Glial targeting mitigation of hyperexcitability in neurodegenerative diseases

Abstract

This disclosure relates to relevant therapeutic agents and methods for alleviating hyperexcitability and for the treatment of neurodegenerative diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 592,271, filed on 23 October 2023. The contents of that application are incorporated herein by reference in their entirety.

[0002] Government interests This invention was made with government support granted by the National Institutes of Health (NIH) under AG072298. The government has certain rights in this invention.

[0003] Electronic sequence listing reference The contents of the electronic sequence listing (161118.04901SeqList.xml, size: (7,578 bytes, and creation date: October 15, 2024) are incorporated herein by reference in their entirety.

[0004] This disclosure relates to relevant therapeutic agents and methods for alleviating hyperexcitability and for the treatment of neurodegenerative diseases. [Background technology]

[0005] Neurodegenerative diseases, characterized by the progressive loss of structure or function of neurons in the brain or peripheral nervous system, affect millions of people worldwide. Neurodegeneration can be found in the brain at many different levels of neural circuits, ranging from molecular to systemic. Aging is the most important risk factor for developing neurodegenerative diseases. Both the incidence and prevalence of neurodegenerative diseases, including Alzheimer's disease and Huntington's disease, frontotemporal dementia (FTD) and FTD-amyotrophic lateral sclerosis (FTD) complications, as well as Parkinson's disease, Lewy body dementia, and multiple system atrophy, all increase with age. Each of these disorders is characterized by progressive neuronal loss, which is closely associated with the progression of symptomatic disease and functional deterioration. In contrast, however, normal aging has been shown to result in synaptic loss without a significant decrease in neuronal density. Despite intensive research in this field, primarily focusing on the potential neurotoxicity of amyloid, tau, and synuclein proteins, as well as the neurotoxicity of neuroinflammation, the pathobiological understanding of neuronal loss in neurodegenerative disorders remains limited. There is an unmet need for novel therapeutic agents and methods to treat neurodegenerative diseases. [Overview of the project]

[0006] This disclosure addresses the aforementioned needs in several aspects.

[0007] In one embodiment, the present disclosure provides a method for (i) reducing brain hyperexcitability or (ii) treating a condition mediated by brain hyperexcitability in a subject requiring treatment. The method involves Na in the glial cells of the subject. + , K + This includes increasing the level or activity of ATPase.

[0008] In another aspect, the disclosure features a method for reducing interstitial potassium levels in subjects requiring such reduction. The method involves reducing Na in the glial cells of the subject. + , K + This includes increasing the level or activity of ATPase.

[0009] In each of the above methods, the subject may have a condition mediated by neuronal hyperexcitability.

[0010] In some embodiments, the brain interstitial potassium level recovers to about ±30% of that of a normal healthy adult brain.

[0011] In each of the above methods, the increasing can include reducing the expression level of the FXYD1 gene in glial cells. In one embodiment, the reducing includes administering to the subject an agent that reduces the expression level of the FXYD1 gene in glial cells. In some embodiments, the agent can comprise or encode an inhibitory nucleic acid or a CRISPR / Cas system. In some embodiments, the inhibitory nucleic acid comprises an RNA molecule such as small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

[0012] In some embodiments, the agent is or comprises an expression cassette or vector comprising a sequence encoding the inhibitory nucleic acid or encoding one or more components of the CRISPR / Cas system. In some embodiments, the sequence is operably linked to a cell type-selective or cell type-specific regulatory sequence. In some embodiments, the cell type-selective or cell type-specific regulatory sequence comprises a promoter, or an enhancer, or both. The promoter can be a glial cell-specific promoter or a regulatable promoter. The vector can be a viral vector.

[0013] In each of the above methods, the glial cells can be astrocytes, glial progenitor cells, or oligodendrocytes. The aforementioned condition can be a neurodegenerative disease. Examples of this condition or neurodegenerative disease include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, Huntington's disease, and schizophrenia.

[0014] In some embodiments, the inhibitory nucleic acid or siRNA molecule comprises or encodes a sequence that is at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100) complementary to a segment of the FXYD1 gene or RNA.

[0015] In some embodiments, the CRISPR / Cas system comprises or encodes a guide RNA (gRNA) sequence that is at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100) complementary to a segment of the FXYD1 gene or RNA.

[0016] In a further aspect, the present disclosure provides an inhibitory nucleic acid or siRNA molecule that comprises or encodes a sequence that is at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100) complementary to a segment of the FXYD1 gene or RNA.

[0017] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (i) the inhibitory nucleic acid or siRNA molecule disclosed herein, and (ii) a pharmaceutically acceptable carrier or excipient.

[0018] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features, objectives, and advantages of the present disclosure will be apparent from the present specification and the claims. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0019] [Figure 1] Detailed information regarding individual data sources of the differentially expressed gene (DEG) list obtained from astrocyte transcriptome data in mouse models of normal aging, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease is shown. [Figure 2]The proposed model is shown in the figure below. Extracellular K+ is elevated in neurodegenerative diseases, in contrast to its typical relative decline in normal, healthy aging. As a key role in K+ buffering, a key gene for dysregulation in astrocytes, e.g., Fxyd1, contributes to increased cortical [K+]e due to impaired buffering in AD and ALS models. Chronic elevation of extracellular K+ generates hyperexcitability and reduces neuronal number in disease models. [Modes for carrying out the invention]

[0020] This disclosure relates to the treatment of neurodegenerative diseases and the mitigation of hyperexcitability in cases where disrupted ion homeostasis may be a driving force of neurodegenerative pathology. Certain aspects of this disclosure are based, at least in part, on the unexpected discovery of astrocyte genes that are dysregulated in astrocytes derived from neurodegenerative diseases but not in healthy, aged astrocytes.

[0021] For example, by meta-analysis of large databases encompassing gene expression from both mouse and human astrocytes, compared to age-matched cells supplied from patients with various neurodegenerative disorders, and derived from both wild-type and senescent cells, the inventors identified astrocyte genes that are dysregulated in astrocytes derived from neurodegenerative diseases but not in healthy, senescent astrocytes. In doing so, the inventors investigated data from both human cell and animal models of amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease.

[0022] 1. Neurodegenerative diseases and hyperexcitability "Neurodegenerative disorder" refers to a disease state involving nerve loss that is at least partially mediated or characterized by deterioration of at least one neural stem cell and / or progenitor cell. Non-limiting examples of neurological diseases and / or disorders of this disclosure include polyglutamine elongation disorders (e.g., HD, dentatorubral-pallidoluysian atrophy, Kennedy disease (also known as spinal-bulbar muscular atrophy), and spinocerebellar ataxia (e.g., types 1, 2, 3 (also known as Machado-Joseph disease), 6, 7, and 17)), other trinucleotide repeat elongation disorders (e.g., Fragile X syndrome, Fragile XE intellectual disability, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), telangiectatic ataxia, and Batten disease (Sp These include ielmeyer-Vogt-Sjögren-Batten disease, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Guillain-Barré syndrome, ischemic stroke, Krabbe disease, Kuru, Lewy body dementia, multiple sclerosis, multiple system atrophy, non-Huntington type chorea, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), Stähr-Richardson-Olzewski disease, schizophrenia, late-onset psychosis, autism spectrum disorder, motor disorders, and spinal fistula. In some contexts, neurodegenerative disorders encompass neurological injuries or damage to the central nervous system (CNS) or primary nervous system (PNS) associated with physical injury (e.g., head injury, mild to severe traumatic brain injury (TBI), diffuse axonal injury, cerebral contusion, acute cerebral edema, etc.).

[0023] In some embodiments, neurodegenerative disorders are disorders associated with neuronal hyperexcitability. A common feature in most neurodegenerative diseases is a condition exhibiting neuronal hyperexcitability, abnormal electrical activity, or an increased likelihood of neural networks being excited or activated. Neuronal hyperexcitability may be involved in spinal cord injury, stroke, traumatic brain injury, hearing loss, epilepsy, painful neurological disorders, attention deficit hyperactivity disorder, autism, central pain syndrome, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, schizophrenia, Rasmussen's encephalitis, Huntington's disease, alcoholism or alcohol withdrawal, and excessively rapid benzodiazepine withdrawal.

[0024] Extracellular potassium concentration in the brain is a powerful modulator of cortical network activity [K + ] e This changes in neurodegeneration compared to normal, healthy aging. For this purpose, the inventors studied various mouse models and found that disrupted extracellular K is altered. + However, we investigated whether it is potentially important for degenerative neuron loss due to its effect on neuronal membrane potential and the activation of pathological hyperexcitability.

[0025] Due to the causal role of dysregulated high interstitial potassium in the hyperexcitability and excitotoxicity of these neurodegenerative disorders, we focused on genes involved in glial potassium transport. These genes are disrupted in each state but are not dysregulated in normal, healthy aging. We identified potassium regulatory genes that were dysregulated in disease-derived astrocytes but not in normally aged astrocytes.

[0026] Among these was FXYD1, which is known to negatively regulate Na / K-ATPase. FXYD1 was found to be abnormally overexpressed in astrocytes originating from each of these disease states, but not abnormally overexpressed in normal wild-type aged astrocytes. Overexpression of FXYD1 in astrocytes is expected to suppress Na / K-ATPase activity, thereby increasing extracellular and interstitial K, which would then increase neuronal network excitation and associated long-term excitotoxicity. Therefore, glial-specific knockdown of FXYD1 gene expression can be used as a therapeutic strategy, in that its glial-targeted suppression can mitigate both the network hyperexcitability and excitotoxicity that characterize these neurodegenerative disorders. As disclosed herein, this can be achieved, for example, by plasmid or viral delivery to central astrocytes, and / or by viral vectors having cell-type specific binding or infection, targeting the latter by glial-specific regulatory sequences of FXYD1 shRNAi or CRISPR-mediated epigenetic knockdown vectors.

[0027] Below are exemplary protein sequences, protein-coding nucleic acid sequences, and corresponding mRNAs for Homo sapiens FXYD1. MASLGHILVFCVGLLTMAKAESPKEHDPFTYDYQSLQIGGLVIAGILFILGILIVLSRRCRCKFNQQQRTGEPDEEEGTFRSSIRRLSTRRR (Sequence ID 1) A. Homo sapiens FXYD domain (FXYD1) containing ion transport regulator 1, transcript variant a, mRNA, NCBI reference sequence: NM_005031.5 (SEQ ID NO: 2) [ka] FXYD domain (FXYD1) containing ion transport regulator 1 of B. Homo sapiens, transcript variant b, mRNA, NCBI reference sequence: NM_021902.4 (SEQ ID NO: 3) [ka] FXYD domain (FXYD1) containing ion transport regulator 1 of C. Homo sapiens, transcript variant c, mRNA, NCBI reference sequence: NM_001278717.2 (SEQ ID NO: 4) [ka] FXYD domain (FXYD1) containing ion transport regulator 1 of D. Homo sapiens, transcript variant d, mRNA, NCBI reference sequence: NM_001278718.2 (SEQ ID NO: 5) [ka]

[0028] One way to address the abnormal overexpression of FXYD1 is through the use of RNA interference to reduce the expression of the FXYD1 gene. This can be achieved with microRNA-based gene therapy. Endogenous mRNA silencing mechanisms can be activated to reduce FXYD1 translation in glial cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) by administering an AAV vector that delivers an expression cassette of a therapeutic miRNA precursor targeting FXYD1 mRNA. Furthermore, the use of AAV vectors with higher targeting of such glial cells can be used to improve safety and therapeutic efficacy. Overexpression of FXYD1 can also be suppressed using antisense oligonucleotide strategies.

[0029] In some embodiments, the Disclosure utilizes a viral vector, such as an AAV vector, to efficiently deliver therapeutic nucleic acids, such as siRNA, to cells, targeting one or more genes or RNAs encoding toxic gain-of-function proteins. In some embodiments, an AAV vector encoding an RNAi molecule, such as the siRNA molecule of the Disclosure, may increase the delivery of the activator to glial cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes). Therapeutic nucleic acids or polynucleotides may significantly inhibit the gene expression (e.g., at the mRNA level) of intracellular gain-of-function proteins, and thus mitigate intracellular protein-induced or induced deficiencies, such as inhibition of sodium / potassium transport ATPase activity and / or protein aggregation and inclusion formation.

[0030] Such inhibitory nucleic acids, such as siRNA, may be used to treat a variety of hereditary and / or acquired neurodegenerative disorders. According to this disclosure, a method for treating and / or improving a disorder in a patient involves administering an effective amount of at least one therapeutic nucleic acid (e.g., a polynucleotide encoding one or more siRNA double strands) to cells in the patient, enabling inhibition / silencing of gene expression.

[0031] 2. Nucleic acid Certain aspects of this disclosure provide one or more inhibitory nucleic acids (e.g., inhibitory RNA molecules), polynucleotides encoding such inhibitory nucleic acids, and transgenes engineered to express such inhibitory nucleic acids. One or more inhibitory nucleic acids may target the same gene (e.g., hybridize to or specifically bind to the same mRNA sequence or different mRNA sequences of the same gene) or different genes (e.g., hybridize to or specifically bind to mRNA of different genes).

[0032] A. Inhibitory nucleic acids Inhibitory nucleic acids refer to nucleic acids that can bind to target nucleic acids (e.g., target RNA) within a cell and reduce or inhibit the level or function of the target nucleic acid within the cell. Examples of inhibitory nucleic acids include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, small interfering (SiO) RNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds, modified base / lock nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimics that specifically hybridize to at least a portion of the target nucleic acid and modulate its level or function.

[0033] In some embodiments, the inhibitory nucleic acid can be antisense RNA, antisense DNA, chimeric antisense oligonucleotide, antisense oligonucleotide including modified ligatures, interfering RNA (iRNA), short or small interfering RNA (siRNA), microRNA or micro-interfering RNA (miRNA), small time-type RNA (stRNA), short hairpin RNA (shRNA), small RNA-inducible gene activator (RNAa), small activating RNA (saRNA), or a combination thereof. In some examples, the inhibitory nucleic acid is an inhibitory RNA molecule that mediates RNA interference.

[0034] RNA interference (RNAi) is a process discovered in 1998 (Fire et al., 1998) in which cells regulate gene expression. Double-stranded RNA (dsRNA) in the cytoplasm of a cell triggers the RNAi pathway, in which the double-stranded RNA is processed into small double-stranded fragments of approximately 21-23 nucleotides in length by the RNAse III-like enzyme DICER. These double-stranded fragments are integrated into a multi-subunit protein called the RNA-Inducible Silencing Complex (RISC). RISC contains an Argonaut protein that unwinds the double-stranded fragments, consisting of a passenger strand to be removed from the complex and a guide strand complementary to a target sequence in a particular mRNA, which instructs the RISC complex to cleave or repress the translation of the specific target mRNA molecule (Kotowska-Zimmer et al., 2021). In this way, the gene encoding the mRNA molecule is essentially inactivated or "silenced".

[0035] RNAi technology can utilize three tools: synthetic siRNA, vector-based shRNA, and artificial miRNA (amiRNA). Synthetic siRNA is exogenous double-stranded RNA that must be delivered into cells and overcome stability and pharmacokinetic challenges. shRNA is an artificial RNA molecule with a tight hairpin loop structure that is delivered to cells using plasmids or viral expression vectors. shRNA is typically transcribed from a strong pol III promoter (e.g., U6 or H1) and enters the RNAi pathway as a hairpin. However, transcription driven by a strong pol III promoter can produce hyperphysiological levels of shRNA that saturate the endogenous miRNA developmental mechanism, leading to toxicity. amiRNA embeds target-specific shRNA insertions into a scaffold based on natural primary miRNA (pri-miRNA). This ensures proper processing and transport similar to endogenous miRNA, while reducing toxicity (Kotowska-Zimmer et al., 2021).

