of any one of claims 25 to 32, wherein expression of the therapeutic RNA transcript and / or protein encoded thereby is not reduced in the target cell compared to expression of the therapeutic RNA transcript and / or protein in the target cell from an otherwise equivalent therapeutic RNA transcript that does not comprise the sequence.

34. 34. The method of any one of claims 25-33, wherein the therapeutic RNA transcript and / or the protein encoded thereby is expressed in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the therapeutic RNA transcript and / or protein in the target cell from an otherwise equivalent therapeutic RNA transcript that does not comprise the sequence.

35. 35. The method of claim 33 or 34, wherein the target cell is a neuronal cell.

36. 36. The method of claim 35, wherein the neural cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

37. 1. A nucleic acid cassette comprising a therapeutic transgene encoding an RNA transcript, wherein the RNA transcript comprises a sequence of (i) any of SEQ ID NOs: 65, 110, and 112; (ii) a variant, functional fragment, or combination thereof; or (iii) a sequence at least 80% identical to (i) or (ii), wherein the sequence reduces expression of mRNA in liver cells.

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

39. The CNS-selective promoter is ２＋ 39. The nucleic acid cassette of claim 38, wherein the promoter is selected from the group consisting of a calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a 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 neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absence, epileptic encephalopathy of sleep with continuous spike and slow waves (CSWS), infantile spasms (West syndrome), young adulthood 41. The nucleic acid cassette of claim 40, wherein the nucleic acid cassette is selected from the group consisting of: chronic myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

42. The RNA transcript is an mRNA, and the mRNA is selected from the group consisting of: (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FO LR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KC NT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNK 42. The nucleic acid cassette of claim 40 or 41, encoding a therapeutic protein selected from: (i) a protein encoded by a gene selected from P, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates expression of a gene from (i).

43. (a) the RNA transcript is mRNA, and the mRNA comprises the sequence; (b) the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter; (c) The mRNA is selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, GA BRAI, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1 , KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG 1, 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 expression of a gene from (i).

44. 44. The nucleic acid cassette of any one of claims 37 to 43, wherein the sequence results in reduced expression of the RNA transcript and / or protein encoded thereby in liver cells to a level that is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than expression of the RNA transcript or protein in liver cells from an otherwise equivalent RNA transcript that does not comprise the sequence.

45. 45. The nucleic acid cassette of any one of claims 37 to 44, wherein the sequence results in expression of the RNA transcript and / or protein encoded thereby in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the RNA transcript or protein in the target cell from an otherwise equivalent RNA transcript that does not comprise the sequence.

46. 46. ​​The nucleic acid cassette of claim 44 or 45, wherein the target cell is a neuronal 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 of any one of claims 37 to 47.

49. 1. A method for reducing hepatic expression of a therapeutic RNA transcript and / or the protein encoded thereby, comprising comprising in said RNA transcript a sequence of (i) any of SEQ ID NOs: 65, 110, and 112, (ii) a variant, functional fragment, or combination thereof, or (iii) a sequence at least 80% identical to (i) or (ii).

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

51. 51. The method of claim 50, wherein the administering is systemic administration.

52. 51. The method of claim 50, wherein the administering is topical administration.

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

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

55. 55. The method of 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 comprising a first sequence that detargets expression in dorsal root ganglion (DRG) cells and a second sequence that detargets expression in liver cells.

57. 57. The nucleic acid cassette of claim 56, wherein the first and second sequences result in decreased expression of the RNA transcript and / or the polypeptide encoded thereby in DRG and liver cells compared to a target tissue.

58. The first and second sequences are a reduction in expression of said RNA transcript or polypeptide encoded thereby 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 expression of said RNA transcript or polypeptide in DRG cells from an otherwise equivalent RNA transcript that does not comprise said first and second sequences, independently; 58. The nucleic acid cassette of claim 56 or 57, which results in reduced expression of the RNA transcript or polypeptide encoded thereby in liver 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 expression of the RNA transcript or polypeptide in liver cells from an equivalent RNA transcript but that does not contain the first and second sequences.

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

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

61. The first sequence of (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 of (iv), (v), or (vi) 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.

61. The nucleic acid cassette of any one of claims 56 to 60, wherein the sequence provides binding sites for hsa-mir-22-3p, hsa-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. 62. The nucleic acid cassette of 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 of the sequences (i) to (vi).

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

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

65. 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 glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, and 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 for treating a neurological disease or disorder.

67. The neurological disease or disorder is Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's drugs), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absence, epileptic encephalopathy of sleep with continuous spike and slow waves (CSWS), infantile spasms (West syndrome), young adulthood 67. The nucleic acid cassette of claim 66, wherein the nucleic acid cassette is selected from the group consisting of: chronic myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, and attention deficit hyperactivity disorder.

68. The RNA transcript is an mRNA, and the mRNA is selected from the group consisting of: (i) ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FO LR1, FOXG1, GABRA1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KC NT1, KV3.1, KV3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNK 68. The nucleic acid cassette of claim 66 or 67, encoding a therapeutic protein selected from: (i) a protein encoded by a gene selected from P, POLG1, PRRT2, PTEN, SCN1A, SCN1B, SCN2A, SCN2B, SCN8A, SHANK3, SLC13A5, SLC25A22, SLC2A1, SLC6A1, SLC6A8, SPTAN1, ST3GAL3, STRADA, STXBP1, SYNGAP1, TBC1D24, UBE3A, and WWOX; (ii) a protein having at least 90% sequence identity to (i); (iii) a functional fragment of (i) or (ii); or (iv) a transcription factor that regulates expression of a gene from (i).

