Glial-targeted mitigation of hyperexcitation in neurodegenerative diseases

Abstract

The present disclosure relates to the alleviation of hyperexcitation and related therapeutic agents and methods for the treatment of neurodegenerative diseases.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 592,271, filed October 23, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Government interests

[0004] This invention was carried out with government support under license number AG072298 granted by the National Institutes of Health. The government owns certain rights to this invention.

[0005] Reference to the electronic sequence list

[0006] The contents of the electronic sequence list (161118.04901SeqList.xml; size: 7,578 bytes; and creation date: October 15, 2024) are incorporated herein by reference in full. Technical Field

[0007] This disclosure relates to the relief of over-excitation and related therapeutic agents and methods for treating neurodegenerative diseases. Background Technology

[0008] Neurodegenerative diseases are characterized by the progressive loss of structure or function of neurons in the brain or peripheral nervous system, affecting millions of people worldwide. Neurodegenerative diseases can be found in neuronal circuits at many different levels in the brain, ranging from molecular to systemic. Aging is the most significant risk factor for the development of 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 complex, as well as Parkinson's disease, Lewy body disease, and multiple system atrophy) increase with age. Each of these conditions is characterized by progressive neuronal loss, which is closely associated with the progression of symptomatic disease and functional deterioration. In contrast, normal aging has been shown to cause synaptic loss without a significant decline in neuronal density. Despite extensive research in this field (primarily focusing on the potential neurotoxicity of amyloid, tau, and synuclein proteins, as well as neuroinflammation), our understanding of the pathobiology of neuronal loss in neurodegenerative diseases remains limited. The need for novel therapeutics and methods for treating neurodegenerative diseases remains unmet. Summary of the Invention

[0009] This disclosure addresses the aforementioned needs in several ways.

[0010] On one hand, this disclosure provides a method for (i) reducing brain hyperexcitation in a subject in need, or (ii) treating symptoms mediated by brain hyperexcitation. The method comprises increasing Na+ in the glial cells of the subject. + ,K + The level or activity of ATPase.

[0011] On the other hand, this disclosure is characterized by a method for reducing interstitial potassium levels in the brain of a subject in need. The method comprises increasing Na+ levels in the glial cells of the subject. + ,K + The level or activity of ATPase.

[0012] In each of the methods described above, the subject may suffer from symptoms mediated by neuronal overexcitation.

[0013] In some implementations, the interstitial potassium level is restored to approximately ±30% of the interstitial potassium level in a normal healthy adult brain.

[0014] In each of the methods described above, the enhancement may include reducing the expression level of the FXYD1 gene in the glial cells. In one embodiment, the reduction includes administering an agent to the subject that reduces the expression level of the FXYD1 gene in the glial cells. In some embodiments, the agent may contain or encode a repressive nucleic acid or a CRISPR / Cas system. In some embodiments, the repressive nucleic acid contains RNA molecules such as small interfering RNA (siRNA), short hammerhead RNA (shRNA), or microRNA (miRNA).

[0015] In some embodiments, the agent is or comprises an expression cassette or vector containing a sequence encoding the inhibitory nucleic acid or one or more components of the CRISPR / Cas system. In some embodiments, the sequence is operatively 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 enhancer or both. The promoter may be a glial cell-specific promoter or a regulated promoter. The vector may be a viral vector.

[0016] In each of the methods described above, the glial cells may be astrocytes, glial progenitor cells, or oligodendrocytes. The conditions mentioned above may be neurodegenerative diseases. Examples of such conditions or neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, Huntington's disease, and schizophrenia.

[0017] In some embodiments, the repressive nucleic acid or siRNA molecule contains or encodes a sequence that is complementary to at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of a segment of the FXYD1 gene or RNA.

[0018] In some embodiments, the CRISPR / Cas system includes or encodes a guide RNA (gRNA) sequence that is complementary to at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of a segment of the FXYD1 gene or RNA.

[0019] In another aspect, this disclosure provides a repressive nucleic acid or siRNA molecule that comprises or encodes a sequence that is complementary to at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) a segment of the FXYD1 gene or RNA.

[0020] In another aspect, this disclosure provides a pharmaceutical composition comprising (i) an inhibitory nucleic acid or siRNA molecule disclosed herein, and (ii) a pharmaceutically acceptable carrier or excipient.

[0021] Details of one or more embodiments of this disclosure are set forth in the following description. Other features, objects, and advantages of this disclosure will be apparent from the specification and claims. Attached Figure Description

[0022] Figure 1 Details of individual data sources for the list of differentially expressed genes (DEGs) are shown, which were obtained from astrocyte transcriptome data in mouse models of normal aging, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.

[0023] Figure 2 This is a diagram illustrating the proposed model: extracellular K+ in neurodegenerative diseases compared to the typical relative decline in normal healthy aging. + Increase. As K + The primary role of buffering is impaired; dysregulated key genes in astrocytes (such as Fxyd1) increase cortical [K+] levels in AD and ALS models through impaired buffering. e In disease models, extracellular K + Chronic elevation of these levels can lead to overexcitation and a decrease in neuron count. Detailed Implementation

[0024] This disclosure relates to the relief of neurodegenerative diseases and hyperexcitability, as disrupted ion homeostasis can be a driving force in neurodegenerative pathologies. Certain aspects of this disclosure are based, at least in part, on the accidental discovery of astrocyte genes that are dysregulated in neurodegenerative disease-derived astrocytes but not in other healthy senescent astrocytes.

[0025] For example, through a meta-analysis of a large database encompassing gene expression in both murine and human astrocytes (e.g., derived from both wild-type and senescent cells), and comparing it with age-matched cells derived from patients with various neurodegenerative diseases, the inventors identified dysregulation of astrocyte genes in neurodegenerative disease-derived astrocytes, but not in other healthy senescent astrocytes. In doing so, the inventors studied data from both human cell and animal models of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Huntington's disease.