[0036] In some embodiments of this disclosure, the inhibitory RNA molecule may be siRNA, miRNA (including amiRNA), or shRNA.

[0037] siRNA is known in the art as a double-stranded RNA molecule approximately 19–25 (e.g., 19–23) base pairs long that induces RNAi in cells. In some embodiments, siRNA sequences can also be inserted into artificial miRNA scaffolds ("shmiRNA").

[0038] shRNA is known in the art as an RNA molecule containing double-stranded RNA of approximately 19-25 (e.g., 19-23) base pairs linked by short loops (e.g., approximately 4-11 nucleotides) that induce RNAi in cells.

[0039] miRNAs are known in the art as RNA molecules that induce RNAi in cells, comprising double-stranded RNA with short (e.g., 19-25 base pairs) sequences linked by loops, and double-stranded RNA with one or more additional sequences containing one or more bulges (e.g., mispaired or unpaired base pairs). As used herein, the term “miRNA” encompasses endogenous miRNAs as well as exogenous or heterologous miRNAs. In some embodiments, “miRNA” may refer to pri-miRNA or pre-miRNA. During miRNA processing, pri-miRNA transcripts are produced. Pri-miRNAs are processed with Drosha-DGCR8 to excise one or more sequences to produce pre-miRNA having a 5' facile region, guide strand, loop region, non-guide strand, and 3' facile region, or by leaving a 5' facile region, non-guide strand, loop region, guide strand, and 3' facile region. Next, the pre-miRNA is exported to the cytoplasm and treated with Dicer to obtain siRNA having a guide strand and a non-guide (or passenger) strand. The guide strand is then used by the RISC complex to catalyze gene silencing, for example, by recognizing a target RNA sequence complementary to the guide strand. Further explanation of miRNA can be found, for example, in WO2008 / 150897. Recognition of the target sequence by miRNA is mainly determined by pairing between the target and the seed sequence of the miRNA, for example, nucleotides 1-8 (from 5' to 3') of the guide strand (see, for example, Boudreau, R. Let al. (2013) Nucleic Acids Res. 41:e9).

[0040] In some embodiments of this disclosure, the inhibitory RNA molecule forms a hairpin structure. Generally, the hairpin-forming RNA is located within a self-complementary “stem-loop” structure comprising a single nucleic acid encoding a double-stranded stem portion including a sense strand (e.g., passenger strand) connected to an antisense strand (e.g., guide strand) by a loop sequence. The passenger strand and guide strand share complementarity. In some embodiments, the passenger strand and guide strand share 100% complementarity. In some embodiments, the passenger strand and guide strand share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% complementarity. The passenger strand and guide strand may lack complementarity due to base pair mismatches. In some embodiments, the passenger strand and guide strand of the hairpin-forming RNA may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. Generally, the first 2–8 nucleotides of the stem (relative to the loop) are called the "seed" residues and play a crucial role in target recognition and binding. The first residue of the stem (relative to the loop) is called the "anchor" residue. In some embodiments, hairpin-forming RNA has a mismatch at the anchor residue.

[0041] In some embodiments, inhibitory RNA molecules are processed within a cell (or subject) to form “mature miRNAs.” Mature miRNAs are the result of a multi-step pathway initiated through the transcription of primary miRNA from its miRNA gene or intron by RNA polymerase II or III, which generates the first precursor molecules in the biological pathway that yields miRNAs. Once transcribed, pri-miRNA (often over 1000 nucleotides long with a hairpin structure) is processed by the Drosha enzyme, which cleaves the pri-miRNA near the junction between the hairpin structure and the ssRNA, yielding a precursor miRNA (pre-miRNA). The pre-miRNA is exported into the cytoplasm and further shortened by the Dicer enzyme in the pre-miRNA loop, yielding a doubled miRNA strand.

[0042] Of the two strands in a miRNA duplex, one arm, the guide strand (miR), is typically found at higher concentrations and binds to and associates with Argonaut proteins, which are eventually loaded into the RNA-induced silencing complex. The guide strand miRNA-RISC complex often helps regulate gene expression by binding to its complementary sequence in mRNA, often at the 3′ UTR of mRNA. The non-guide strand of the miRNA duplex, known as the passenger strand, is often degraded but may persist and acts either intact or partially degraded to play a functional role in gene expression.

[0043] In some embodiments, the transgene is engineered to express an inhibitory nucleic acid (e.g., miRNA) having a guide chain that targets a human gene. "Targeting" refers to the hybridization or specific binding of the inhibitory nucleic acid to its congenerate (e.g., complementary) sequence on the target gene (e.g., the mRNA transcript of the target gene). In some embodiments, the inhibitory nucleic acid targeting the gene transcript shares a complementary region with the target gene that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the complementary region is 30 or more nucleotides long.

[0044] Typically, the guide strand may target a human gene transcript associated with a disease or disorder. An example is one for FXYD1. In some embodiments, the guide strand targeting any of the gene transcripts may be complementary to a segment of the sequence described above, such as SEQ ID NOs: 2–4.

[0045] In some embodiments, the inhibitory nucleic acid is 5 to 300 nucleotides long (e.g., nucleotide lengths such as 10 to 30, 15 to 25, 19 to 22, 25 to 50, 40 to 90, 60 to 90, 75 to 100, 90 to 150, 110 to 200, 150 to 250, 200 to 300). The inhibitory nucleic acid sequence encoding pre-miRNA or mature miRNA may be 10 to 50 or 5 to 50 nucleotides long.

[0046] scaffold In certain embodiments, an inhibitory RNA molecule may be encoded by an inhibitory nucleic acid comprising a molecular scaffold. As used herein, “molecular scaffold” is a framework or starting molecule that forms a sequence or structural basis for designing or fabricating subsequent molecules.

[0047] In some embodiments, the molecular scaffold includes at least one 5' facile region, or one 3' facile region, or both. In non-limiting examples, the 5' or 3' facile region may be of any length, may be derived from all or part of a wild-type microRNA sequence, or may be a completely artificial sequence. In some embodiments, one or both of the 5' and 3' facile regions may be absent. In some embodiments, the 5' and 3' facile regions may be the same length or of different lengths. In some embodiments, the 5' or 3' facile region may be 1 to 10 nucleotides long, 5 to 15 nucleotides long, 10 to 30 nucleotides long, 20 to 50 nucleotides long, greater than 40 nucleotides long, greater than 50 nucleotides long, greater than 100 nucleotides long, or greater than 200 nucleotides long.

[0048] In some embodiments, the inhibitory nucleic acid sequence containing or encoding a pri-miRNA scaffold is at least 200, 250, 260, 270, 280, 290, or 300 nucleotides long. In some embodiments, the inhibitory nucleic acid contains, or consists of, a sequence of nucleotides that are at least 80% or 90% complementary to, for example, at least 5, 10, 15, 20, 25, or 30 nucleotides, or up to 30 or 40 nucleotides, of the target nucleic acid, or a sequence of nucleotides having up to 3 mismatches (e.g., up to 1 or up to 2 mismatches) over 10, 15, 20, 25, or 30 nucleotides of the target nucleic acid.

[0049] In some embodiments, the inhibitory nucleic acid is an artificial miRNA (amiRNA). amiRNAs are induced by modifying a natural miRNA, replacing the natural targeting region of the pre-mRNA with the targeting region of interest. For example, a naturally occurring and expressed miRNA can be used as a scaffold or skeleton (e.g., a pri-miRNA scaffold) with its stem sequence replaced by the stem sequence of the miRNA targeting the gene of interest. Artificial precursor microRNAs (pre-amiRNAs) are typically processed to preferentially produce a single, stable, small RNA.

[0050] Forming a stem of a stem-loop structure is a minimum requirement for the inhibitory nucleic acid encoding at least one siRNA, miRNA, shRNA, or other RNAi agent described herein. In some embodiments, the siRNA, miRNA, shRNA, or other RNAi agent described herein comprises at least one nucleic acid sequence that is partially complementary to or hybridizes with a target sequence. In some embodiments, the 5' arm of the stem-loop structure of the inhibitory nucleic acid comprises a nucleic acid sequence encoding an antisense sequence (i.e., a guide sequence / strand). In some other embodiments, the 3' arm of the stem-loop structure of the inhibitory nucleic acid comprises a nucleic acid sequence encoding an antisense / guide sequence.

[0051] In certain embodiments, the sense and antisense sequences of the stem-loop structure of the inhibitory nucleic acid are separated by a loop sequence (also known as a loop motif, linker, or linker motif). The loop sequence may be of any length and may consist of 4–30 nucleotides, 4–20 nucleotides, 4–15 nucleotides, 5–15 nucleotides, 6–12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, and / or 15 nucleotides.

[0052] Some aspects of this disclosure relate to nucleic acid sequences encoding guide strands targeting human genes, which are inserted into human or non-human (e.g., mouse) pri-miRNA scaffolds. In some embodiments, the pri-miRNA scaffold can be selected from mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155, or miR-451. In some embodiments, the pri-miRNA scaffold is adjacent to an inhibitory nucleic acid or its target sequence that targets human mRNA (e.g., FXYD1).

[0053] Therefore, gene silencing, specifically FXYD1, can be mediated by inhibitory nucleic acids, either through interaction with RNA transcripts or, alternatively, through interaction with specific gene sequences, such interactions, at the transcriptional or post-transcriptional level, for example, at RNAi, or using target nucleotide sequences, by modifying the target chromatin structure or methylation pattern, thereby preventing transcription of the target gene and resulting in gene silencing through cellular processes that mediate silencing.

[0054] These inhibitory nucleic acids may include short double-stranded regions of RNA. A double-stranded RNA molecule can be symmetric or asymmetric and may include two distinct, complementary strands, i.e., two single-stranded RNA molecules, or it may include a single-stranded molecule in which two complementary regions, e.g., a sense region and an antisense region, are base-paired and covalently linked by one or more single-stranded "hairpin" regions (i.e., loops), resulting in, for example, a single-stranded short hairpin polynucleotide or a cyclic single-stranded polynucleotide.

[0055] The linker can be a polynucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotide linker. In some embodiments, the hairpin or cyclic inhibitory nucleic acid molecule contains one or more loop motifs, and at least one of the loop portions of the molecule is biodegradable. For example, a single-stranded hairpin molecule can be designed so that in vivo degradation of the loop portion of the molecule produces a double-stranded siRNA molecule having a 3' terminal overhang, such as a 3' terminal nucleotide overhang containing 1, 2, 3, or 4 nucleotides. Alternatively, a cyclic inhibitory nucleic acid molecule can be designed so that in vivo degradation of the loop portion of the molecule produces a double-stranded siRNA molecule having a 3' terminal overhang, such as a 3' terminal overhang containing about 2 nucleotides.

[0056] In symmetric inhibitory nucleic acid molecules, each strand, the sense (passenger) strand, and the antisense (guide) strand can independently have a nucleotide length of approximately 15 to approximately 40 (e.g., approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40).

[0057] In asymmetric inhibitory nucleic acid molecules, the antisense region or chain of the molecule can have a nucleotide length of approximately 15 to approximately 30 (e.g., approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), and the sense region has a nucleotide length of approximately 3 to approximately 25 (e.g., approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25).

[0058] In yet another embodiment, the inhibitory nucleic acid molecules described herein may include single-stranded hairpin siRNA molecules, the molecules may have a nucleotide length of about 25 to about 70 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 40, 45, 50, 55, 60, 65, or 70).

[0059] In other embodiments, the molecule may include a single-stranded cyclic siRNA molecule having a nucleotide length of about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70).

[0060] In various symmetrical embodiments, the inhibitory nucleic acid double strands described herein may independently comprise about 15 to about 40 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40).

[0061] In yet another embodiment in which the inhibitory nucleic acid molecule described herein is asymmetric, the molecule may contain about 3 to 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs.

[0062] In other embodiments where the inhibitory nucleic acid molecule has a hairpin or cyclic structure, the molecule may contain about 3 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs.

[0063] The sense strand and antisense strand, or sense region and antisense region, of an inhibitory nucleic acid molecule can be complementary. Furthermore, the antisense strand or antisense region can be complementary to the nucleotide sequence or a portion thereof of the target RNA (e.g., FXYD1). The sense strand or sense region of the inhibitory nucleic acid may contain the nucleotide sequence or a portion thereof of the target gene.

[0064] In some embodiments, inhibitory nucleic acids can be optimized (based on sequence) or chemically modified to minimize degradation before and / or during delivery to the tissue of interest. Commercial sources of these interfering nucleic acids include, but are not limited to, Thermo-Fisher Scientific / Ambion, Origene, Qiagen, Dharmacon, and Santa Cruz Biotechnology. In some embodiments, such optimization and / or modification may be performed to ensure that a sufficient payload of the inhibitory nucleic acid is delivered to the tissue of interest. Other embodiments include the use of small molecules, aptamers, or oligonucleotides designed to reduce the expression of a target gene by either binding to the DNA of the gene to restrict the expression of an antisense oligonucleotide, or by imposing post-transcriptional gene silencing (PTGS) through mechanisms including, but not limited to, directly binding to a target transcript or gene product or one or more other proteins in a manner that reduces the expression of the gene, or the use of other small molecule decoys that reduce the expression of a particular gene.

[0065] Any inhibitory nucleic acid molecule or construct described herein may contain one or more chemical modifications. These modifications can be used to improve in vivo or in vivo characteristics such as stability, activity, toxicity, immune response (e.g., preventing stimulation of interferon response, inflammatory or pro-inflammatory cytokine response, or Toll-like receptor (TlF) response), and / or bioavailability.

[0066] Chemically modified molecules exhibit improved RNAi activity compared to their corresponding unmodified or minimally modified molecules. The chemically modified motifs disclosed herein provide nuclease resistance and pharmacokinetic properties suitable for use in therapeutic applications, while simultaneously maintaining RNAi activity substantially similar to that of unmodified or minimally modified active siRNAs.

[0067] In various embodiments, the inhibitory nucleic acid molecules described herein may include modifications in which any (e.g., one or more or all) nucleotides present in the sense and / or antisense strands are modified nucleotides. In some embodiments, the molecule may be partially modified by chemical modification (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80 nucleotides are modified). In other embodiments, the molecule may be completely modified by chemical modification (e.g., 100% modification).

[0068] Chemical modifications within a single molecule may be the same or different. In some embodiments, at least one chain has at least one chemical modification. In other embodiments, each chain has at least one chemical modification, which may be the same or different, such as sugar, base, or skeletal (i.e., internucleotide bond) modifications. In other embodiments, a molecule may contain at least two, three, four, five, or more different chemical modifications.

[0069] Non-limiting examples of suitable chemical modifications include, for example, those disclosed in U.S. Patent No. 8,202,979 and U.S. 2005 / 0266422, which include sugars, bases, and phosphates, non-nucleotide modifications, and / or any combination thereof.

[0070] In various embodiments, the majority of pyrimidine nucleotides present in double-stranded inhibitory nucleic acid molecules are sugar-modified. In yet another embodiment, the majority of purine nucleotides present in double-stranded molecules are sugar-modified. In certain examples, purines and pyrimidines are differentially modified at the 2' sugar position (i.e., at least one purine has a modification at the 2' sugar position that is different from at least one pyrimidine in the same or different chains).