69. the nucleic acid cassette comprises a promoter selected from the group consisting of a Ca2+ / calmodulin-dependent kinase subunit alpha (CaMKII) promoter, a synapsin I promoter, a 67 kDa glutamic acid 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 Tα1 α-tubulin promoter, a decarboxylase promoter, a dopamine β-hydroxylase promoter, an NCAM promoter, a HES-5 promoter, an α-internexin promoter, a peripherin promoter, a GAP-43 promoter, and a PaqR4 promoter; The RNA transcripts are selected from the group consisting of ALDH7A1, ARHGEF9, ARX, BRAT1, CACNA1A, CACNA1D, CACNB4, CDKL5, CHD2, CHRNA2, CHRNA4, CHRNB2, CLCN2, CLN, CLN2, DEPDC5, DNM1, FGF13, FMR1, FOLR1, FOXG1, and GABR. A1, GABRB3, GABRD, GABRG2, GRIN2A, GRIN2B, HCN1, HCN4, KCNQ2, KCNQ3, KCNT1, KV3.1, KV 3.2, KV3.3, LGI1, MECP2, MEF2C, myoclonin 1 / EFHC1, NPRL2, PCDH19, PLCB1, PNKP, POLG1, PR 69. The nucleic acid cassette of any one of claims 56 to 68, which is a therapeutic protein encoded by a gene selected from RT2, 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) an mRNA encoding a transcription factor that regulates expression of a gene from (i).

70. 70. The nucleic acid cassette of any one of claims 56 to 69, wherein the first and second sequences result in expression of the RNA transcript or polypeptide encoded thereby 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 of the RNA transcript or polypeptide in the target cell from an otherwise equivalent RNA transcript lacking the first and second sequences.

71. 71. The nucleic acid cassette of claim 70, wherein the target cell is a neuronal cell.

72. 72. The nucleic acid cassette of claim 71, wherein the neural cell is a cerebral 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. 74. The nucleic acid cassette of claim 73, wherein the viral vector is an adeno-associated viral (AAV) vector.

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

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

77. The first and second sequences are a reduction in expression of said therapeutic RNA transcript and / or polypeptide encoded thereby 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 expression of said therapeutic RNA transcript or polypeptide in DRG cells from an otherwise equivalent therapeutic RNA transcript that does not comprise said first and second sequences, and independently, 77. The method of claim 76, wherein the method results in reduced expression of the therapeutic RNA transcript and / or polypeptide encoded thereby in liver 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 expression of the therapeutic RNA transcript or polypeptide in liver cells from an otherwise equivalent therapeutic RNA transcript that does not comprise the first and second sequences.

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

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

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

81. 80. The method of claim 79, wherein the administering is topical administration.

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

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

84. The subject is selected from the group consisting of Alpers-Huttenlocher syndrome, Angelman syndrome, CDKL5 deficiency, Dravet syndrome, Rett syndrome, Parkinson's disease and Parkinson's disease LIDS (side effects of Parkinson's medications), Alzheimer's disease, creatine transporter deficiency, FOXG1 syndrome, fragile X syndrome, Phelan-McDermid syndrome, childhood absence epilepsy, childhood epilepsy with central temporal lobe spikes (benign rolandic epilepsy), early myoclonic encephalopathy (EME), epileptic eyelid myoclonia (Jeavons syndrome), infantile epilepsy with migrating focal seizures, epileptic myoclonic absences, epileptic encephalopathy of sleep with continuous spike and slow waves (CSWS), infantile spasms (West syndrome), juvenile 84. The method of claim 83, wherein the patient has myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome (LGS), myoclonic epilepsy in infancy, Otahara syndrome, Panayiotopoulos syndrome, progressive myoclonic epilepsy, reflex epilepsy, self-limited familial and non-familial neonatal infantile seizures, self-limited late-onset occipital lobe epilepsy, Gastaut syndrome, epileptic generalized tonic-clonic seizures alone, genetic epilepsy with febrile seizures plus, juvenile absence epilepsy, myoclonic atonic epilepsy (Douse syndrome), sleep-related hyperkinetic epilepsy (SHE), febrile seizures, focal epilepsy, West syndrome, early-onset epilepsy, benign familial infantile epilepsy, or attention deficit hyperactivity disorder.

85. 85. The method of any one of claims 79-84, wherein the therapeutic RNA transcript and / or protein encoded thereby is expressed in DRG and liver 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 expression of the therapeutic RNA transcript or protein in DRG and liver cells from an otherwise equivalent mRNA that does not comprise the first and second sequences.

86. 86. The method of any one of claims 79-85, wherein the therapeutic RNA transcript and / or the protein encoded thereby is expressed in a target cell at a level that is at least at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the expression of the therapeutic RNA transcript or protein in the target cell from an otherwise equivalent therapeutic RNA transcript that does not comprise the first and second sequences.

87. 87. The method of claim 86, wherein the target cell is a neuronal cell.

88. 88. The method of claim 87, wherein the neural cells are cerebral cells, brain stem cells, hippocampal cells, or cerebellar cells.

89. 89. The method of claim 88, wherein the neuronal cell is a GABAergic cell.

90. 90. The method of claim 89, wherein the GABAergic cells express parvalbumin.

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