[0026] 1. Neurodegenerative diseases and overexcitation

[0027] "Neurodegenerative disease" refers to a disease symptom involving at least one of the deterioration of neural stem cells and / or progenitor cells that mediates or characterizes neural loss. Non-limiting examples of neurological diseases and / or conditions disclosed herein include polyglutamine expansion disorder (e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also known as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., types 1, 2, 3 (also known as Machado-Joseph disease), 6, 7, and 17)), and other trinucleotide repeat expansion disorders (e.g.,Fragile X syndrome, Fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, Alexander disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia-telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Guillain-Barré syndrome, ischemic stroke, Krabbe disease. Diseases including kuru, Lewy body dementia, multiple sclerosis, multiple system atrophy, non-Huntingtonian type of Chorea, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff's disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), Steele Richardson-Olszewski disease, schizophrenia, late-onset psychosis, autism spectrum disorder, motor disorders, and tabes dorsalis. In some cases, neurodegenerative conditions encompass damage or impairment of the CNS or PNS associated with bodily injury (e.g., head trauma, mild to severe traumatic brain injury (TBI), diffuse axonal injury, brain contusion, acute brain edema, etc.).

[0028] In some implementations, neurodegenerative diseases are conditions associated with neuronal overexcitation. A common feature of most neurodegenerative diseases is neuronal overexcitation, abnormal electrical activity, or a state in which neural networks exhibit an increased likelihood of being excited or activated. Neuronal overexcitation can be associated with spinal cord injury, stroke, traumatic brain injury, hearing loss, epilepsy, painful neuropathy, attention deficit hyperactivity disorder (ADHD), 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 abstinence, and rapid withdrawal from benzodiazepines.

[0029] Extracellular concentration of brain potassium [K] + ] e It is a potent regulator of cortical network activity, and its concentration changes in neurodegenerative diseases (compared to concentrations in normal healthy aging). To this end, the inventors studied various mouse models and examined extracellular K+. + Whether damage to neurons plays a potentially key role in the loss of degenerative neurons (due to its influence on neuronal membrane potential and pathological overexcitation).

[0030] Because dysregulated high interstitial potassium plays a causal role in network hyperexcitability and excitotoxicity in these neurodegenerative diseases, the inventors focused on genes involved in glial potassium transport that are disrupted in each disease but not dysregulated during normal healthy aging. The inventors identified potassium-regulating genes that are dysregulated in disease-related astrocytes but not in normally aging astrocytes.

[0031] FXYD1 is one of these, known to negatively regulate Na / K-ATPase. FXYD1 was found to be aberrantly overexpressed in astrocytes derived from each of these disease symptoms, but not in normal wild-type senescent astrocytes. Astrocyte overexpression of FXYD1 is expected to inhibit Na / K-ATPase activity, thus increasing extracellular and interstitial K, which in turn increases neuronal network excitability and its associated long-term excitotoxicity. Therefore, glial-specific knockdown of FXYD1 gene expression could be used as a therapeutic strategy, as its glial-targeted inhibition could allow for the alleviation of both network hyperexcitability and excitotoxicity characteristic of these neurodegenerative conditions. As disclosed herein, this can be achieved, for example, by delivering FXYD1 shRNAi or CRISPR-mediated epigenetic knockdown vector plasmids or viruses to central astrocytes (targeting the latter via glial-specific regulatory sequences and / or using viral vectors with cell-type-specific binding or infection).

[0032] The following shows an exemplary protein sequence of Homo sapiens FXYD1 and the nucleic acid sequence encoding the protein and the corresponding mRNA.

[0033] MASLGHILVFCVGLLTMAKAESPKEHDPFTYDYQSLQIGGLVIAGILFILGILIVLSRRCRCKFNQQQRTGEPDEEEGTFRSSIRRLSTRRR (SEQ ID NO: 1)

[0034] A. Homo sapiens containing the FXYD domain of ion transport regulator 1, (FXYD1), transcriptomorph a, mRNA; NCBI reference sequence: NM_005031.5 (SEQ ID NO: 2)

[0035]

[0036] B. Homo sapiens containing the FXYD domain of ion transport regulator 1, (FXYD1), transcript variant b, mRNA; NCBI reference sequence: NM_021902.4 (SEQ ID NO: 3)

[0037]

[0038] C. Homo sapiens containing the FXYD domain of ion transport regulator 1, (FXYD1), transcriptomorph c, mRNA; NCBI reference sequence: NM_001278717.2 (SEQ ID NO: 4)

[0039]

[0040] D. Homo sapiens containing the FXYD domain of ion transport regulator 1, (FXYD1), transcriptomorph d, mRNA; NCBI reference sequence: NM_001278718.2 (SEQ ID NO: 5)

[0041]

[0042] One approach to addressing aberrant FXYD1 overexpression is to reduce FXYD1 gene expression through RNA interference. This can be achieved through microRNA-based gene therapy. For example, administering an AAV vector carrying an expression cassette delivering a therapeutic miRNA precursor targeting FXYD1 mRNA can activate endogenous mRNA silencing mechanisms to reduce FXYD1 translation in glial cells (e.g., glial progenitors, astrocytes, or oligodendrocytes). Furthermore, using AAV vectors with high tropism for these glial cells can improve safety and therapeutic efficacy. Antisense oligonucleotide strategies can also be used to inhibit FXYD1 overexpression.

[0043] In some embodiments, this disclosure employs a viral vector (such as an AAV vector) to efficiently deliver therapeutic nucleic acids (such as siRNA) targeting one or more genes or RNA encoding toxic gain-of-function proteins into cells. In some embodiments, the AAV vector encoding RNAi molecules (e.g., the siRNA molecules of this disclosure) can increase the delivery of the active agent to glial cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes). Therapeutic nucleic acids or polynucleotides may be able to significantly inhibit gene expression (e.g., mRNA levels) of toxic gain-of-function proteins within cells; thus improving intracellular protein-induced or induced defects, such as inhibition of sodium / potassium transporter ATPase activity and / or protein aggregation and inclusion formation.