[0071] In certain embodiments, at least one modified nucleotide is a 2'-deoxy-2-fluoronucleotide, a 2'-deoxynucleotide, or a 2'-O-alkyl (e.g., 2'-O-methyl) nucleotide. In yet other embodiments, at least one nucleotide has a ribo-like, northern, or A-type helix configuration (see, for example, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Non-limiting examples of nucleotides having a Northern configuration include Loc nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl)nucleotide); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl nucleotide, 2'-deoxy-2'-fluoronucleotide, 2'-deoxy-2'-chloronucleotide, 2'-azido nucleotide, 2'-O-trifluoromethyl nucleotide, 2'-O-ethyl-trifluoromethoxy nucleotide, 2'-O-difluoromethoxy-ethoxy nucleotide, 4'-thionucleotide, and 2'-O-methyl nucleotide.

[0072] The inhibitory nucleic acids described herein can be obtained using several techniques known to those skilled in the art. For example, inhibitory nucleic acids can be chemically synthesized or encoded by plasmids (e.g., transcribed as sequences that automatically fold into double strands with hairpin loops). siRNA can also be produced by cleaving longer dsRNAs.

[0073] In some embodiments, inhibitory nucleic acids are chemically synthesized. Oligonucleotides (e.g., certain modified oligonucleotides or parts of oligonucleotides lacking ribonucleotides) can be synthesized using protocols known in the art, such as those described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19; Thompson et al., International Patent Application No. 99 / 54459; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74, 59; Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45; and Brennan, U.S. Patent No. 6,001,311. The synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as a 5'-terminus dimethoxytrityl and a 3'-terminus phosphoramidite.

[0074] Alternatively, inhibitory nucleic acids can be synthesized separately and then joined together after synthesis, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International Patent Application No. 93 / 23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization after synthesis and / or deprotection.

[0075] In some embodiments, inhibitory nucleic acids can be delivered from a transcription unit that is expressed and inserted into a recombinant DNA or RNA vector. The recombinant vector can be a DNA plasmid or a viral vector. The viral vector can be constructed based on, but is not limited to, adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses.

[0076] B.CRISPR / Cas system In one embodiment, the repression or knockdown of one or more of the genes described herein can be achieved via CRISPR-Cas-induced nucleases using the CRISPR / Cas system and related methods known in the art. See, for example, US11225659B2, WO2021 / 168799A1, WO2022 / 188039A1, WO2022 / 188797A1, WO2020 / 068912A1, and WO2022 / 047624A1. See also Gimenez et al., “CRISPR-on System for the Activation of the Endogenous human INS gene,” Gene Therapy 23:543-547 (2016), Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012), Zhang et al., “Multiplex Genome Engineering Using CRISPR / Cas Systems,” Science 339(6121):819-23 (2013), and Gaj et al., “ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), which are incorporated herein by reference in their entirety.

[0077] The CRISPR-Cas system is a genetic technique that enables sequence-specific control of gene expression in prokaryotic and eukaryotic cells through induced nuclease double-strand DNA cleavage. It is based on the CRISPR (clustered regular-spacing palindromic repetition) pathway derived from the bacterial immune system.

[0078] In another embodiment, the application provides a complex comprising (i) a protein composition comprising a Cas protein, or an orthologue, homolog, derivative, conjugate, functional fragment thereof, conjugate thereof, or fusion thereof, and (ii) a polynucleotide composition comprising a CRISPR RNA and a programmable spacer sequence or guide sequence complementary to at least a portion of a target RNA or DNA. The programmable guide RNA, CRISPR RNA, and Cas protein together form a CRISPR / Cas-based module for sequence targeting and recognition.

[0079] The target RNA can be any RNA molecule of interest, including naturally occurring RNA molecules and engineered RNA molecules. The target RNA can be mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, microzyme, or viral RNA.

[0080] In some embodiments, the target nucleic acids are associated with conditions or disorders mediated by disrupted ion homeostasis and / or hyperexcitability, as well as conditions or diseases such as the related disorders described herein. Therefore, in some embodiments, the systems described herein can be used to treat such conditions or diseases by targeting these nucleic acids.

[0081] For example, a target nucleic acid associated with a condition or disease may be an RNA molecule overexpressed in affected cells, old or older cells, or senescent cells. The target nucleic acid may also be toxic RNA and / or mutant RNA (e.g., mRNA molecules with splicing defects or mutations). The target nucleic acid may also be a miRNA. For example, the increased activity of the target nucleic acid may contribute to hyperexcitability and neuronal loss in neurodegenerative diseases, loss of [K+]e homeostasis, and extracellular K+ + This could be due to a gene linked to the pathological increase.

[0082] Various Cas proteins can be used in this invention. Interchangeably used Cas proteins, CRISPR-related proteins, or CRISPR proteins refer to CRISPR-Cas class 1 or class 2 proteins or proteins derived therefrom, including type I, type II, type III, type IV, type V, or type VI systems that possess RNA-induced DNA binding. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas13, Cas13e, Cas13f, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or C Examples include asB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. For example, see US11225659B2, WO2021 / 168799A1, WO2022 / 188039A1, WO2022 / 188797A1, WO2022 / 068912A1, WO2022 / 047624A1, WO2014 / 144761, WO2014 / 144592, WO2013 / 176772, US2014 / 0273226, and US2014 / 0273233, the contents of which are incorporated herein by reference in their entirety.

[0083] C. Recombinant nucleic acids The recombinant nucleic acids of this disclosure include the inhibitory nucleic acids described above, as well as plasmids and vector genomes containing inhibitory nucleic acids. Recombinant nucleic acids, plasmids, or vector genomes may include regulatory sequences for regulating replication (e.g., plasmids) and / or controlling the expression of a transgene (e.g., inhibitory nucleic acid). Recombinant nucleic acids may also be provided as components of viral vectors (e.g., rAAV vectors). Generally, viral vectors include a vector genome containing recombinant nucleic acids packaged in a capsid.

[0084] D. Adjustment element This disclosure includes recombinant nucleic acids comprising transgenes (e.g., those encoding RNA) and various regulatory or control elements (e.g., Woodchuck hepatitis post-transcriptional regulatory elements). Typically, regulatory elements are nucleic acid sequences that affect the expression of operably linked polynucleotides. The exact nature of regulatory elements useful for gene expression, including, for example, promoters, enhancers, and introns, varies from organism to organism and cell to cell type, intended to facilitate the transcription and / or translation of appropriate heterologous polynucleotides. Regulatory control may be affected at levels such as transcription, translation, splicing, and message stability. Typically, regulatory elements that regulate transcription are juxtaposed near the 5' end (i.e., upstream) of the polynucleotide being transcribed. Regulatory elements may also be located at the 3' end (i.e., downstream) of the transcription sequence or within the transcript (e.g., within an intron). Regulatory elements can be located at distances from the transcription sequence (e.g., 1-100, 100-500, 500-1000, 1000-5000, 5000-10000, or more nucleotides). However, due to the length of the vector genome (e.g., AAV vector genome), regulatory elements are typically within 1-1000 nucleotides of a polynucleotide.

[0085] promoter As used herein, the term “promoter,” such as “eukaryotic promoter,” refers to a nucleotide sequence that initiates transcription of a particular gene or one or more coding sequences in a eukaryotic cell (e.g., glial progenitor cells, astrocytes, or oligodendrocytes). Promoters can work in conjunction with other regulatory elements or regions to direct the transcription level of a gene or coding sequence. These regulatory elements include, for example, transcription binding sites, repressors, and activating protein binding sites, as well as other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from a promoter, including, for example, attenuators, enhancers, and silencers. Promoters are often located on the same strand, near the transcription start site, 5', of the gene or coding sequence to which they are operably linked. Promoters are generally 100–1000 nucleotides long. Promoters typically increase gene expression compared to the expression of the same gene in the absence of the promoter.

[0086] As used herein, “core promoter” or “minimal promoter” refers to the minimum portion of a promoter sequence necessary for proper initiation of transcription. This may include a transcription start site, an RNA polymerase binding site, and a general transcription factor binding site. The promoter may also include other primary regulatory elements (e.g., enhancers, silencers, boundary elements, insulators) and a proximal promoter sequence (5' of the core promoter) containing a distal promoter sequence (3' of the core promoter).

[0087] Examples of suitable promoters include adenovirus promoters such as the major late adenovirus promoter; heterologous promoters such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoters; Rous sarcoma virus (RSV) promoter; albumin promoter; inducible promoters such as the mouse mammary cancer virus (MMTV) promoter; metallothionein promoter; heat shock promoter; α-1 antitrypsin promoter; hepatitis B surface antigen promoter; transferrin promoter; apolipoprotein A-1 promoter; chicken β-actin (CBA) promoter; elongation factor 1a promoter (EF1a), a hybrid form of the CBA promoter (CBh promoter), and the CAG promoter (cytomegalovirus early enhancer element and promoter, as well as the first exon and first intron of the tribetaactin gene, and the splice acceptor of the rabbit betaglobin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2).

[0088] Promoter may be constitutive, tissue-specific, or regulated. A constitutive promoter is one that consistently expresses a activatably linked gene. In some embodiments, constitutive promoters are active in most eukaryotic tissues under most physiological and developmental conditions.

[0089] A regulated promoter is one that can be activated or deactivated. Regulated promoters include inductive promoters, which are normally "off" but can be induced to be "on," and "repressive" promoters, which are normally "on" but can be "off." Many different regulators are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute, and constitutive promoters are often regulated to some extent. In some cases, for example, using promoters that are spontaneously downregulated when a pathological condition improves, it may be possible to utilize endogenous pathways to provide regulation of transgene expression.

[0090] A tissue-specific promoter is a promoter that is active only in a specific type of tissue, cell, or organ. Typically, a tissue-specific promoter is recognized by a transcriptional activation element specific to a particular tissue, cell, and / or organ. For example, a tissue-specific promoter may be more active in one or more specific tissues (e.g., two, three, or four) than in other tissues. In some embodiments, the expression of a gene regulated by a tissue-specific promoter is much higher in the tissue to which the promoter is specific than in other tissues. In some embodiments, the promoter may be little to substantially inactive in any tissue other than the tissue to which it is specific.

[0091] Enhancer In another embodiment, the recombinant nucleic acids described herein may further include enhancers that increase the expression of a transgene (e.g., an RNA molecule disclosed herein). Typically, the enhancer element is located upstream of the promoter element, but may be located downstream or within another sequence (e.g., the transgene). The enhancer may be located 100, 200, 300, or more nucleotides upstream or downstream of the modified nucleic acid. The enhancer typically increases the expression of the transgene (e.g., encoding an inhibitory nucleic acid) beyond the increased expression provided by the promoter element alone.

[0092] Many enhancers are known in the art, including, but not limited to, the major early enhancer of cytomegalovirus. More specifically, the CMV MIE promoter includes three regions: a modulator, a unique region, and an enhancer (Isomura and Stinski (2003) J. Virol. 77(6):3602-3614). The CMV enhancer region can be combined with another promoter, or a part thereof, to form a hybrid promoter, further increasing the expression of nucleic acids operably bound to it. For example, the CBA promoter, or a part thereof, can be combined with the CMV promoter / enhancer, or a part thereof, to create a version of the CBA called the "CBh" promoter, representing the chicken beta-actin hybrid promoter, as described by Gray et al. (2011, Human Gene Therapy 22:1143-1153). Like promoters, enhancers may be constitutive, tissue-specific, or modulated.

[0093] Fillers, spacers, and stuffers As disclosed herein, recombinant nucleic acids can be used in rAAV vectors. In this case, the recombinant nucleic acid may contain additional nucleic acid elements to adjust the length of the nucleic acid to a size close to, or to, the normal size of the viral genome sequence acceptable for AAV packaging into the rAAV vector (e.g., approximately 4.7–4.9 kilobases) (Grieger and Samulski (2005) J. Virol. 79(15):9933–9944). Such sequences may be synonymously referred to as fillers, spacers, or stuffers. In some embodiments, the filler DNA is an untranslated (non-protein-coding) segment of the nucleic acid. In some embodiments, the filler or stuffer polynucleotide sequence is a sequence of approximately 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000, or longer.

[0094] AAV vectors typically accept DNA inserts having sizes ranging from approximately 4 kb to approximately 5.2 kb or approximately 4.1 to 4.9 kb for optimal packaging of nucleic acids into the AAV capsid. In some embodiments, the rAAV vector includes a vector genome having a full length between approximately 3.0 kb to approximately 3.5 kb, approximately 3.5 kb to approximately 4.0 kb, approximately 4.0 kb to approximately 4.5 kb, approximately 4.5 kb to approximately 5.0 kb, or approximately 5.0 kb to approximately 5.2 kb. In some embodiments, the rAAV vector includes a vector genome having a full length of approximately 4.7 kb. In some embodiments, the rAAV vector includes a self-complementary vector genome. The total length of the self-complementary (sc) vector genome in an rAAV vector is equal to that of the single-stranded (ss) vector genome (i.e., approximately 4kb to 5.2kb), but the nucleic acid sequence encoding the sc vector genome (i.e., including the transgene, regulatory elements, and ITR) must be half the length of the nucleic acid sequence encoding the ss vector genome in order for the sc vector to be packaged within the capsid.

[0095] Introns and Exons In some embodiments, the recombinant nucleic acids disclosed herein include, for example, introns, exons, and / or portions thereof. Introns may function as filler or stuffer polynucleotide sequences to achieve an appropriate length for vector genome packaging into rAAV vectors. Intron and / or exon sequences may also enhance the expression of a transgene (e.g., RNA disclosed herein) compared to expression in the absence of intron and / or exon elements (Kurachi et al. (1995) J. Biol. Chem. 270(10):576-5281, WO2017 / 074526). Furthermore, filler / stuffer polynucleotide sequences (also referred to as “insulators”) are well known in the art and include, but are not limited to, those described in WO2014 / 144486 and WO2017 / 074526.

[0096] Polyadenylation signal sequence (polyA) Further regulatory elements may include, but are not limited to, stop codons, termination sequences, and polyadenylation (polyA) signal sequences, such as the bovine growth hormone polyA signal sequence (BHG polyA). PolyA signal sequences drive the efficient addition of a polyadenosine "tail" at the 3' end of eukaryotic mRNA, inducing the termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23):16330-16334). The polyA signal functions as a signal for endonucleotide cleavage of the newly formed precursor mRNA at its 3' end and for the addition of an RNA stretch consisting solely of adenine bases to this 3' end. The polyA tail is important for mRNA nuclear export, translation, and stability. In some embodiments, polyA can be an early SV40 polyadenylation signal, a late SV40 polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5 E1b polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal, or a polyadenylation signal designed in silico.

[0097] 3. Expression cassettes and expression vectors This disclosure also provides expression cassettes comprising, or comprising, recombinant nucleic acids encoding the inhibitory nucleic acids described above. If such recombinant nucleic acids do not yet contain a promoter, the expression cassette may further include a promoter. Thus, the expression cassette according to the present invention comprises, in the 5' to 3' direction, a promoter, a coding sequence, and optionally a terminator or other elements. The expression cassette enables the easy transfer of the nucleic acid sequence of interest into an organism, preferably a cell, preferably a diseased cell.

[0098] The expression cassettes of the present disclosure are preferably contained in a vector. Therefore, the vectors of the present disclosure enable the transformation of cells having a nucleic acid sequence of interest. Correspondingly, the present disclosure provides host cells containing an expression cassette or recombinant nucleic acid according to the present disclosure. The recombinant nucleic acid may also contain a promoter or enhancer that enables the expression of the nucleic acid sequence of interest.