[0044] Such inhibitory nucleic acids (e.g., siRNA) can be used to treat a variety of hereditary and / or acquired neurodegenerative diseases. According to this disclosure, a method for treating and / or improving the condition in a patient comprises administering an effective amount of at least one therapeutic nucleic acid (e.g., a polynucleotide encoding one or more siRNA duplexes) to the patient's cells, and allowing for the suppression / silencing of gene expression.

[0045] 2. Nucleic acid

[0046] Certain aspects of this disclosure provide one or more repressive nucleic acids (e.g., repressive RNA molecules), polynucleotides encoding such repressive nucleic acids, and transgenes engineered to express such repressive nucleic acids. The one or more repressive nucleic acids may target the same gene (e.g., hybridizing or specifically binding to the same mRNA sequence of the same gene or different mRNA sequences) or different genes (e.g., hybridizing or specifically binding to the mRNA of different genes).

[0047] A. Inhibitory nucleic acids

[0048] Inhibitory nucleic acids are nucleic acids that can bind to target nucleic acids (e.g., target RNA) in a cell and reduce or inhibit the level or function of the target nucleic acid in the cell. Examples of inhibitory nucleic acids include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, small interfering (si)RNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds, modified base / locked 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 regulate its level or function.

[0049] In some implementations, the repressive nucleic acid can be antisense RNA, antisense DNA, chimeric antisense oligonucleotides, modified linked antisense oligonucleotides, interfering RNA (iRNA), short or small interfering RNA (siRNA), microRNA or small interfering RNA (miRNA), sequential small RNA (stRNA), short hairpin RNA (shRNA), small RNA-induced gene activator (RNAa), small activating RNA (saRNA), or combinations thereof. In some examples, the repressive nucleic acid is a repressive RNA molecule that mediates RNA interference.

[0050] RNA interference (RNAi) is a process discovered in 1998 (Fire et al., 1998) through which cells regulate gene expression. Double-stranded RNA (dsRNA) in the cytoplasm triggers the RNAi pathway, in which the double-stranded RNA is processed by the RNase III-like enzyme DICER into small double-stranded fragments approximately 21-23 nucleotides in length. These double-stranded fragments integrate into a multi-subunit protein called the RNA-induced silencing complex (RISC). The RISC contains the Argonaute protein, which unwinds the double-stranded fragments into a passenger strand removed from the complex and a guide strand complementary to a target sequence in a specific mRNA. The Argonaute protein then guides the RISC complex to cleave or inhibit the translation of a specific target mRNA molecule (Kotowska-Zimmer et al., 2021). In this way, the gene encoding the mRNA molecule essentially becomes inactive or “silenced.”

[0051] RNAi technology can employ three tools: synthetic siRNA, vector-based shRNA, and artificial miRNA (amiRNA). Synthetic siRNA is an exogenous double-stranded RNA that must be delivered into cells and must overcome stability and pharmacokinetic challenges. shRNA is an artificial RNA molecule with a tight hairpin loop structure, delivered to cells using plasmid or viral expression vectors. shRNA is typically transcribed from strong pol III promoters (e.g., U6 or H1) and enters the RNAi pathway as a hairpin. However, transcription driven by strong pol III promoters can produce supraphysiological levels of shRNA, saturating endogenous miRNA biogenesis and leading to toxicity. AmiRNA embeds a target-specific shRNA insert into a scaffold based on natural primary miRNA (pri-miRNA). This ensures proper processing and transport similar to endogenous miRNA, thereby reducing toxicity (Kotowska-Zimmer et al., 2021).

[0052] In some embodiments of this disclosure, the repressive RNA molecule may be siRNA, miRNA (including amiRNA), or shRNA.

[0053] siRNA is known in the art as a double-stranded RNA molecule of approximately 19-25 (e.g., 19-23) base pairs in length that induces RNAi in cells. In some embodiments, the siRNA sequence may also be inserted into an artificial miRNA scaffold (“shmiRNA”).

[0054] shRNA is known in the art as an RNA molecule containing a double-stranded RNA of about 19-25 (e.g., 19-23) base pairs, said double-stranded RNA being linked by a short loop (e.g., about 4-11 nucleotides) that induces RNAi in the cell.

[0055] miRNA is referred to in the art as an RNA molecule that induces RNAi in cells, wherein the RNAi comprises a short (e.g., 19-25 base pairs) sequence of a double-stranded RNA linked by a loop, and contains one or more additional sequences of the double-stranded RNA comprising one or more bumps (e.g., mismatched or unpaired base pairs). As used herein, the term “miRNA” encompasses both endogenous miRNA and exogenous or heterologous miRNA. In some embodiments, “miRNA” may refer to pri-miRNA or pre-miRNA. During miRNA processing, pri-miRNA transcripts are produced. pri-miRNA is processed by Drosha-DGCR8 to produce pre-miRNA, specifically by removing one or more sequences, leaving pre-miRNA with a 5' flanking region, a guide strand, a loop region, a non-guide strand, and a 3' flanking region; or pre-miRNA with a 5' flanking region, a non-guide strand, a loop region, a guide strand, and a 3' flanking region. The pre-miRNA is then exported to the cytoplasm and processed by Dicer to produce siRNA with a guide strand and a non-guided (or guest) strand. The RISC complex then uses the guide chain to catalyze gene silencing, for example, by recognizing a target RNA sequence complementary to the guide chain. Further descriptions of miRNAs can be found, for example, WO 2008 / 150897. The recognition of a target sequence by a miRNA is primarily determined by the pairing between the target sequence and the miRNA seed sequence, for example, nucleotides 1–8 (5' to 3') of the guide chain (see, for example, Boudreau, RL et al. (2013) Nucleic Acids Res. 41:e9).

[0056] In some embodiments of this disclosure, the repressive RNA molecule forms a hairpin structure. Typically, the hairpin-forming RNA is arranged in a self-complementary "stem-loop" structure, comprising a single nucleic acid encoding a stem portion having a double helix containing a sense strand (e.g., a guest strand) connected to an antisense strand (e.g., a guide strand) via a loop sequence. The guest strand and the guide strand share complementarity. In some embodiments, the guest strand and the guide strand share 100% complementarity. In some embodiments, the guest strand and the 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 guest strand and the guide strand may lack complementarity due to base pair mismatches. In some embodiments, the guest strand and the 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. Typically, 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 implementations, the RNA forming the hairpin has a mismatch at the anchor residue.

[0057] In some implementations, repressive RNA molecules are processed in cells (or subjects) to form "mature miRNAs." Mature miRNAs are produced via a multi-step pathway that begins with the transcription of an initial miRNA from its miRNA gene or intron by RNA polymerase II or III, generating an initial precursor molecule in the biological pathway of miRNA production. Once transcribed, the pri-miRNA (typically longer than one thousand nucleotides 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, producing the pre-miRNA. The pre-miRNA is exported to the cytoplasm, where it is further reduced in a loop by the Dicer enzyme to produce a double-stranded miRNA chain.

[0058] Of the two strands of a miRNA duplex, one arm, the guide strand (miR), is typically present in a higher concentration and binds to and associates with the Argonaute protein, which is ultimately loaded into the RNA-induced silencing complex. The guide strand miRNA-RISC complex helps regulate gene expression by binding to its complementary sequence on the mRNA (typically in the 3' UTR). The non-guide strand of the miRNA duplex, called the transit strand, is usually degraded but can persist and function in gene expression, either intact or partially degraded.

[0059] In some embodiments, the transgene is engineered to express a repressive nucleic acid (e.g., miRNA) having a guide strand that targets a human gene. "Targeting" refers to the hybridization or specific binding of the repressive nucleic acid to a homologous (e.g., complementary) sequence on the target gene (e.g., the mRNA transcript of the target gene). In some embodiments, the repressive nucleic acid of the target gene transcript shares a complementary region with the target gene of 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 in length. In some embodiments, the complementary region is longer than 30 nucleotides.

[0060] Typically, the guide strand can target human gene transcripts associated with a disease or condition. Examples include the human gene transcripts of FXYD1. In some embodiments, the guide strand targeting any gene transcript can be complementary to a segment of the sequence shown above (such as SEQ ID NO: 2-4).

[0061] In some implementations, the length of the repressive nucleic acid is 5 to 300 bases (e.g., 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 nucleotides, etc.). The length of the repressive nucleic acid sequence encoding pre-miRNA or mature miRNA may be 10 to 50 bases or 5 to 50 bases.

[0062] support

[0063] In some implementations, repressive RNA molecules can be encoded in repressive nucleic acids containing a molecular scaffold. As used herein, a “molecular scaffold” is a framework or initiator molecule that forms the sequence or structural basis for designing or manufacturing subsequent molecules.

[0064] In some embodiments, the molecular scaffold comprises at least one 5' flanking region or one 3' flanking region, or both. As a non-limiting example, the 5' or 3' flanking region may comprise a 5' or 3' flanking sequence, which may have any length and may be wholly or partially derived from a wild-type microRNA sequence or entirely an artificial sequence. In some embodiments, one or both of the 5' and 3' flanking sequences may be absent. In some embodiments, the 5' and 3' flanking sequences may have the same or different lengths. In some embodiments, the length of the 5' or 3' flanking sequence may be 1 to 10 nucleotides, 5 to 15 nucleotides, 10 to 30 nucleotides, 20 to 50 nucleotides, more than 40 nucleotides, more than 50 nucleotides, more than 100 nucleotides, or more than 200 nucleotides.

[0065] In some embodiments, the repressive nucleic acid sequence comprising or encoding a pri-miRNA scaffold has a length of at least 200, 250, 260, 270, 280, 290, or 300 bases. In some embodiments, the repressive nucleic acid comprises or is composed of a base sequence that is at least 80% or 90% complementary to the target nucleic acid (e.g., at least 5, 10, 15, 20, 25, or 30 bases, or up to 30 or 40 bases of the target nucleic acid), or comprises a base sequence having at most 3 mismatches (e.g., at most 1 or at most 2 mismatches) on 10, 15, 20, 25, or 30 bases of the target nucleic acid.

[0066] In some implementations, the repressive nucleic acid is an artificial miRNA (amiRNA). amiRNAs are derived by modifying natural miRNAs to replace the natural target region of the pre-mRNA with the target region of interest. For example, naturally occurring, expressed miRNAs can be used as scaffolds or backbones (e.g., pri-miRNA scaffolds), where the stem sequence is replaced by the stem sequence of a miRNA targeting the gene of interest. Artificial precursor microRNAs (pre-amiRNAs) are typically processed in a manner that preferentially produces a single, stable small RNA.

[0067] The stem forming the stem-loop structure is a minimal repressive 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 will hybridize with the target sequence. In some embodiments, the 5' arm of the stem-loop structure of the repressive nucleic acid contains 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 repressive nucleic acid contains a nucleic acid sequence encoding the said antisense / guide sequence.

[0068] In some embodiments, the circular sequence (also referred to as a circular motif, adapter, or adapter motif) that separates the sense and antisense sequences of the stem-loop structure of the repressive nucleic acid is a circular sequence. The circular sequence can have any length, including 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.

[0069] Some aspects of this disclosure relate to a nucleic acid sequence encoding a guide strand targeting a human gene, said guide strand being inserted into a human or non-human (e.g., mouse) pri-miRNA scaffold. In some embodiments, the pri-miRNA scaffold may 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 located on the repressive nucleic acid flanking a target human mRNA (such as the mRNA of FXYD1) or its target sequence.

[0070] Therefore, repressive nucleic acids can be used to mediate gene silencing by interacting with RNA transcripts or, alternatively, by interacting with specific gene sequences, particularly FXYD1, where such interactions enable gene silencing at the transcriptional or post-transcriptional level (e.g., but not limited to RNAi) or by regulating the chromatin structure or methylation pattern of the target and preventing transcription of the target gene, wherein the nucleotide sequence of the target thereby mediates silencing.