[0099] Exogenous genetic material (e.g., nucleic acids, expression cassettes, or expression vectors encoding one or more therapeutic or inhibitory RNAs) can be introduced in vivo into target cells of interest by gene transfer methods such as transfection or transduction to provide genetically modified cells. Various expression vectors (i.e., vehicles for facilitating the delivery of exogenous genetic material to target cells) are known to those skilled in the art. As used herein, “exogenous genetic material” refers to nucleic acids or oligonucleotides, whether natural or synthetic, that are not found naturally in cells, or, if found naturally in cells, are not transcribed or expressed by cells at biologically significant levels. Therefore, “exogenous genetic material” includes, for example, nucleic acids that are not naturally occurring and can be transcribed into RNA.

[0100] As used herein, “cellular transfection” refers to the acquisition of new genetic material by a cell through the incorporation of added nucleic acid (DNA, RNA, or a hybrid thereof) without the use of a viral delivery vehicle. Therefore, transfection refers to the introduction of nucleic acid into a cell using physical or chemical methods. Several transfection techniques are known to those skilled in the art and include calcium phosphate nucleic acid coprecipitation, strontium phosphate nucleic acid coprecipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, and tungsten particle-enhanced microparticle impaction. In contrast, “cellular transduction” refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. RNA viruses (e.g., retroviruses) used for transferring nucleic acid into a cell are referred to herein as transduction chimeric viruses. The exogenous genetic material contained within the virus can be incorporated into the genome of the transduced cell. Cells transduced with a chimeric DNA virus (for example, an adenovirus carrying DNA encoding a therapeutic agent) may not possess exogenous genetic material integrated into their genome, but they may be able to express exogenous genetic material that is held outside the chromosome within the cell.

[0101] Typically, exogenous genetic material may contain a heterologous gene (encoding therapeutic RNA or protein) along with a promoter that controls the transcription of the new gene. The promoter characteristically has a specific nucleotide sequence required to initiate transcription. Optionally, the exogenous genetic material may further contain additional sequences (i.e., enhancers) required to obtain the desired gene transcriptional activity. The exogenous genetic material may be introduced into the cellular genome immediately downstream of the promoter so that the promoter and coding sequence are operably ligated to enable transcription of the coding sequence. Retroviral expression vectors may also contain exogenous promoter elements to control the transcription of the inserted exogenous gene. Such exogenous promoters may include both constitutive and inductive promoters.

[0102] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes under the control of constitutive promoters are expressed under all conditions of cell growth. Exemplary constitutive promoters include those for the following genes encoding specific constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase, dihydrofolate reductase, adenosine deaminase, phosphoglycerol kinase, pyruvate kinase, phosphoglycerol mutase, actin promoter, ubiquitin, elongation factor-1, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long-terminal repeats (LTRs) of Moloney's leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Therefore, any of the above constitutive promoters can be used to control the transcription of heterologous insertors.

[0103] Genes under the control of an inductive promoter are expressed only in the presence of an inducer, or are greatly regulated by it (for example, transcription under the control of a metallothionein promoter is greatly increased in the presence of certain metal ions). Inductive promoters contain responsive elements (REs) that stimulate transcription when their inducers bind to them. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain an inductive response, and in some cases, the RE itself can be bound to a different promoter, thereby conferring inductivity to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive vs. inductive, strong vs. weak), it is possible to control both the presence and expression level of therapeutic agents in recombinant cells. If the gene encoding a therapeutic agent is under the control of an inductive promoter, in-situ delivery of the therapeutic agent can be induced, for example, by injecting a specific inducer of the inductive promoter that controls the transcription of the drug, or by exposing the recombinant cells in situ to conditions that enable the transcription of the therapeutic agent. For example, in-situ expression of a therapeutic agent encoded by a gene under the control of the metallothionein promoter by recombinant cells can be enhanced by contacting the recombinant cells in situ with a solution containing the appropriate (i.e., inducing) metal ions.

[0104] Therefore, the amount of therapeutic agent delivered in situ is regulated by controlling the following factors: (1) the nature of the promoter used to direct the transcription of the inserted gene (i.e., whether the promoter is constitutive or inductive, strong or weak); (2) the number of copies of the exogenous gene inserted into the cell; (3) the number of transduced / transfected cells administered to the patient (e.g., transplanted); (4) the size of the implant (e.g., graft or encapsulated expression system); (5) the number of implants; (6) the length of time the transduced / transfected cells or implants remain in place; and (7) the rate of therapeutic agent production by the genetically modified cells. The selection and optimization of these factors for the delivery of a specific therapeutic agent in a therapeutically effective dose is considered to be within the scope of the art without excessive experimentation, taking into account the factors disclosed above and the patient's clinical profile.

[0105] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may include a selection gene, such as a neomycin resistance gene or a fluorescent protein gene, to facilitate the selection of cells transfected or transduced with the expression vector. Alternatively, cells may be transfected with two or more expression vectors, where at least one vector contains a gene encoding a therapeutic agent and the other vector contains a selection gene. The selection of suitable promoters, enhancers, selection genes, and / or signal sequences is considered within the scope of those skilled in the art without excessive experimentation.

[0106] The coding sequences of this disclosure can be inserted into any type of target or host cell. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method of the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0107] 4. Delivery of carrier / polynucleotide As disclosed herein, the polynucleotides or nucleic acid molecules described above can be used to treat disorders in a subject. Accordingly, this disclosure provides systems and methods for delivering polynucleotides to target cells or a subject.

[0108] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0109] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents No. 5,350,674 and No. 5,585,362.

[0110] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0111] The polynucleotides or nucleic acids described herein (e.g., inhibitory nucleic acids, those encoding the CRISPR-Cas system, expression cassettes, and expression vectors) can be directly attached, complexed with cationic lipids and packaged in liposomes, or delivered to target cells or tissues as recombinant plasmids or viral vectors. Methods for delivering nucleic acid molecules are known in the art. For example, see U.S. Patent No. 6,395,713, WO94 / 02595; Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et al., 1999, Mol.Membr.Biol., 16, 129-140; Hofland and Huang, 1999, Handb.Exp.Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp.Ser., 752, 184-192. These protocols can be used for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by various methods known to those skilled in the art, including, but not limited to, ion electrophoresis, or by incorporation into other vehicles such as biodegradable polymers, hydrogels, cyclodextrins (see, e.g., Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074, WO03 / 47518 and WO03 / 46185), poly(lactic acid-coglycol) acid (PLGA) and PLCA microspheres (see, e.g., U.S. Patent No. 6,447,796 and U.S. 2002 / 130430), biodegradable nanocapsules, and bioadhesive microspheres, or by protein vectors (see, e.g., WO00 / 53722), including encapsulation within liposomes.

[0112] In one embodiment, this application provides a carrier system containing nucleic acid molecules as described herein. In some embodiments, the carrier system is a lipid-based carrier system, a cationic lipid, or a liposomal nucleic acid complex, liposomes, micelles, viromosomes, lipid nanoparticles, or a mixture thereof. In other embodiments, the carrier system is a polymer-based carrier system, such as a cationic polymer nucleic acid complex. In additional embodiments, the carrier system is a cyclodextrin-based carrier system, such as a cyclodextrin polymer-nucleic acid complex. In further embodiments, the carrier system is a protein-based carrier system, such as a cationic peptide-nucleic acid complex. Preferably, the carrier system is a lipid nanoparticle formulation. The lipid nanoparticle ("LNP") formulations described herein can be applied to any nucleic acid molecule (e.g., RNA molecule) or a combination of nucleic acid molecules as described herein.

[0113] In certain embodiments, the nucleic acid molecules described herein are formulated as lipid nanoparticle compositions as described in U.S. Patents 7,514,099 and 7,404,969. In some embodiments, this application features compositions comprising nucleic acid molecules formulated as one of the formulations described in U.S.2012 / 0029054, such as LNP-051, LNP-053, LNP-054, LNP-069, LNP-073, LNP-077, LNP-080, LNP-082, LNP-083, LNP-060, LNP-061, LNP-086, LNP-097, LNP-098, LNP-099, LNP-100, LNP-101, LNP-102, LNP-103, or LNP-104.

[0114] In other embodiments, the present disclosure features conjugates and / or complexes of nucleic acid molecules as described herein. Such conjugates and / or complexes can be used to facilitate the delivery of nucleic acid molecules to living systems such as cells. The conjugates and complexes provided herein can impart therapeutic activity by transferring therapeutic compounds across cell membranes, altering pharmacokinetics, and / or modulating the localization of the nucleic acid molecules of the present invention. Non-limiting examples of such conjugates are described, for example, in U.S. Patents No. 7,833,992, No. 6,528,631, No. 6,335,434, No. 6,235,886, No. 6,153,737, No. 5,214,136, and No. 5,138,045.

[0115] In various embodiments, polyethylene glycol (PEG) can be covalently attached to nucleic acid molecules as described herein. The attached PEG can have any molecular weight, preferably about 100 to about 50,000 daltons (Da). Accordingly, this disclosure features compositions or formulations comprising poly(ethylene glycol) lipids (PEG-modified or long-circulating liposomes or stealth liposomes) and surface-modified liposomes containing nucleic acid molecules as described herein. (See, for example, WO96 / 10391, WO96 / 10390, and WO96 / 10392).

[0116] In some embodiments, nucleic acid molecules may also be formulated or complexed with polyethyleneimines and their derivatives, such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, nucleic acid molecules may be formulated in the manner described in US2003 / 0077829.

[0117] In other embodiments, the nucleic acid molecules described herein may be complexed with membrane disruptors such as those described in US2001 / 0007666. In yet another embodiment, the membrane disruptors and molecules may be complexed with cationic lipids or helper lipid molecules such as the lipids described in U.S. Patent No. 6,235,310.

[0118] In certain embodiments, the nucleic acid molecules described herein may be complexed with delivery systems described in U.S. Patent Publications 2003 / 077829, 2005 / 0287551, 2005 / 0164220, 2005 / 0191627, 2005 / 0118594, 2005 / 0153919, 2005 / 0085486, and 2003 / 0158133, as well as IWO00 / 03683 and WO02 / 087541.

[0119] In some embodiments, the liposome formulations described herein are subject to U.S. Patent Nos. 6,858,224, 6,534,484, 6,287,591, 6,835,395, 6,586,410, 6,858,225, 6,815,432, 6,586,001, 6,120,798, 6,977,223, 6,998,115, 5,981,501, 5,976,567, 5,705,385, and U.S. Patent Application Publications 2006 / 0019912 and 2006 / 001925. This specification may include nucleic acid molecules (e.g., inhibitory nucleic acids) formulated or complexed with compounds and compositions described in No. 8, No. 2006 / 0008909, No. 2005 / 0255153, No. 2005 / 0079212, No. 2005 / 0008689, No. 2003 / 0077829, No. 2005 / 0064595, No. 2005 / 0175682, No. 2005 / 0118253, No. 2004 / 0071654, No. 2005 / 0244504, No. 2005 / 0265961, and No. 2003 / 0077829.

[0120] As disclosed herein, the nucleic acid molecules described above can be used to treat disorders in a subject. The vectors described above (such as recombinant plasmids and viral vectors) can be used to deliver inhibitory nucleic acids or therapeutic agents such as the CRISPR-Cas system described herein. Delivery of the vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from the subject and subsequently reintroduced into the subject, or by any other means that would enable delivery to the desired target cells. Such recombinant vectors can also be administered directly or in conjunction with suitable delivery reagents, such as Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), or liposomal lipid-based carrier systems, cationic lipids, or liposomal nucleic acid complexes, micelles, viromosomes, or lipid nanoparticles.

[0121] A. Viral vectors In some embodiments, the polynucleotide encoding the therapeutic agent (e.g., an RNA molecule) may be inserted into or encoded by a vector such as a plasmid or a viral vector. Preferably, the polynucleotide is inserted into or encoded by a viral vector. The viral vector may be a herpesvirus (HSV) vector, a retroviral vector, an adenovirus vector, an AAV vector, a lentiviral vector, and the like. In some specific embodiments, the viral vector is an AAV vector. In some embodiments, the RNA may be encoded by a retroviral vector (see, for example, U.S. Patents 5,399,346, 5,124,263, 4,650,764, and 4,980,289, the contents of each of these are incorporated herein by reference in their entirety).

[0122] lentiviral vectors Lentiviruses, such as HIV, are "slow viruses." Vectors derived from lentiviruses can be expressed for extended periods in host cells after several administrations to a patient, for example, via ex vivo transdextrinsed stem cells or progenitor cells. For most diseases and disorders, including genetic disorders, cancer, and neurological disorders, long-term expression is essential for successful treatment. Regarding the safety of lentiviral vectors, several strategies for eliminating the replication ability of lentiviral vectors are now known in the art. See, for example, US2021 / 0401868 and US2021 / 0403517, which are incorporated herein by reference in their entirety. For example, deletion of promoter and enhancer elements from the U3 region of a long terminal repeat (LTR) is considered to result in an LTR without the indicated transcription. The resulting vector is called "self-inactivated" (SIN).

[0123] Lentiviral vectors are particularly suitable for achieving long-term gene transfer because they allow for the long-term and stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from onchoretroviruses such as mouse leukemia virus in that they can transduce non-proliferating cells such as CNS cells. They also have the additional advantage of lower immunogenicity. Generally, suitable vectors contain a functional origin of replication, a promoter sequence, a convenient restriction nuclease site, and one or more selection markers (e.g., WO01 / 96584 and WO01 / 29058, and U.S. Patent No. 6,326,193) in at least one organism. Several vector promoter sequences are available for transgene expression. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence ligated to it. Another example of a suitable promoter is EF1a. However, other constitutive promoter sequences may also be used, but are not limited to, the Simian virus 40 (SV40) initial promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate initial promoter, Roussarcoma virus promoter, and, as well as, but are not limited to, human gene promoters such as actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0124] This disclosure provides recombinant lentiviruses that can infect dividing and non-dividing cells, such as oligodendrocytes, astrocytes, or glial progenitor cells. The viruses are useful for in vivo and ex vivo transfer and expression of nucleic acid sequences. The lentiviral vectors of this disclosure may be lentiviral transfer plasmids or infectious lentiviral particles. Constructions of lentiviral vectors, helper constructs, envelope constructs, etc., for use in lentiviral transfer systems are described, for example, in US2021 / 0401868 and US2021 / 0403517, which are each incorporated herein in their entirety by reference.

[0125] Adenovirus Adenoviruses are eukaryotic DNA viruses that can be modified in vivo to efficiently deliver nucleic acids to various cell types and are widely used in gene therapy protocols, including those for targeting genes in nerve cells and glial cells. Various replication-deficient adenoviruses and minimal adenovirus vectors have been described for nucleic acid therapeutics (see, for example, PCT Patent Publications 1994 / 26914, 1995 / 02697, 1994 / 28152, 1994 / 12649, 1995 / 02697, and 1996 / 22378, the contents of each of these being incorporated together by reference). Such adenovirus vectors can also be used to deliver the therapeutic molecules of the present disclosure to cells.

[0126] 4. AAV Adeno-associated viruses are widely used gene therapy vectors due to their clinical safety record, non-pathogenic nature, ability to infect non-dividing cells (such as neurons), and ability to provide long-term gene expression after a single dose (Hocquemiller et al., 2016). Many human and non-human primate AAV serotypes have now been identified (Gao et al., 2004). AAV vectors have demonstrated safety in hundreds of clinical trials worldwide, and clinical efficacy has been shown in trials for hemophilia B, spinal muscular atrophy, alpha-1 anti-trypsone, and Leber congenital amaurosis (Keeler et al., 2017). Three AAV-based gene therapies have been approved. The first, Glybera, was approved by the European Medicines Agency (EMA) in 2012 (and revoked in 2017, primarily due to commercial failure). Luxturna was approved by the FDA in 2017 for a rare hereditary retinal dystrophy, and Zolgensma was approved by the FDA in 2019 for spinal muscular atrophy.

[0127] Due to their safety, non-pathogenic nature, and ability to infect neurons, AAVs, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9, are commonly used gene therapy vectors for CNS application. However, after direct CNS injection, these serotypes exhibit dominant neuronal orientation, particularly when gene expression is driven by constitutive promoters, with low expression in glial cells. AAV1 / 2, AAV2, and AAV8 have been shown to transduce oligodendrocytes only when oligodendrocyte-specific promoters are used (Chen et al., 1998; Lawlor et al., 2009; Li et al., 2019). Dependence on cell-specific promoters for expression specificity allows for non-selective cell uptake in non-oligodendrocyte lineage cells and the possibility of leaky expression of transgenes via latent promoter activity.

[0128] Approaches described herein to mitigate these challenges include the use of AAV serotypes with high directionality to glial cells. Recently, a chimeric AAV capsid, AAV / Olig001, with strong selectivity for oligodendrocytes has been described using DNA shuffling and directional evolution (Powell et al., 2016). Subsequently, AAV / Olig001 has been shown to transduce neonatal oligodendrocytes in a mouse model of Canavan disease (Francis et al., 2021). Capsid libraries can be generated that can be screened for directionality and selectivity to glial progenitor cells, astrocytes, or oligodendrocytes using other approaches such as random mutagenesis and peptide library insertion.

[0129] As stated above, the terms “adeno-associated virus” and / or “AAV” refer to parvoviruses and their variants that have a linear, single-stranded DNA genome. This term encompasses all subtypes, as well as both naturally occurring and recombinant forms, unless otherwise required. Parvoviruses, including AAVs, are useful as gene therapy vectors because they can enter cells and introduce nucleic acids (e.g., transgenes) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms a circular concatemer that persists as an episome within the nucleus of the transduced cell. In some embodiments, the transgene is inserted into a specific site in the host cell genome, for example, a site on human chromosome 19. Site-directed insertion, in contrast to random insertion, is considered more likely to result in a predictable long-term expression profile. The insertion site of AAV into the human genome is referred to as AAVS1. Once introduced into cells, the RNA or polypeptide encoded by the nucleic acid can be expressed by the cells. Since AAV is not associated with any pathogenic disease in humans, the nucleic acids delivered by AAV can be used to express therapeutic RNA or polypeptides for the treatment of diseases, disorders, and / or conditions in human subjects.

[0130] Multiple serotypes of AAV exist naturally, and at least 15 wild-type serotypes (i.e., AAV1–AAV15) have been identified in humans to date. Naturally occurring and variant serotypes are distinguished by having serologically different protein capsids from other AAV serotypes. Examples include, in particular, AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74 (see WO2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as variants produced by recombination (e.g., capsid variants with insertions, deletions, and substitutions), such as variants designated as AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, and RHM4-1. For example, "primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals.

[0131] Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies against a given AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences and antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAVs or artificial AAV mutations (e.g., recombinant AAVs) may not exhibit serological differences from any of the currently known serotypes. These viruses may then be considered a subgroup of the corresponding type, or more simply, a variant AAV. Thus, as used herein, the term “serotype” refers to both serologically distinct viruses and viruses that are serologically indistinguishable but may fall within a subgroup or variant of a given serotype.

[0132] A comprehensive list and alignment of amino acid sequences of known AAV serotype capsids is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909. Genomic sequences of various serotypes of AAV, as well as sequences of native ITRs, 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. For example, see GenBank accession numbers NC_002077(AAV1), AF063497(AAV1), NC_001401(AAV2), AF043303(AAV2), NC_001729(AAV3), NC_001863(AAV3B), NC_001829(AAV4), U89790(AAV4), NC_006152(AAV5), NC_001862(AAV6), AF513851(AAV7), AF513852(AAV8), and NC_006261(AAV8), which are incorporated herein by reference. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71:6823, Chiorini et al. (1999) J. Virology 73:1309, Bantel-Schaal et al. (1999) J. Virology 73:939, Xiao et al. al. (1999) J. Virology 73:3994, Muramatsu et al. (1996) Virology 221:208, Shade et al. (1986) J. Virol. 58:921, Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854, Morris et al. (2004) Virology See also 33:375-383, International Patent Publications 00 / 28061, 99 / 61601, 98 / 11244, 2013 / 063379, 2014 / 194132, 2015 / 121501, and U.S. Patents 6,156,303 and 7,906,111.

[0133] As discussed herein, “recombinant adeno-associated virus” or “rAAV” is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with non-natural sequences. Incorporating non-natural sequences into the virus defines the viral vector as a “recombinant” vector, and therefore, an “rAAV vector.” An rAAV vector may contain heterologous polynucleotides encoding a desired RNA or protein or polypeptide (e.g., the RNA molecules disclosed herein). The recombinant vector sequence may be capsidized or packaged in an AAV capsid and referred to as an “rAAV vector,” an “rAAV vector particle,” an “rAAV viral particle,” or simply “rAAV.”

[0134] For the production of rAAV vectors, the desired ratio of VP1:VP2:VP3 can be in the range of about 1:1:1 to about 1:1:100, preferably in the range of about 1:1:2 to about 1:1:50, and more preferably in the range of about 1:1:5 to about 1:1:20. The desired ratio of VP1:VP2 is 1:1, but the range of the VP1:VP2 ratio can vary from 1:50 to 50:1.

[0135] This disclosure provides rAAV vectors containing polynucleotide sequences that are not of AAV origin (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotides may be flanked by at least one, and possibly two, AAV terminal repeat sequences (e.g., reverse terminal repeats). The heterologous polynucleotides flanked by the ITRs, also referred to herein as the “vector genome,” typically encode the RNA or polypeptide of interest, or the gene of interest, e.g., a target for therapeutic treatment. Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the encoded RNA / protein / peptide to the subject. Thus, rAAV vectors can be used to transcribe / deliver heterologous polynucleotides for expression, for example, to treat various diseases, disorders, and conditions.

[0136] rAAV vector genomes generally retain a 145-base ITR cis relative to heterologous nucleic acid sequences that replace the viral rep and cap genes. While such ITRs are useful for producing recombinant AAV vectors, modified AAV ITRs and non-AAV terminal repeats containing partially or completely synthesized sequences can also serve this purpose. ITRs form hairpin structures and function, for example, as primers for host cell-mediated synthesis of complementary DNA strands after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements required cis for AAV genome replication and packaging into rAAV vectors. rAAV vector genomes optionally contain two ITRs commonly present at the 5' and 3' ends of the vector genome containing heterologous sequences (e.g., transgenes encoding the gene of interest, or nucleic acid sequences of interest including but not limited to antisense and siRNA, and CRISPR molecules, among many others). The 5' and 3' ITRs may both contain the same sequence, or each may contain a different sequence. AAV ITR may originate from any AAV, including but not limited to serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV.

[0137] The rAAV vectors of this disclosure may contain an ITR from an AAV serotype (e.g., wild-type AAV2, a fragment or variant thereof) different from the serotype of the capsid (e.g., AAV8, Olig001). Such an rAAV vector containing at least one ITR from one serotype but a capsid from a different serotype may be referred to as a hybrid viral vector (see U.S. Patent No. 7,172,893). The AAV ITR may contain the entire wild-type ITR sequence, or a variant, fragment, or modification thereof, while retaining functionality.

[0138] In some embodiments, the rAAV vector genome is a linear single strand adjacent to an AAV ITR. Before transcription and translation of heterologous genes, the approximately 4700 nucleotide single-stranded DNA genome must be converted to a double-stranded form by DNA polymerase (e.g., DNA polymerase in the transduced cell) using a free 3'-OH from one of the self-priming ITRs to initiate the synthesis of the second strand. In some embodiments, the full-length single-stranded vector genomes (i.e., sense and antisense) anneal to produce a full-length double-stranded vector genome. This can occur when multiple rAAV vectors carrying genomes of opposite polarity (i.e., sense or antisense) are transduced into the same cell simultaneously. Regardless of how they are produced, once a double-stranded vector genome is formed, the cell can transcribe and translate the double-stranded DNA and express heterologous genes.

[0139] The efficiency of transgene expression from rAAV vectors can be hindered by the need to convert single-stranded rAAV genomes (ssAAV) to double-stranded DNA before expression. This step can be avoided by using self-complementary AAV genomes (scAAV) that can package reverse-repetitive genomes that can fold into double-stranded DNA without requiring DNA synthesis or base pairing between multiple vector genomes. See, for example, U.S. Patent No. 8,784,799; McCarty, (2008) Molec. Therapy 16(10):1648-1656, and McCarty et al., (2001) Gene Therapy 8:1248-1254, and McCarty et al., (2003) Gene Therapy 10:2112-2118.

[0140] The viral capsid of the rAAV vector may be derived from wild-type AAV or variant AAV, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: 5 of WO2015 / 013313), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV29G, AAV2.8G9, AAV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, goat AAV, shrimp AAV, ovine AAV, and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4 th ed., Lippincott-Raven Publishers). The capsid may be derived from several AAV serotypes disclosed in U.S. Patent No. 7,906,111, Gao et al. (2004) J. Virol. 78:6381, Morris et al. (2004) Virol. 33:375, WO2013 / 063379, WO2014 / 194132, and may include the true type AAV (AAV-TT) variants disclosed in WO2015 / 121501, as well as RHM4-1, RHM15-1 to RHM15-6, and variants thereof disclosed in WO2015 / 013313. The full complement of AAV cap proteins includes VP1, VP2, and VP3. The ORF comprising the nucleotide sequence encoding the AAV VP capsid protein may comprise less than the full complement of AAV Cap proteins, or the full complement of AAV cap proteins may be provided.

[0141] In some embodiments, an rAAV vector containing a capsid protein encoded by nucleotide sequences derived from two or more AAV serotypes (e.g., wild-type AAV serotype, variant AAV serotype) is referred to as a “chimeric vector” or “chimeric capsid” (see U.S. Patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid protein is encoded by nucleic acid sequences derived from two, three, four, five, six, seven, eight, nine, ten or more AAV serotypes. In some embodiments, recombinant AAV vectors include capsid sequences derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variants thereof, resulting in a chimeric capsid protein containing a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may include a VP1 from one serotype, a VP2 from a different serotype, a VP3 from yet another different serotype, and a mixture of combinations thereof. For example, a chimeric viral capsid may include an AAV1 cap protein or subunit, as well as at least one AAV2 cap protein or subunit. A chimeric capsid may include, for example, an AAV capsid having one or more B19cap subunits, where, for example, an AAV cap protein or subunit may be replaced by a B19cap protein or subunit. For example, in one embodiment, the VP3 subunit of an AAV capsid may be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is an Olig001 capsid, such as those described in WO2021 / 221995 and WO2014 / 052789, which are incorporated herein by reference.

[0142] In some embodiments, chimeric vectors are engineered to exhibit altered directivity or directivity to specific tissues or cell types. The term “directivity” refers to the preferential entry of a virus into a particular cell type (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) or tissue type, and / or preferential interaction with the cell surface that facilitates entry into a particular cell type or tissue type. AAV directivity is generally determined by specific interactions between different viral capsid proteins and their homologous cell receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, once the virus or viral vector enters a cell, sequences (e.g., heterologous sequences such as transgenes) carried by the vector genome (e.g., rAAV vector genome) are expressed.

[0143] A "directional profile" refers to the pattern of transduction of one or more target cells in various tissues and / or organs. For example, a chimeric AAV capsid may have a directional profile characterized by efficient transduction of astrocytes, with only low transduction of neurons, oligodendrocytes, and other CNS cells.

[0144] Such chimeric capsids may be considered "astroglial-specific" if, when administered directly to the CNS, they preferentially transduce astrocytes over neurons, oligodendrocytes, and other CNS cell types, exhibiting astrocyte-specificity. In some embodiments, at least about 80% of the cells transduced by an astrocyte-specific capsid are astrocytes, for example, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are astrocytes.

[0145] 5. Virus particles and production A viral vector (e.g., an rAAV vector) having a transgene (e.g., one encoding the RNA disclosed herein) can be assembled from polynucleotides encoding the transgene, suitable regulatory elements, and elements necessary for the production of a viral protein that mediates cell transduction. Examples of viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, herpesviruses, and AAV vectors, particularly rAAV vectors.

[0146] The vector genome components of an rAAV vector produced according to the methods of this disclosure include at least one transgene (e.g., a polynucleotide encoding an RNA molecule) and associated expression regulatory sequences for controlling RNA expression. In preferred embodiments, the vector genome includes a portion of a parvovirus genome, such as an AAV genome deleting rep and cap, and / or replaced by a transgene and its associated expression regulatory sequences. The transgene is typically inserted adjacent to (i.e., flanked to) one or two AAV ITR or ITR elements sufficient for viral replication, in place of the nucleic acids encoding the viral rep and cap proteins. Other regulatory sequences suitable for use in promoting tissue-specific expression of the transgene in target cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) may also be included.

[0147] A. Packaging cells Those skilled in the art will understand that an rAAV vector containing a transgene but lacking viral proteins necessary for viral replication (e.g., cap and rep) cannot replicate because such proteins are required for viral replication and packaging. The cap and rep genes may be supplied to the cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid separate from the plasmid supplying the transgene to the vector genome.

[0148] Packaging cells or producer cells mean cells or cell lines that can be transfected with a vector, plasmid, or DNA construct and that transfect the lost functions necessary for the complete replication and packaging of the viral vector. Genes required for the assembly of the rAAV vector include the vector genome (e.g., RNA-coding transgenes, regulatory elements, and ITRs), the AAV rep gene, the AAV cap gene, and certain helper genes from other viruses (e.g., adenoviruses). Those skilled in the art will understand that the genes necessary for AAV production can be introduced into packaging cells by various methods, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, helper) may already be present in the packaging cells, be incorporated into the genome, or be carried on episomes. In some embodiments, the packaging cells express one or more lost viral functions in a constitutive or inductive manner.

[0149] Any suitable packaging cells known in the art may be used to produce packaged viral vectors. Mammalian cells or insect cells are preferred. Examples of cells useful for producing packaging cells in practice of this disclosure include, for example, PER.C6, WI38, MRC5, A549, HEK293 cells (expressing functional adenovirus E1 under the control of a constitutive promoter), B-50, or any other HeLa cells, human cell lines such as HepG2, Saos-2, HuH7, and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC, or CHO cells.

[0150] In some embodiments, packaging cells can be grown in suspension culture. In some embodiments, packaging cells can be grown in serum-free medium. For example, HEK293 cells are grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells, such as those described in U.S. Patent No. 9,441,206, deposited in the American Type Culture Collection (ATCC) under number PTA13274. Numerous rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO2002 / 46359.

[0151] Examples of cell lines for use as packaging cells include insect cell lines. Any insect cell line that can enable AAV replication and be maintained in culture can be used in accordance with this disclosure. Examples include Spodoptera frugiperda, e.g., Sf9 or Sf21 cell lines, Drosophila cell lines, or mosquito cell lines, e.g., Aedes albopictus cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein with respect to their teachings regarding the use of insect cells for the expression of heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al. (1989) J. Virol. 63:3822-3828; Kajigaya et al. (1991) Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al. (1992) J. Virol. 66:6922-6930; Kimbauer et al. (1996) Virol. 219:37-44; Zhao et al. al. (2000) Virol. 272:382-393, and U.S. Patent No. 6,204,059.