[0071] These repressive nucleic acids can contain short double-stranded regions of RNA. A double-stranded RNA molecule can contain two distinct and independent strands, which can be symmetrical or asymmetrical and complementary, i.e., two single-stranded RNA molecules, or it can contain a single-stranded molecule in which two complementary regions (e.g., sense and antisense regions) are base-paired and covalently linked by one or more single-stranded "hairpin" regions (i.e., loops), producing, for example, single-stranded short hairpin polynucleotides or cyclic single-stranded polynucleotides.

[0072] 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 circular repressive nucleic acid molecule contains one or more circular motifs, wherein at least one circular portion of the molecule is biodegradable. For example, single-stranded hairpin molecules can be designed such that the degradation of the circular portion of the molecule in vivo can produce double-stranded siRNA molecules with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising 1, 2, 3, or 4 nucleotides. Alternatively, circular repressive nucleic acid molecules can be designed such that the degradation of the circular portion of the molecule in vivo can produce, for example, double-stranded siRNA molecules with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.

[0073] In symmetrical repressive nucleic acid molecules, the length of each strand (sense strand and antisense strand) can be independently from about 15 to about 40 nucleotides (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).

[0074] In asymmetric repressive nucleic acid molecules, the antisense region or chain of the molecule can be about 15 to about 30 nucleotides long (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30), of which the sense region is about 3 to about 25 nucleotides long (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).

[0075] In other embodiments, the repressive nucleic acid molecule described herein may comprise a single-stranded hairpin siRNA molecule, wherein the length of the molecule may be from 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) nucleotides.

[0076] In other embodiments, the molecule may comprise a single-stranded circular siRNA molecule, wherein the length of the molecule is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65 or 70) nucleotides.

[0077] In each of the various symmetrical embodiments, the inhibitory nucleic acid duplexes 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).

[0078] In other embodiments, when 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.

[0079] In other embodiments, when the inhibitory nucleic acid molecule is a hairpin or ring 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.

[0080] The sense and antisense strands, or sense and antisense regions, of a repressive nucleic acid molecule can be complementary. Furthermore, the antisense strand or antisense region can be complementary to the nucleotide sequence of the target RNA (e.g., the target RNA of FXYD1) or a portion thereof. If the repressive nucleic acid can contain the nucleotide sequence of the target gene or a portion thereof, then the sense strand or sense region can contain the nucleotide sequence of the target gene or a portion thereof.

[0081] In some embodiments, the repressive nucleic acid may be optimized (sequence-based) or chemically modified to minimize degradation before and / or after delivery to the tissue of interest. Commercially available 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 repressive 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 target genes by: binding to the gene's DNA to restrict expression, such as antisense oligonucleotides; or by applying posttranscriptional gene silencing (PTGS) through mechanisms including, but not limited to, mechanisms that directly bind to the target transcript or gene product or one or more other proteins to reduce the expression of the gene; or the use of other small molecule decoys that reduce the expression of specific genes.

[0082] Any inhibitory nucleic acid molecule or construct described herein may contain one or more chemical modifications. Modifications may be used to improve in vitro or in vivo properties such as stability, activity, toxicity, immune response (e.g., prevention of stimulation of interferon response, inflammatory or pro-inflammatory cytokine response, or Toll-like receptor (TIF) response) and / or bioavailability.

[0083] Compared to their unmodified or minimally modified counterparts, the chemically modified molecules exhibit enhanced RNAi activity. The chemically modified motifs disclosed herein provide the ability to maintain RNAi activity substantially similar to that of unmodified or minimally modified active siRNAs, while also providing nuclease resistance and pharmacokinetic properties suitable for therapeutic applications.

[0084] In various embodiments, the repressive nucleic acid molecules described herein may contain modifications in which any (e.g., one or more or all) nucleotides present in the sense strand and / or antisense strand are modified nucleotides. In some embodiments, the molecule may be partially modified by chemical modifications (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, or 80 nucleotides are modified). In other embodiments, the molecule may be fully modified (e.g., 100% modified) by chemical modification.

[0085] The chemical modifications within a single molecule can 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 can be the same or different, such as modifications to sugars, bases, or backbones (i.e., internucleotide bonds). In other embodiments, molecule A can contain at least two, three, four, five, or more different chemical modifications.

[0086] Non-limiting examples of suitable chemical modifications include, for example, those disclosed in U.S. Patent Nos. 8202979 and 20050266422, and include sugars, bases and phosphates, non-nucleotide modifications and / or any combination thereof.

[0087] In various embodiments, most pyrimidine nucleotides present in the double-stranded repressive nucleic acid molecule contain sugar modifications. In other embodiments, most purine nucleotides present in the double-stranded molecule contain sugar modifications. In some cases, purines and pyrimidines are differentially modified at the 2'-sugar position (i.e., at least one purine and at least one pyrimidine have different modifications in the same or different strands at the 2'-sugar position).

[0088] In some specific 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 other embodiments, at least one nucleotide has a ribose-like, Northern, or A-helical configuration (see, for example, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Non-limiting examples of nucleotides having a Northern conformation include locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-furanose)nucleotides); 2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoronucleotides, 2'-deoxy-2'-chloronucleotides, 2'-azidonucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides.

[0089] The repressive nucleic acids described herein can be obtained using a variety of techniques known to those skilled in the art. For example, repressive nucleic acids can be chemically synthesized or can be encoded by plasmids (e.g., transcribed into sequences that automatically fold into double strands with hairpin loops). siRNAs can also be produced by cleaving longer dsRNAs.

[0090] In some embodiments, the repressive nucleic acid is chemically synthesized. Oligonucleotides (e.g., certain modified oligonucleotides or oligonucleotide moieties lacking ribonucleotides) can be synthesized using methods known in the art, such as those described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19; Thompson et al., International PCT Publication WO 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 dimethoxytriphenylmethyl at the 5' end and phosphoramidite at the 3' end.

[0091] Alternatively, repressive nucleic acids can be synthesized individually and conjugated together post-synthesis, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT Publication No. WO 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.

[0092] In some implementations, the repressive nucleic acid can be expressed and delivered from a transcription unit 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 virus, retrovirus, adenovirus, or alphavirus.

[0093] B. CRISPR / Cas system

[0094] On the one hand, the inhibition or knockdown of one or more genes described herein can also be achieved using the CRISPR / Cas system and related methods known in the art via CRISPR-Cas-guided nucleases. See, for example, US11225659B2, WO 2021168799A1, WO 2022188039A1, WO 2022188797A1, WO 2022068912A1 and WO2022047624A1. 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 / CasSystems,” 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), all of which are hereby incorporated by full citation.

[0095] The CRISPR-Cas system is a genetic technology that allows for sequence-specific control of gene expression in prokaryotic and eukaryotic cells by guiding the cleavage of double-stranded DNA by nucleases. It is based on the CRISPR (clustered regularly spaced palindromic repeats) pathway derived from the bacterial immune system.

[0096] On the other hand, this application provides a complex comprising: (i) a protein composition comprising a Cas protein or its ortholog, homolog, derivative, conjugate, functional fragment, conjugate, or fusion thereof; and (ii) a polynucleotide composition comprising 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.

[0097] Target RNA can be any RNA molecule of interest, including naturally occurring and engineered RNA molecules. Target RNA can be mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), interfering RNA (siRNA), ribozymes, riboswitches, satellite RNA, microswitches, yeast or viral RNA.

[0098] In some embodiments, the target nucleic acid is associated with symptoms or diseases, such as those mediated by disrupted ion homeostasis and / or hyperexcitability, as described herein, and related conditions. Therefore, in some embodiments, the system described herein can be used to treat such symptoms or diseases by targeting these nucleic acids.

[0099] For example, target nucleic acids associated with symptoms or diseases can be RNA molecules overexpressed in diseased cells, older or less aged cells, or senescent cells. Target nucleic acids can also be toxic RNAs and / or mutant RNAs (e.g., mRNA molecules with splicing defects or mutations). Target nucleic acids can also be miRNAs. For example, a target nucleic acid can be a gene whose increased activity is associated with the loss of [K+]e homeostasis and extracellular K+. + This is related to a pathological increase, which leads to overexcitation and neuronal loss in neurodegenerative diseases.

[0100] Various Cas proteins can be used in this invention. Interchangeable Cas proteins, CRISPR-related proteins, or CRISPR proteins refer to proteins of CRISPR-Cas class 1 or 2 or proteins derived from CRISPR-Cas class 1 or 2, including type I, II, III, IV, V, or VI systems, which have RNA-guided 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 CasB), 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. See, for example, US11225659B2, WO 2021168799A1, WO 2022188039A1, WO 2022188797A1, WO 2022068912A1, WO 2022047624A1, WO 2014144761, WO 2014144592, WO 2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entirety.

[0101] C. Recombinant nucleic acids

[0102] The recombinant nucleic acids disclosed herein include the repressive nucleic acids described above, as well as plasmids and vector genomes containing the repressive nucleic acids. The recombinant nucleic acid, plasmid, or vector genome may contain regulatory sequences to regulate proliferation (e.g., plasmid proliferation) and / or control the expression of transgenes (e.g., repressive nucleic acids). Recombinant nucleic acids may also be provided as components of viral vectors (e.g., rAAV vectors). Typically, viral vectors comprise a vector genome containing recombinant nucleic acids packaged in a capsid.

[0103] D. Control element

[0104] This disclosure includes recombinant nucleic acids, including transgenes (e.g., transgenes encoding RNA) and various regulatory or control elements (e.g., post-transcriptional regulatory elements for guinea pig hepatitis). Typically, a regulatory element is one or more nucleic acid sequences that affect the expression of an operatively linked polynucleotide. The precise nature of the regulatory element used for gene expression will vary depending on the organism and cell type, and includes, for example, promoters, enhancers, introns, etc., with the aim of promoting proper heteropolynucleotide transcription and / or translation. Regulatory control can be affected at the levels of transcription, translation, splicing, message stability, etc. Typically, regulatory control elements regulating transcription are located near the 5' end (i.e., upstream) of the transcribed polynucleotide. Regulatory control elements can also be located at the 3' end (i.e., downstream) of the transcribed sequence or within the transcript (e.g., in an intron). Regulatory control elements can be located at a certain distance from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of the vector genome (e.g., the AAV vector genome), regulatory control elements are typically located within 1 to 1000 nucleotides of the polynucleotide.

[0105] promoter

[0106] As used herein, the term "promoter" (e.g., "eukaryotic promoter") refers to a nucleotide sequence or one or more coding sequences in a eukaryotic cell (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) that initiates the transcription of a specific gene. Promoters may function in conjunction with other regulatory elements or regulatory regions to direct the transcriptional level of the gene or the one or more coding sequences. These regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to function directly or indirectly to regulate the amount of transcription by the promoter, including, for example, attenuators, enhancers, and silencers. Promoters are most commonly located on the same strand and near the transcription start site, i.e., at the 5' of the gene or coding sequence to which they are operatively linked. Promoter length is typically 100–1000 nucleotides. Promoters generally increase gene expression relative to the expression of the same gene in the absence of a promoter.

[0107] As used herein, a “core promoter” or “minimum promoter” refers to the smallest portion of the promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, an RNA polymerase binding site, and a universal transcription factor binding site. The promoter may also contain a proximal promoter sequence (5' of the core promoter) and a distal promoter sequence (3' of the core promoter) containing other major regulatory elements (e.g., enhancers, silencers, boundary elements, insulators).