[0152] As a further alternative, the viral vectors of this disclosure may be produced in insect cells using a baculovirus vector for delivering the rep / cap gene and rAAV template, as described, for example, by Urabe et al. (2002) Human Gene Therapy 13:1935-1943. When baculovirus production is used for AAV, in some embodiments the vector genome is self-complementary. In some embodiments, the host cell is a baculovirus-infected cell (e.g., an insect cell) that optionally contains additional nucleic acids encoding baculovirus helper functions, thereby promoting the production of the viral capsid.

[0153] Packaging cells generally contain one or more viral vector functions, along with helper functions and packaging functions sufficient to result in viral vector replication and packaging. These various functions can be supplied to packaging cells together or separately using gene constructs such as plasmids or amplicons, and they may be extrachromosomal within the cell line or integrated into the chromosomes of the host cell.

[0154] B. Helper function AAV cannot replicate within cells without co-infection with a helper virus. Helper function includes helper viral elements necessary to establish active infection of the packaging cell, which is required to initiate the packaging of the viral vector. Helper viruses typically include adenoviruses or herpes simplex viruses. Adenovirus helper function typically includes adenovirus components, adenovirus initial region 1A (E1a), E1b, E2a, E4, and virus-associated (VA) RNA. Helper function (e.g., E1a, E1b, E2a, E4, and VA RNA) can be provided to the packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements. Alternatively, the host cell (e.g., the packaging cell) may contain nucleic acids encoding helper proteins. For example, HEK293 cells are generated by transforming human cells with adenovirus 5 DNA and currently express several adenovirus genes, including but not limited to E1 and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72). Thus, these helper functions can be provided by HEK293 packaging cells without the need to supply them to the cells, for example, by encoding plasmids.

[0155] In some embodiments, packaging cells are transfected with at least (i) a plasmid comprising a vector genome containing a transgene and AAV ITR, and further comprising at least one of the regulatory elements of enhancers, promoters, exons, introns, and polyA; (ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., Olig001 cap); and (iii) a plasmid comprising helper function.

[0156] Any method can be used to introduce a nucleotide sequence having helper function into a cell host for replication and packaging, including, but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals. In some embodiments, the helper function is provided by transfection using a viral vector or by infection using a helper virus, and standard methods for producing viral infection may be used.

[0157] The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct, and may be single-stranded, double-stranded, or double-stranded (i.e., self-complementary as described in WO2001 / 92551).

[0158] C. Production of packaged viral vectors Viral vectors can be prepared by several methods known to those skilled in the art (see, for example, WO2013 / 063379). A preferred method is described in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated herein by reference for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, and adherent HEK293 cell lines from a qualified clinical master cell bank are grown under animal component-free suspension conditions in shaker flasks and WAVE bioreactors, enabling rapid and scalable rAAV production. Using a triple transfection method (e.g., WO96 / 40240), the HEK293 cell line suspension yields 1 × 10⁶ cells when collected 48 hours after transfection. 5 Ultraparticle-containing vector genome (vg) / cell, or 1 × 10⁻⁶ 14It is possible to generate a vg / L (cell culture) of more than . More specifically, triple transfection refers to a method of transfecting packaging cells with three plasmids (one plasmid encoding AAV rep and cap genes, another plasmid encoding various helper functions (e.g., adenovirus or HSV proteins, e.g., E1a, E1b, E2a, E4, and VA RNA), and another plasmid encoding a transgene (e.g., RNA as described herein) and various elements that control the expression of the transgene).

[0159] Single-stranded vector genomes are packaged in capsids in approximately equal proportions as either the positive or negative strand. In some embodiments of rAAV vectors, the vector genome is positive-strand polar (i.e., the sense or coding sequence of the DNA strand). In some embodiments of rAAV vectors, the vector is negative-strand polar (i.e., the antisense or template DNA strand). Given the nucleotide sequence of the positive strand with a 5' to 3' orientation, the nucleotide sequence of the negative strand with a 5' to 3' orientation can be determined as the reverse complement of the nucleotide sequence of the positive strand.

[0160] To achieve the desired yield, several variables are optimized, including the selection of a suitable serum-free suspension medium that supports both growth and transfection, the selection of transfection reagents, transfection conditions, and cell density.

[0161] rAAV vectors can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include those described in Clark et al. (1999) Human Gene Therapy 10(6):1031-1039, Schenpp and Clark (2002) Methods Mol. Med. 69:427-443, U.S. Patent No. 6,566,118, and WO98 / 09657.

[0162] A universal purification strategy based on ion exchange chromatography may be used to produce high-purity vector preparations of AAV serotypes 1-6, 8, 9, and various chimeric capsids. In some embodiments, this process can be completed within one week, resulting in a high total capsid-to-empty capsid ratio (>90% total capsid) and a post-purification yield (>1 × 10⁻¹⁶) suitable for clinical use. 13 The method provides the rAAV vector (vg / L) and purity. In some embodiments, such a method is universal for all serotypes and chimeric capsids. Extensible manufacturing techniques may be used to produce GMP clinical and commercial grade rAAV vectors (e.g., for the treatment of hereditary and acquired neurodegenerative disorders).

[0163] After the rAAV vectors of this disclosure are produced and purified, they may be titrated (for example, the amount of rAAV vector in a sample may be quantified) in order to prepare compositions for administration to subjects such as human subjects with hereditary or acquired neurodegenerative disorders. The titration of rAAV vectors can be achieved using methods known in the art.

[0164] In some embodiments, the number of viral particles containing vector genomes and "empty" capsids without vector genomes can be determined by electron microscopy, for example, transmission electron microscopy (TEM). Such TEM-based methods can provide the number of vector particles (or viral particles in the case of wild-type AAV) in a sample.

[0165] In some embodiments, the rAAV vector genome can be titrated using quantitative PCR (qPCR) with primers targeting sequences within the vector genome, e.g., ITR sequences, and / or sequences within the transgene or regulatory elements. By performing qPCR in parallel against dilutions of known concentrations of standard substances, such as plasmids containing the vector genome sequence, a standard curve can be generated that allows for the calculation of the rAAV vector concentration as the number of vector genomes (vg) per unit volume, such as microliters or milliliters. For example, the number of empty capsids can be determined by comparing the number of vector particles measured by electron microscopy with the number of vector genomes in the sample. Since the vector genome contains the therapeutic transgene, the vg / kg or vg / ml of the vector sample may represent a therapeutic dose greater than the number of vector particles the subject would receive, some of which may be empty and not contain the vector genome. Once the concentration of the rAAV vector genome in the stock solution is determined, it can be diluted to a buffer suitable for use in preparing a composition for administration to a subject (e.g., a subject with hereditary or acquired neurodegenerative disorder), or dialyzed against it.

[0166] 6. Method of Use and Treatment Nucleic acids (e.g., RNA molecules or polynucleotides encoding RNA molecules) disclosed herein may be used for the treatment and / or prevention of gene therapy for diseases, disorders, or conditions. In particular, it can be used to treat or prevent diseases, disorders, or conditions associated with deficiency or dysfunction of a specific target gene (e.g., FXYD1), as well as any other conditions and / or diseases where reducing the expression of the relevant target gene may result in therapeutic benefit or improvement, for example, diseases, disorders, or conditions mediated by or associated with an increase in the level or function of the relevant protein (e.g., FXYD1) compared to the level or function of the protein in an otherwise healthy individual. The vector genomes and / or rAAV vectors described herein may be used for the treatment and / or prevention of gene therapy for the same diseases, disorders, or conditions.

[0167] In some embodiments, the methods of the Disclosure include the use of an rAAV vector or a pharmaceutical composition thereof in the treatment of a disease, disorder, or condition of interest. In some embodiments, the methods of the Disclosure include the use of an rAAV vector or a pharmaceutical composition thereof in a subject requiring a reduction in the level of a gene of interest (e.g., FXYD1).

[0168] The nucleic acids, vector genomes, and / or rAAV vectors described above can be used in the preparation of pharmaceuticals for the treatment and / or prevention of diseases, disorders, or conditions associated with or caused by deficiencies or dysfunctions (e.g., neurodegenerative diseases), and any other conditions or diseases in which downmodulation of associated proteins may result in therapeutic benefits or improvements.

[0169] In certain embodiments, the compositions described herein may be administered in combination with cognitive enhancers (nootropes). Exemplary agents include any drugs, supplements, or other substances that improve cognitive function, particularly executive function, memory, creativity, or motivation, in healthy individuals. Non-limiting examples include racetams (e.g., piracetam, oxyracetam, and aniracetam), dietary supplements (e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA), stimulants (e.g., amphetamine drugs, methylphenidate, eugeroics, xanthines, and nicotine), L-theanine, tolcapone, levodopa, atomoxetine, and desipramine.

[0170] The overall dosage of a therapeutic agent (e.g., RNA molecules, polynucleotides encoding RNA molecules, vector genomes, or vectors, e.g., rAAV, or cells) is a therapeutically effective dose that depends on several factors, including the subject's overall health, the subject's disease state, the severity of the condition, observation of improvement, and the prescription and route of administration of the selected drug. Determining the therapeutically effective dose is within the scope of the skills of those skilled in the art. The exact prescription, route of administration, and dosage can be selected by individual physicians, taking into account the subject's condition.

[0171] In other embodiments, the cell or nucleotide composition may be administered in an amount effective in promoting the survival of CNS neurons in a subject by increasing the number of viable neurons by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to untreated CNS neurons or the number of viable neurons in the subject.

[0172] In some embodiments, the subject is also administered a second agent to treat or prevent a neurological disorder or impairment. In some embodiments, the first and second agents are co-formulated. In some embodiments, the first and second agents are administered simultaneously. In some embodiments, the first and second agents are administered at different times to produce overlapping therapeutic effects in the patient. When the first and second agents are administered simultaneously or at different times to produce overlapping therapeutic effects, the agents may be administered by the same or different routes of administration (e.g., oral versus infusion).

[0173] 7. Pharmaceutical compositions This disclosure provides pharmaceutical compositions or medicinal products for preventing or treating hereditary or acquired neurodegenerative disorders. In some embodiments, the pharmaceutical composition comprises one or more of the above-mentioned RNA molecules, polynucleotides, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and host cells.

[0174] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and / or other pharmaceutical agent. The pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other pharmaceutical agent is not biologically or otherwise undesirable, for example, the material can be administered to a subject without causing undesirable biological effects that outweigh the advantageous biological effects of the material. Any suitable pharmaceutically acceptable carrier or excipient can be used in the preparation of the pharmaceutical composition according to the present invention (see, for example, Remington The Science and Practice of Pharmacy, Adeboye Adejare (Editor), Academic Press, November 2020).

[0175] Pharmaceutical compositions are typically sterile under manufacturing and storage conditions, pyrogenic, and stable. They may be formulated as solutions (e.g., water, saline, dextrose solution, buffer solution, or other pharmaceutically sterile fluids), microemulsions, liposomes, or other ordered structures suitable for accommodating high product concentrations (e.g., viral vector particles, microparticles, or nanoparticles).

[0176] In some embodiments, pharmaceutical compositions comprising the above-mentioned RNA molecules, polynucleotides, expression cassettes, expression vectors, vector genomes, host cells, or rAAV vectors of this disclosure are formulated in water or buffered saline solution. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. In some embodiments, it may be preferable to include an isotonic agent, such as a sugar, a polyhydric alcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Sustained adsorption of the injectable composition can be brought about by including an absorption-delaying agent, such as monostearate and gelatin, in the composition. In some embodiments, the nucleic acids, vectors, and / or host cells of this disclosure may be administered in a composition comprising a controlled-release formulation, such as an implant and a microencapsulation delivery system, a sustained-release polymer, or other carrier that protects the product from rapid release.

[0177] In some embodiments, the pharmaceutical compositions of the present disclosure are parenteral pharmaceutical compositions, including compositions suitable for intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and / or intracisional (ICM) administration. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for administration by ICV injection. In some embodiments, the rAAV vector is formulated in 350 mM NaCl and 5% D-sorbitol in PBS.

[0178] 8. Method of administration The above-mentioned RNA molecules, or polynucleotides, or vectors (e.g., vector genomes, rAAV vectors) may be administered to a subject (e.g., a patient) to treat the subject. Administration of the vector to a human subject or animal requiring vector administration can be carried out by any means known in the art for vector administration. Target cells of the vectors of this disclosure include cells of the CNS, preferably glial progenitor cells, astrocytes, or oligodendrocytes.

[0179] The vector may be administered in addition to and as an adjunct to standard care treatment. That is, the vector may be administered simultaneously, concurrently, or at predetermined dosing intervals with another agent, compound, drug, treatment, or treatment regimen, as determined by those skilled in the art using conventional methods. Uses disclosed herein include the administration of the rAAV vector of this disclosure in addition to and / or concurrently with standard care for diseases known in the art.

[0180] In some embodiments, the combination composition comprises one or more immunosuppressants. In some embodiments, the combination composition comprises an rAAV vector containing a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressants. In some embodiments, the method comprises administering or delivering the rAAV vector containing the transgene to a target, and administering an immunosuppressant to a target prophylactically before administration of the vector or after administration of the vector (i.e., before or after the vector and / or proteins are provided and thereby symptoms of a response to them become apparent).

[0181] In one embodiment, the vector of the Disclosure (e.g., the rAAV vector) is administered systemically. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic, and intramuscular administration, as well as direct injection into tissue or organ. Those skilled in the art will understand that systemic administration can deliver nucleic acids to all tissues. In some embodiments, direct administration into tissue or organ includes administration to areas directly affected by the deficiency (e.g., the brain and / or central nervous system). In some embodiments, the vector of the Disclosure and its pharmaceutical composition are administered into the brain parenchyma (i.e., by intraparenchymal administration), into the spinal canal or subarachnoid space (i.e., by intrathecal administration) to reach the cerebrospinal fluid (CSF), into the ventricles of the brain (i.e., by intraventricular administration), and / or into the cisterna magnum of the brain (i.e., by intracisional administration).

[0182] Accordingly, in some embodiments, the vectors of the Disclosure are administered by direct injection into the brain (e.g., parenchyma, ventricles, cisterna magna, etc.) and / or into the CSF (e.g., spinal canal or subarachnoid space) to treat neurodegenerative disorders. Target cells of the vectors of the Disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, the target cells of the vectors of the Disclosure are glial cells (glial progenitor cells, astrocytes, or oligodendrocytes). Additional administration routes may also include topical application of the vector under direct visualization, e.g., surface cortical application, or other stereotactic application.

[0183] In some embodiments, the vectors of this disclosure are administered by at least two routes. For example, the vectors are administered systemically and directly to the brain. When administered via at least two routes, the administrations of the vectors may, but do not have to, be simultaneous or concurrent. Alternatively, administrations via different routes may be carried out separately with a time interval between each administration.

[0184] The above-mentioned RNA molecule, or polynucleotide encoding the RNA molecule, or rAAV vectors containing a vector genome or polynucleotide may be used for ex vivo cell transduction or direct administration to a subject (e.g., directly to the CNS of a patient with a disease). In some embodiments, the transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, disorder, or condition (e.g., cell therapy for disease). The rAAV vector containing the therapeutic nucleic acid (e.g., encoding RNA) is preferably administered to cells in a bioeffective amount. In some embodiments, a bioeffective amount of the vector is sufficient to result in a reduction of the expression of the relevant gene in the target cell.