[0108] Examples of suitable promoters include adenovirus promoters, such as the adenovirus major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoters; Rous sarcoma virus (RSV) promoters; albumin promoters; inducible promoters, such as the mouse mammary tumor virus (MMTV) promoter; metallothionein promoters; heat shock promoters; α-1-antitrypsin promoters; hepatitis B surface antigen promoters; transferrin promoters; apolipoprotein A-1 promoters; chicken β-actin (CBA) promoters; elongation factor 1a promoters (EF1a); hybrid forms of CBA promoters (CBh promoters); and CAG promoters (cytomegalovirus promoters). c ytomegalovirus) early enhancer elements and chicken β-actin ( a The promoter, first exon, and first intron of the ctin gene, as well as rabbit β-globulin ( g (The splice acceptor of the lobin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2).

[0109] Promoters can be constitutive, tissue-specific, or regulated. Constitutive promoters are those that consistently enable the operative expression of linked genes. In some implementations, constitutive promoters are active in most eukaryotic tissues under most physiological and developmental conditions.

[0110] Regulated promoters are those that can be activated or inactivated. Regulated promoters include inducible promoters and "repressive" promoters; inducible promoters are typically "off" but can be induced to "on," while repressive promoters are typically "on" but can be "off." Many different regulatory factors are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute; constitutive promoters can often be regulated to some extent. In some cases, endogenous pathways can be used to regulate transgene expression, for example, by using promoters that are naturally downregulated when pathological symptoms improve.

[0111] Tissue-specific promoters are promoters that are active only in specific types of tissues, cells, or organs. Typically, tissue-specific promoters are recognized by transcriptional activator elements specific to a particular tissue, cell, and / or organ. For example, a tissue-specific promoter may be more active in one or more (e.g., two, three, or four) specific tissues than in others. In some embodiments, the expression of a gene regulated by a tissue-specific promoter is much higher in the tissue specifically targeted by that promoter than in other tissues. In some embodiments, the promoter may have little or no activity in any tissue other than its specifically targeted tissue.

[0112] enhancer

[0113] On the other hand, the recombinant nucleic acids described herein may further include enhancers to increase the expression of transgenes (e.g., the RNA molecules disclosed herein). Typically, enhancer elements are located upstream of promoter elements, but they can also be located downstream or within another sequence (e.g., the transgene). Enhancers can be located 100, 200, 300, or more nucleotides upstream or downstream of the modified nucleic acid. Enhancers typically increase the expression of transgenes (e.g., encoding repressive nucleic acids) beyond the expression increase provided by the promoter element alone.

[0114] Many enhancers are known in the art, including but not limited to the cytomegalovirus major immediate early enhancer. More specifically, the CMV MIE promoter comprises three regions: a regulator, 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 portion thereof to form a heterozygous promoter to further increase the expression of the nucleic acid operatively linked thereto. For example, the CBA promoter or a portion thereof can be combined with the CMV promoter / enhancer or a portion thereof to form a CBA version of the promoter called the “CBh” promoter, which represents the chicken β-actin heterozygous promoter (…). c Hicken b eta-actin h Hybrid promoters, as described by Gray et al. (2011, Human Gene Therapy 22:1143-1153). Like promoters, enhancers can be constitutive, tissue-specific, or regulated.

[0115] Filler, spacer, and stuffer sequences.

[0116] As disclosed herein, recombinant nucleic acids can be used in rAAV vectors. In this case, the recombinant nucleic acid may include additional nucleic acid elements to adjust the length of the nucleic acid to near or to the normal size of an acceptable viral genome sequence (e.g., approximately 4.7 to 4.9 kilobases) in order to package AAV into the rAAV vector (Grieger and Samulski (2005) J. Virol. 79(15):9933-9944). Such sequences may be interchangeably referred to as filler sequences, spacer sequences, or plugging sequences. In some embodiments, the filler DNA is a non-translated (non-protein-coding) nucleic acid segment. In some implementations, the filling or plugging polynucleotide sequence is a sequence of length about 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.

[0117] AAV vectors typically accept DNA inserts ranging in size from about 4 kb to about 5.2 kb or from about 4.1 kb to 4.9 kb to optimally package nucleic acids into the AAV capsid. In some embodiments, the rAAV vector comprises a vector genome of total length between about 3.0 kb and about 3.5 kb, about 3.5 kb to about 4.0 kb, about 4.0 kb to about 4.5 kb, about 4.5 kb to about 5.0 kb, or about 5.0 kb to about 5.2 kb. In some embodiments, the rAAV vector comprises a vector genome of total length about 4.7 kb. In some embodiments, the rAAV vector comprises a self-complementary vector genome. Although the total length of the self-complementary (sc) vector genome in the rAAV vector is equal to that of the single-stranded (ss) vector genome (i.e., approximately 4kb to approximately 5.2kb), the nucleic acid sequence encoding the sc vector genome (i.e., containing transgenes, regulatory elements, and ITRs) must be only half the length of the nucleic acid sequence encoding the ss vector genome in order to encapsulate the sc vector genome.

[0118] Introns and exons

[0119] In some embodiments, the recombinant nucleic acids disclosed herein include, for example, introns, exons, and / or portions thereof. Introns can act as filler or plugging polynucleotide sequences to achieve the appropriate length for packaging the vector genome into the rAAV vector. Intron and / or exon sequences can also enhance the expression of transgenes (e.g., the RNA disclosed herein) compared to expression without intron and / or exon elements (Kurachi et al. (1995) J. Biol. Chem. 270 (10):576-5281; WO 2017 / 074526). Furthermore, filler / plugging polynucleotide sequences (also known as “insulators”) are well known in the art and include, but are not limited to, those described in WO 2014 / 144486 and WO 2017 / 074526.