[0185] In some embodiments, the Disclosure includes methods for reducing the level and / or activity of genes in cells by administering polynucleotides encoding the RNA molecules described herein to cells (in vivo, in vitro, or ex vivo), either alone or in a vector (including plasmids, viral vectors, nanoparticles, liposomes, or any known method for delivering nucleic acids to cells).

[0186] The dosage of rAAV vectors depends, for example, on the mode of administration, the disease or condition being treated, the stage and / or invasiveness of the disease, the condition of the individual subject (age, sex, weight, etc.), the specific viral vector, the stability of the expressed protein, the host immune response to the vector, and / or the gene being delivered. Generally, the dose should be at least 1 × 10¹⁶ per kilogram (kg) of the subject's body weight to achieve a therapeutic effect. 8 Or more, for example, 1 × 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 This is the range of a vector genome (vg) or larger.

[0187] In some embodiments, polynucleotides encoding the RNA molecules described herein may be administered as components of a DNA molecule (e.g., recombinant nucleic acid) having regulatory elements (e.g., promoters) suitable for expression in target cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes). The polynucleotides may be administered as components of plasmids or viral vectors, such as rAAV vectors. rAAV vectors may be administered in vivo by direct delivery of the vector to a patient in need of treatment (e.g., directly to the CNS). rAAV vectors may also be administered ex vivo to a patient by in vitro administration of the vector to cells from a donor patient in need of treatment, followed by reintroduction of the transduced cells into the donor (e.g., cell therapy).

[0188] 9. Kit This disclosure provides a kit comprising packaging materials and one or more components. The kit typically includes a label or accompanying document containing a description of the components, or instructions for the in vitro, in vivo, or ex vivo use of the components. The kit may include such components, for example, the above-mentioned RNA molecules, polynucleotides, nucleic acids, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and host cells, as well as optionally a second activator, for example, a compound, therapeutic agent, drug, or assembly of compositions.

[0189] A kit refers to a physical structure containing one or more components of a kit. Packaging materials can be made from materials commonly used for such purposes, such as paper, glass, plastic, foil, ampoules, vials, tubes, etc., that can maintain the sterility of the components.

[0190] The label or package insert may include information identifying one or more of its components, dosage, mechanism of action, pharmacokinetics, and clinical pharmacology of the active ingredient, including pharmacodynamics. The label or package insert may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or package insert may include information about diseases for which the kit components may be used (e.g., hereditary or acquired neurodegenerative disorders). The label or package insert may include instructions for clinicians or patients for the use of one or more of the kit components in a method, use, or treatment protocol or treatment regimen. The instructions may include dosage, frequency of duration, and instructions for implementing any of the methods, use, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0191] Labels or accompanying information may include information about potential side effects, complications, or reactions, such as warnings to the subject or clinician regarding situations in which the use of a particular composition is inappropriate.

[0192] 10. Definition As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are synonymous with any molecule composed of or containing monomeric nucleotides linked by phosphodiester bonds. A nucleic acid may be an oligonucleotide or a polynucleotide. Nucleic acid sequences are presented herein in the 5' to 3' direction. Nucleic acid sequences (i.e., polynucleotides) in this disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules, and refer to all forms of nucleic acids, e.g., double-stranded molecules, single-stranded molecules, small or short hairpin RNA (shRNA), microinterfering RNA or microRNA (miRNA), small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, and ribosomal RNA. If a polynucleotide is a DNA molecule, that molecule may be a gene, cDNA, an antisense molecule, or a fragment of any of the aforementioned molecules. Nucleotides are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Nucleotide sequences may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholino and roch nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these sequences is distinguished from naturally occurring DNA or RNA by alterations to the molecular backbone. Phosphothioate nucleotides may also be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'-P5'-phosphoramidates, and oligoribonucleotide phosphorothioates, as well as their 2'-O-allyl analogs, and 2'-O-methylribonucleotide methylphosphonate, which may be used in the nucleotide sequences of this disclosure.

[0193] In some embodiments, the nucleic acid may, in various embodiments, comprise one or more non-standard nucleotides that may or may not be naturally occurring (i.e., are artificial and not found in nature), and / or comprise modified sugars or modified skeletal linkages. Nucleic acid modifications (e.g., bases, sugars, and / or skeletal modifications), non-standard nucleotides, or nucleosides, such as those known in the art as useful in the context of RNA interference (RNAi), aptamers, CRISPR technology, polypeptide production, reprogramming, or antisense-based molecules for research or therapeutic purposes, may be incorporated in various embodiments. Such modifications may, for example, increase stability (e.g., by reducing sensitivity to nuclease cleavage), decrease in vivo clearance, increase cellular uptake, improve translation, potency, efficacy, specificity, or otherwise confer other properties that make the nucleic acid more suitable for its intended use.Various non-limiting examples of nucleic acid modification include, for example, Deleavey GF, et al., Chemical modification of siRNA. Curr. Protoc. Nucleic Acid Chem. 2009;39:16.3.1-16.3.22; Crooke, ST (ed.) Antisense drug technology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; and Kurreck, J. (ed.) Therapeutic oligonucleotides, RSC biomolecular sciences. Cambridge: Royal Society of This information is described in Chemistry, 2008, U.S. Patent Nos. 4,469,863, 5,536,821, 5,541,306, 5,637,683, 5,637,684, 5,700,922, 5,717,083, 5,719,262, 5,739,308, 5,773,601, 5,886,165, 5,929,226, 5,977,296, 6,140,482, 6,455,308, and / or PCT publications WO00 / 56746 and WO01 / 14398. Different modifications may be used on the two strands of a double-stranded nucleic acid. Nucleic acids may be uniformly modified, partially modified, and / or may contain multiple different modifications. When the length of a nucleic acid or nucleic acid region is given in terms of the number of nucleotides (nt), it should be understood that, unless otherwise indicated, this number refers to the number of nucleotides in each strand of a single-stranded or double-stranded nucleic acid. "Oligononucleotides" are relatively short nucleic acids, typically about 5 to 100 nt in length.

[0194] In some embodiments, proteins or nucleic acids are isolated. As used herein, the term “isolated” means artificially produced. As used herein with respect to nucleic acids, the term “isolated” means (i) amplified in vitro, for example, by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified by cleavage and gel separation, or (iv) synthesized, for example, by chemical synthesis. Isolated nucleic acids are readily manipulable by recombinant DNA techniques well known in the art. Thus, nucleotide sequences contained in a vector whose 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences are disclosed are considered isolated, but nucleic acid sequences that exist in their native state in their native host are not isolated. Isolated nucleic acids may be substantially purified, but do not necessarily need to be purified. For example, isolated nucleic acids in a cloning or expression vector are not pure in that they may contain a small percentage of the material present in the cell in which they exist. However, such nucleic acids are readily manipulable by standard techniques known to those skilled in the art, and are therefore isolated as the term is used herein. Where used herein in relation to proteins or peptides, the term “isolated” refers to a protein or peptide isolated from its natural environment or artificially produced (e.g., by chemical synthesis, recombinant DNA technology, etc.). In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequences provided herein may be replaced with any other nucleotides suitable for base pairing with adenosine nucleotides (e.g., via Watson-Crick base pairing). For example, T may be replaced with U, and U may be replaced with T.

[0195] "Heterogeneous" means that it originates from an entity that is genotype-different from the rest of the entity being compared to, or from, the entity into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterogeneous polynucleotide (which, when expressed, can encode a heterogeneous polypeptide). Similarly, a cellular sequence incorporated into a viral vector (e.g., a gene or part thereof) is a heterogeneous nucleotide sequence relative to the vector.

[0196] The term "transgene" refers to a heterologous polynucleotide that is introduced into a cell, transcribed into RNA, and can be optionally translated and / or expressed under appropriate conditions. In one embodiment, it confers a desired characteristic to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic outcome. In another embodiment, it may be transcribed into RNA interference-mediating molecules such as miRNA, siRNA, or shRNA.

[0197] As used herein, the term “recombinant” refers to vectors, polynucleotides (e.g., recombinant nucleic acids), polypeptides, or cells, and / or other procedures that result in a construct different from naturally occurring products, which are products of various combinations of cloning, restriction, or ligation steps. Recombinant viruses or vectors (e.g., rAAV vectors) include a vector genome containing recombinant nucleic acids (e.g., nucleic acids containing a transgene and one or more regulatory elements). The term includes replicas of the original polynucleotide construct and offspring of the original viral construct, respectively.

[0198] As used herein, the term “operably linked” refers to the linking of functionally related nucleic acid sequence (or polypeptide) elements. A nucleic acid is operably linked when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence (e.g., an enhancer) is operably linked to a coding sequence if it affects the transcription of the coding sequence. In some embodiments, operably linked means that the linked nucleic acid sequences are contiguous. In some embodiments, operably linked does not mean that the nucleic acid sequences are contiguous, but rather that intervening sequences exist between those linked nucleic acid sequences.

[0199] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV nucleic acid sequences) adjacent to at least one, and in embodiments, two, AAV reverse-terminal repeat sequences. Such an rAAV vector can be replicated and packaged into infectious viral particles if present in a host cell that is infected with a suitable helper virus (or expresses suitable helper function) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., a chromosome, or another vector such as a plasmid used for cloning or transfection), the rAAV vector may be called a "pro-vector" that can be "rescued" by replication and capsidation in the presence of AAV packaging function and suitable helper function. An rAAV vector may be any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexation with lipids, encapsulation within liposomes, and capsidation within viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV virus capsids to produce "recombinant adeno-associated virus particles (rAAV particles)."

[0200] As used herein, the term “vector” refers to a plasmid, virus (e.g., rAAV), cosmid, or other vehicle that can be manipulated by the insertion or incorporation of a nucleic acid (e.g., recombinant nucleic acid). Vectors can be used for a variety of purposes, including, for example, genetic engineering (e.g., cloning vectors), to introduce / transfer nucleic acids into cells and transcribe or translate the inserted nucleic acid within the cell. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for proliferation in the cell. In some embodiments, the vector nucleic acid includes a heterologous nucleic acid sequence, expression regulatory elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and / or ITRs. In some embodiments, upon delivery to a host cell, the nucleic acid sequence is propagated. In some embodiments, upon delivery to a host cell, either in vitro or in vivo, the cell expresses a polypeptide encoded by the heterologous nucleic acid sequence. In some embodiments, upon delivery to a host cell, the nucleic acid sequence or a portion of the nucleic acid sequence is packaged in a capsid. The host cell may be an isolated cell or a cell within a host organism. In addition to nucleic acid sequences encoding RNA, polypeptides, or proteins (e.g., transgenes), additional sequences (e.g., regulatory sequences) may be present within the same vector (i.e., cis relative to the gene) and adjacent to the gene. In some embodiments, the regulatory sequences may be located on a separate (e.g., second) vector acting trans to regulate gene expression. Plasmid vectors may be referred to herein as “expression vectors.”

[0201] As used herein, the term “vector genome” refers to a recombinant nucleic acid sequence that is packaged or capsidized to form an rAAV vector. Typically, a vector genome includes heterologous polynucleotide sequences, such as transgenes, regulatory elements, and ITRs that are not originally present in the capsid. When a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g., an rAAV vector), the vector genome does not include the entire plasmid, but rather only the sequence intended for delivery by the viral vector. This non-vector genome portion of the recombinant plasmid is typically referred to as the “plasmid backbone,” which is crucial for plasmid cloning, selection, and amplification—processes required for the propagation of recombinant viral vector production—but is not packaged or capsidized into an rAAV vector by itself.

[0202] As used herein, the term “viral vector” generally refers to a viral particle (i.e., a viral particle containing a vector genome, e.g., a transgene instead of nucleic acids encoding AAV rep and cap) packaged within a viral particle (i.e., a capsid), and includes, for example, lentiviruses and parvoviruses containing AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not contain a vector genome containing rep and / or cap genes.

[0203] As used herein, “miRNA scaffold” may refer to a polynucleotide comprising (i) a double-stranded sequence targeting a gene of interest for RNAi knockdown, and (ii) an additional sequence that forms a stem-loop structure similar to that of an endogenous miRNA. The gene-targeting sequence for RNAi (e.g., a short sequence of about 20 nt) may be ligated to a sequence that creates a miRNA-like stem-loop and a sequence that base-pairs with the sequence of interest to form a double-stranded structure when the polynucleotide is assembled into a miRNA-like secondary structure. As described herein, this double-stranded structure may be imperfectly hybridized, for example, it may contain one or more unpaired or mispaired bases. Upon cleavage of this polynucleotide by Dicer, this double-stranded structure containing the gene-targeting sequence of interest may be unwound and incorporated into the RISC complex. The miRNA scaffold may refer to the miRNA itself or a DNA polynucleotide encoding miRNA. An example of a miRNA scaffold is the miR-155 sequence (Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9). Commercial kits for cloning sequences into miRNA scaffolds are known in the art (e.g., the INVITROGEN BLOCK-IT Pol II miR RNAi expression vector kit from Life Technologies, Thermo Fisher Scientific, Waltham, Mass.).

[0204] A functional variant or equivalent of a reference peptide, polypeptide, or protein refers to a polypeptide derivative of the reference peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of the reference peptide, polypeptide, or protein. Generally, a functional equivalent is at least 60% (e.g., any number between 60% and 100% (including both extreme values), e.g., 60%, 70%, 80%, 85%, 90%, 95%, and 99%) identical to the reference peptide, polypeptide, or protein. In certain embodiments, point mutations may be conservative modifications.

[0205] As used herein, the term “conservative modification” refers to an amino acid modification that does not significantly affect or alter the biological characteristics of a polypeptide or protein. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into polypeptides or proteins by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions involve exchanging one member of one of these classes with another.

[0206] As used herein, the terms “to treat,” “to treat,” or “treatment” refer to the administration of a therapy that partially or completely reduces, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition.

[0207] The terms “reduce,” “decrease,” “reduced,” “decrease,” “decrease,” or “inhibit” are all used herein to generally mean a reduction of a statistically significant amount compared to a reference. However, to eliminate any doubt, “reduce,” “decrease,” or “decrease,” or “inhibit” typically mean a reduction of at least 10% compared to a reference level, and may include, for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, for example, up to and including the complete non-existence of a given entity or parameter compared to a reference level, or any reduction of 10–99% compared to the non-existence of a given treatment.

[0208] As used herein, the term “ameliorate” means a detectable or measurable improvement of the disease, disorder, or condition, or its symptoms, or the underlying cellular response. Detectable or measurable improvement includes subjective or objective reduction, decrease, inhibition, suppression, limitation, or control of the onset, frequency, severity, progression, or duration of the disease, disorder, or condition, or the cause of complications caused by or associated with them, or improvement of their symptoms, or reversal thereof.

[0209] As used herein, the term “associated with” means that the presence, level, and / or form of one correlate with the presence, level, and / or form of the other. For example, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of that disease, disorder, or condition (e.g., across a whole population of the subject).

[0210] As used herein, the terms “prevent” or “prevention” refer to the delay in the onset, and / or reduction in the frequency and / or severity, of one or more signs or symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is evaluated on a population basis such that a drug is considered to “prevent” a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency and / or intensity of one or more signs or symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed by a given period.

[0211] As used herein, the term “agent” means any compound or substance, including but not limited to small molecules, nucleic acids, polypeptides, peptides, drugs, ions, viruses, cells, etc. “A drug” can be any chemical substance, entity, or part, including but not limited to synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, a drug is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, nucleic acid, amino acid, or carbohydrate oligomer, including but not limited to proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and their modifications and combinations. In some embodiments, a drug is selected from the group consisting of nucleic acids, small molecules, polypeptides, and peptides. In some embodiments, a drug is an oligonucleotide, protein, or small molecule. In some embodiments, a drug comprises one or more oligonucleotides. In some embodiments, the oligonucleotide is a splice-switching oligonucleotide. In certain embodiments, the oligonucleotide is an antisense oligonucleotide (ASO). In certain embodiments, a drug is a small molecule having a chemical part. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties, including macrolides, leptomycin, and related natural products or their analogues. The compound may be known to have the desired activity and / or properties, or it may be selected from a diverse library of compounds. In some embodiments, the drug is a genome modification system (e.g., CRISPR / Cas, zinc finger nuclease, or TALEN system). The CRISPR / Cas system can use various Cas proteins (Haft et al. PLos Comput Biol. 2005;1(6)e60). In some embodiments, the CRISPR / Cas system is a CRISPR type I system. In some embodiments, the CRISPR / Cas system is a CRISPR type II system.In some embodiments, the CRISPR / Cas system is a CRISPR V system.

[0212] "Low molecular weight" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Examples of low molecular weight include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetic compounds, and antibody mimetic compounds. As therapeutic agents, low molecular weight may be more permeable to cells, less susceptible to degradation, and less likely to induce an immune response than macromolecules.

[0213] As used herein, the terms “polypeptide” or “protein” are used to refer to a series of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The term “polypeptide” refers to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. The term “peptide” is often used in reference to smaller polypeptides, but its use in the art overlaps with that of “protein” or “polypeptide.” Exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments, and other equivalents, as well as both naturally occurring and non-naturally occurring variants, fragments, and analogs of the foregoing.

[0214] As used herein, the term “subject” refers to an organism, e.g., a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, dog). In some embodiments, the subject is a non-human disease model. In some embodiments, the human subject is an adult, adolescent, or child subject. In some embodiments, the subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject suffers from a disease, disorder, or condition related to neuronal hyperexcitability. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, a susceptible subject is more likely to have and / or exhibit an increased risk of developing the disease, disorder, or condition (compared to the mean risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit a specific symptom (e.g., clinical symptoms of the disease) or feature of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual to be diagnosed and / or treated.

[0215] As used herein, the term “pharmaceutically acceptable” means a drug, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0216] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material involved in transporting or delivering the drug of the subject from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) Oil, glycol, e.g., propylene glycol, (11) polyol, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG), (12) ester, e.g., ethyl oleate and ethyl laurate, (13) agar, (14) buffer, e.g., magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) water free of pyrogens, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solution, (21) polyester, polycarbonate, and / or polyacid anhydride, (22) fillers, e.g., polypeptides and amino acids, (23) serum components, e.g., serum albumin, HDL, and LDL, (22) C2-C 12Examples include alcohols, such as ethanol, and (23) other non-toxic, suitable substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as “excipient,” “carrier,” and “pharmaceutically acceptable carrier” are used interchangeably herein.

[0217] As used herein, the term “therapeutic dose” refers to the amount that produces the desired therapeutic effect for which the administration is intended. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered according to a therapeutic administration regimen to a population that is suffering from or susceptible to the disease, disorder, or condition. In some embodiments, the therapeutic dose is an amount that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition, and / or delays their onset. Those skilled in the art will understand that the term “therapeutic dose” does not actually require that the success of the treatment be achieved in a particular individual. Rather, the therapeutic dose may be an amount that, when administered to patients in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects.

[0218] As used herein, the term “administer” means to place a drug or composition in a subject by a method or route that results in at least partial localization of the drug or composition at a desired site to produce the desired effect. Suitable routes of administration for the methods of the present invention include both topical and systemic routes. Generally, topical administration results in more of the administered drug being delivered to a specific location compared to systemic administration, while systemic administration results in the drug being delivered essentially to the entire body of the subject.

[0219] The compositions and agents disclosed herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. Exemplary modes of administration include, but are not limited to, injection, infusion, drip infusion, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracranial, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In preferred embodiments of the embodiments described herein, the compositions are administered by intravenous infusion or injection.

[0220] As used herein, the term “glial cell” refers to a population of non-neuronal cells that provide support and nutrition, maintain homeostasis, promote myelin formation or myelin sheathing, and participate in signal transduction in the nervous system. As used herein, “glial cell” includes fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, as well as glial progenitor cells. Glial progenitor cells are cells that have the potential to differentiate into cells of the glial lineage, such as oligodendrocytes and astrocytes.

[0221] The glial progenitor cells described herein may be derived from any suitable source of pluripotent stem cells, such as, but not limited to, human induced pluripotent stem cells (iPSCs) and embryonic stem cells, as described in more detail below. In one embodiment, the glial progenitor cells may be rejuvenated cells derived from the glial progenitor cells described herein or their offspring.

[0222] In some embodiments, in order to do so in a subject requiring treatment, the glial progenitor cells or rejuvenated cells are young glial or glial progenitor cells, or younger than the counterpart of the subject being treated.

[0223] As used herein, the terms “about” or “approximately” refer to measurable values ​​such as the amount of biological activity, the homology or length of a polynucleotide or polypeptide sequence, dose, time, temperature, and unless otherwise specified, unless evident from the context, or unless such number exceeds 100% of the possible value, are intended to include variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or even 0.1% in either direction of the specified amount (above or below).

[0224] As used herein, the terms “homologous” or “homonymy” refer to two or more reference entities (e.g., nucleic acid or polypeptide sequences) that share at least partial identity over a given region or portion. For example, two peptides are homologous at a position if the amino acid positions in those peptides are occupied by the same amino acids. In particular, homologous peptides retain the activity or function associated with the unmodified peptide or reference peptide, while modified peptides generally have an amino acid sequence that is “substantially homologous” to the amino acid sequence of the unmodified sequence. Where referring to polypeptides, nucleic acids, or fragments thereof, “substantially homologous” or “substantially similar” means that, when optimally aligned with another polypeptide, nucleic acid (or its complementary chain), or fragment thereof, including appropriate insertions or deletions, there is sequence identity of at least about 70% to 99% of the sequence. The degree of homology (identity) between two sequences can be determined using computer programs or mathematical algorithms known in the art. Such algorithms for calculating percent sequence homology (or identity) generally account for sequence gaps and mismatches over the comparison region or area. [Examples]

[0225] Example 1 A meta-analysis was conducted to identify astrocyte genes that are dysregulated in astrocytes derived from neurodegenerative diseases but not in healthy, aged astrocytes. The analysis was performed using a large database encompassing gene expression from both mouse and human astrocytes, derived from both wild-type and aged cells, and compared with age-matched cells supplied from patients with various neurodegenerative disorders.

[0226] More specifically, six transcriptome datasets were used to study the effects of aging (Boisvert, MM, Erikson, GA, Shokhirev, MN & Allen, NJ: The Aging Astrocyte Transcriptome from Multiple Regions of the Mouse Brain. Cell Rep. 22, 269-285 (2018)), Alzheimer's disease (Zeng, H., et al.: Integrative in situ mapping of single-cell transcriptional states and tissue histopathology in a mouse model of Alzheimer's disease. Nat. Neurosci. 26, 430-446 (2023), Habib, N., et al.: Disease-associated astrocytes in Alzheimer's disease and aging. Nat. Neurosci. 23, 701-706 (2020), and Park, H., et al.: Single-cell RNA-sequencing identifies disease-associated The data was collected from publicly available studies investigating oligodendrocytes in male APP NL-GF and 5XFAD mice. Nat Commun 14, 802 (2023)), ALS (Liu, W., et al. Single-cell RNA-seq analysis of the brainstem of mutant SOD1 mice reveals perturbed cell types and pathways of amyotrophic lateral sclerosis. Neurobiol. Dis. 141, 104877 (2020)), or Huntington's disease (Benraiss, A., et al. Cell-intrinsic glial pathology is conserved across human and murine models of Huntington's disease. Cell Rep. 36, 109308 (2021)).These six datasets enabled the inventors to complete a comparison of disease states with wild-type differential expression. Particular focus was placed on astrocytes, and it was ensured that all included datasets were mouse-derived. These datasets encompassed both bulk and single-cell resolution RNA sequencing experiments. Using the given datasets, the inventors were able to access or complete six different state / wild-type crosses.

[0227] Where available, we used a publicly available list of differentially expressed genes (DEGs). If such a list is unavailable, the inventors used STAR to align publicly available FastQ files with the GRCm39 ens106 reference and performed RNA-Seq by Expectation Maximization (RSEM) (Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. Aug 4 2011;12:323.doi:10.1186 / 1471-2105-12-323) on bulk RNA and STARsolo (Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. Jan 1 2013;29(1):15-21.doi:10.1093 / bioinformatics / bts635) on cell-resolution datasets. Next, to enrich the astrocyte dataset, the cell-resolution dataset was limited to clusters expressing GFAP and AQP4. A DEG list was generated by comparing a given state to the wild type using Wilcoxon rank sum, which requires an adjusted p-value of 0.05 or less.Next, when these DEG lists were filtered, the Enrichr database (Chen EY, Tan CM, Kou Y, et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. Apr 15 2013;14:128.doi:10.1186 / 1471-2105-14-128, Kuleshov MV, Jones MR, Rouillard AD, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. Jul 8 2016;44(W1):W90-7.doi:10.1093 / nar / gkw377, and Xie Z, Bailey A, Kuleshov MV, et al. Gene Set Knowledge Discovery with Enrichr. Curr Protoc. Mar) The study included only genes included in the list of 280 genes associated with either a potassium-related gene ontology (GO) term or pathway, as found in 2021;1(3):e90.doi:10.1002 / cpz1.90). Finally, we identified genes that appeared in at least three of the six datasets and created a concise list of genes that were commonly and significantly dysregulated in astrocytes of mice with our target neurodegenerative state.

[0228] Cortical [K + ] eThe consistent changes suggested the existence of a common molecular pathway mediating dysregulated potassium homeostasis. Based on this, we mined six publicly available transcriptome databases covering astrocyte gene expression in the AD, ALS, and HD mouse models we studied (Figures 1-2). Using (where available) publicly available differentially expressed gene (DEG) lists, or by constructing them ourselves (where unavailable), we identified common DEGs present in at least three of the six datasets.

[0229] Notably, there were numerous shared DEGs between disease states, but no shared DEGs in astrocytes during normal aging. + / K + Fxyd1, a negative regulator of ATPase activity, was selectively upregulated in all three AD and ALS models, but remained unchanged during normal aging. In contrast, its expression was reduced in Huntington's disease (HD) model mice, similar to the compensatory mechanism for the downregulation of K channel expression in HD glial cells previously described (Osipovitch et al., 2019).

[0230] Fxyd1 is particularly interesting due to its common positive expression. Fxyd1 (FXYD domain-containing ion transport regulator-1) transports Na from cells. + And K in the cell + It regulates the activity of sodium / potassium transport ATPase (NKA) that transports Na + / K + -Because it plays a role in downregulating ATPase activity, relative overexpression of Fxyd1 is associated with astrocytes [K] in AD and ALS. + ] e This suggests disruption of uptake and buffering, and the high extracellular K in these states. + This results in... Notably, Fxyd1 is Na only in the non-phosphorylated state. + / K +It has been reported to inhibit ATPase activity, and its activity in the CNS when phosphorylated remains unknown. Therefore, overexpression of Fxyd1 may have the effect of increasing extracellular K+.

[0231] Astrocytes are traditionally K + It is considered the main cell type responsible for buffering (Verkhratsky, A., Nedergaard, M. & Hertz, L. Why are astrocytes important? Neurochem. Res. 40, 389-401 (2015), Parpura, V., et al. Glial cells in (patho)physiology. J Neurochem 121, 4-27 (2012), and Walz, W. Role of astrocytes in the clearance of excess extracellular potassium. Neurochemistry international 36, 291-300 (2000)). + / K + -ATPase is the extracellular potassium after neuronal activity. + Since it contributes to the clearance (Larsen, BR, et al. Glia 62, 608-622 (2014)), its negative regulation by Fxyd1 is [K + ] e This can increase the elevated cortical [K] in both ALS and AD models. + ] e and enhanced startle-inducing [K + ] e It brings about both.

[0232] The descriptions of the above-mentioned examples and preferred embodiments should be construed as illustrative, not limiting, the disclosure as defined by the claims. For ease of understanding, numerous variations and combinations of the features described above can be utilized without departing from the disclosure as defined in the claims. Such variations shall not be deemed to deviate from the scope of the disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated in their entirety by reference.

Claims

1. (i) a method for treating a subject in need of reducing brain hyperexcitability or (ii) a condition mediated by brain hyperexcitability, wherein the Na of the glial cells of the subject ＋ _K ＋ A method comprising increasing the level or activity of ATPase.

2. A method for reducing interstitial potassium levels in the brain of a subject requiring a reduction in such levels, wherein the Na in the glial cells of the subject ＋ _K ＋ A method comprising increasing the level or activity of ATPase.

3. The method according to claim 1 or 2, wherein the subject has a state mediated by neuronal hyperexcitation.

4. The method according to claim 2 or 3, wherein the brain interstitial potassium level is restored to ±30% of that of a normal, healthy adult brain.

5. The method according to any one of the prior claims, wherein the increase includes reducing the expression level of the FXYD1 gene in the glial cells.

6. The method according to claim 5, comprising administering to the subject a drug that reduces the expression level of the FXYD1 gene in the glial cells.

7. The method according to claim 6, wherein the agent comprises or codes for an inhibitory nucleic acid or a CRISPR / Cas system.

8. The method according to claim 7, wherein the inhibitory nucleic acid comprises an RNA molecule (small interfering RNA (siRNA), short hammerhead RNA (shRNA), or microRNA (miRNA).

9. The method according to claim 7, wherein the drug is an expression cassette or vector comprising a sequence encoding the inhibitory nucleic acid or one or more components of the CRISPR / Cas system.

10. The method according to claim 9, wherein the sequence is operably linked to a cell type-selective or cell type-specific regulatory sequence.

11. The method according to claim 10, wherein the cell type-selective or cell type-specific regulatory sequence comprises a promoter, an enhancer, or both.

12. The method according to claim 11, wherein the promoter is a glial cell-specific promoter or a regulatory promoter.

13. The method according to any one of claims 9 to 12, wherein the vector is a viral vector.

14. The method according to any one of the prior claims, wherein the glial cells are astrocytes.

15. The method according to any one of the prior claims, wherein the glial cells are glial progenitor cells.

16. The method according to any one of claims 1 and 3 to 15, wherein the condition is a neurodegenerative disease.

17. The method according to any one of claims 1 and 3 to 15, wherein the condition is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, Huntington's disease, or schizophrenia.

18. The method according to any one of claims 7 to 17, wherein the inhibitory nucleic acid or siRNA molecule comprises or encodes a sequence that is at least 75% complementary to the FXYD1 gene or RNA segment.

19. The method according to any one of claims 7 to 17, wherein the CRISPR / Cas system includes or encodes a guide RNA (gRNA) sequence that is at least 75% complementary to the FXYD1 gene or RNA segment.

20. An inhibitory nucleic acid or siRNA molecule containing, or encoding, a sequence that is at least 75% complementary to the FXYD1 gene or RNA segment.

21. (i) an inhibitory nucleic acid or siRNA molecule as described in claim 20, and (ii) a pharmaceutically acceptable carrier or excipient, comprising a pharmaceutical composition.

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