[0120] Polyadenylation signal sequence (polyA)

[0121] Other regulatory elements may include stop codons, stop sequences, and polyadenylation (polyA) signaling sequences, such as, but not limited to, the bovine growth hormone polyA signaling sequence (BHG polyA). The polyA signaling sequence drives the efficient addition of a polyadenylated "tail" to the 3' end of eukaryotic mRNA, which guides the termination of gene transcription (see, for example, Goodwin and Rottman J. Biol. Chem. (1992) 267(23):16330-16334). The polyA signal acts as a signal for endonuclease at the 3' end of newly formed precursor mRNA and the addition of an RNA strand consisting only of adenine bases to this 3' end. The polyA tail is important for nuclear export, translation, and stability of mRNA. In some implementations, poly A can be an early polyadenylation signal of SV40, a late polyadenylation signal of SV40, a polyadenylation signal of HSV thymidine kinase, a polyadenylation signal of protamine gene, a polyadenylation signal of adenovirus 5 E1b, a polyadenylation signal of growth hormone, a polyadenylation signal of PBGD, or a computer-designed polyadenylation signal.

[0122] 3. Expression box and expression carrier

[0123] This disclosure also provides an expression cassette comprising or composed of a recombinant nucleic acid encoding or consisting of the repressive nucleic acid as described above. Where the recombinant nucleic acid may not yet contain a promoter, the expression cassette may additionally contain a promoter. Thus, the expression cassette according to the invention comprises a promoter, a coding sequence, and optionally a terminator or other elements in the 5' to 3' direction. The expression cassette allows for the easy transfer of the nucleic acid sequence of interest into an organism (preferably a cell, and preferably a diseased cell).

[0124] The expression cassette of this disclosure is preferably contained in a vector. Therefore, the vector of this disclosure allows transformation of cells with the nucleic acid sequence of interest. Correspondingly, this disclosure provides host cells containing the expression cassette according to this disclosure or the recombinant nucleic acid according to this disclosure. The recombinant nucleic acid may also contain a promoter or enhancer to allow expression of the nucleic acid sequence of interest.

[0125] Exogenous genetic material (e.g., nucleic acids, expression cassettes, or expression vectors encoding one or more therapeutic or repressive RNAs) can be introduced into target cells of interest via genetic transfer methods (such as transfection or transduction) to provide genetically modified cells. Various expression vectors (i.e., agents 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 natural or synthetic nucleic acids or oligonucleotides that are not naturally present in cells; or, if naturally present in cells, are not transcribed or expressed by cells at a biologically significant level. Therefore, “exogenous genetic material” includes, for example, non-naturally present nucleic acids that can be transcribed into RNA.

[0126] As used herein, “cell transfection” refers to the acquisition of new genetic material into cells by incorporation of added nucleic acids (DNA, RNA, or hybrids thereof) without the use of viral delivery agents. Therefore, transfection refers to the introduction of nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those skilled in the art, including: calcium phosphate coprecipitation, strontium phosphate coprecipitation, DEAE-glucan, electroporation, cationic liposome-mediated transfection, and tungsten particle-promoted microparticle bombardment. In contrast, “cell transduction” refers to the process of transferring nucleic acids into cells using DNA or RNA viruses. RNA viruses used to transfer nucleic acids into cells (e.g., retroviruses) are referred to herein as transducing chimeric viruses. The exogenous genetic material contained within the virus can be incorporated into the genome of the transduced cell. Cells transduced with chimeric DNA viruses (e.g., adenoviruses carrying DNA encoding therapeutic agents) may not have the exogenous genetic material integrated into their genome, but may be able to express the exogenous genetic material retained extrachromosomally within the cell.

[0127] Typically, exogenous genetic material may include a heterologous gene (encoding therapeutic RNA or protein) and a promoter that controls the transcription of the novel gene. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material may further include additional sequences (i.e., enhancers) required to obtain the desired gene transcriptional activity. The exogenous genetic material may be introduced directly downstream of the promoter into the cell genome so that the promoter and coding sequence are operatively linked, thereby allowing transcription of the coding sequence. Retroviral expression vectors may include exogenous promoter elements to control the transcription of the inserted exogenous gene. Such exogenous promoters include constitutive and inducible promoters.

[0128] Naturally occurring constitutive promoters control the expression of essential cellular functions. As a result, genes controlled by constitutive promoters are expressed under all cell growth conditions. Exemplary constitutive promoters include promoters encoding genes with certain constitutive or "housekeeping" functions, such as hypoxanthine phosphoribosyltransferase, dihydrofolate reductase, adenosine deaminase, glycerol phosphokinase, pyruvate kinase, glycerol phosphotransmutase, actin promoter, ubiquitin, elongation factor-1, and other constitutive promoters known to those skilled in the art. Additionally, many viral promoters function constitutively in eukaryotic cells. These promoters include early and late promoters of SV40; long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus, etc. Therefore, any of the constitutive promoters mentioned above can be used to control the transcription of heterologous gene insertions.

[0129] Genes under the control of inducible promoters are expressed only in the presence of an inducer or are primarily controlled by the presence of an inducer (e.g., transcription under the control of metallothionein promoters increases dramatically in the presence of certain metal ions). Inducible promoters include response elements (REs) that stimulate transcription when their inducible factors bind. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMPs. Promoters containing specific REs can be selected to obtain an inducible response, and in some cases, the REs themselves can be linked to different promoters, thereby conferring inducibility to recombinant genes. Therefore, the presence and expression levels of therapeutic agents in genetically modified cells can be controlled by selecting appropriate promoters (constitutive promoters versus inducible promoters; strong promoters versus weak promoters). If the gene encoding a therapeutic agent is under the control of an inducible promoter, then in situ delivery of the therapeutic agent can be triggered by in situ exposure of the genetically modified cells to conditions that allow transcription of the therapeutic agent (e.g., by injection of a specific inducer of the inducible promoter that controls the transcription of the therapeutic agent). For example, by in situ contacting genetically modified cells with a solution containing appropriate (i.e., induced) metal ions, the in situ expression of gene-encoded therapeutic agents controlled by the metallothionein promoter in genetically modified cells was enhanced.

[0130] Therefore, the amount of therapeutic agent delivered in situ is modulated by controlling the following factors: (1) the nature of the promoter used to guide the transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak); (2) the copy number of the exogenous gene in the inserted cell; (3) the number of transduced / transfected cells administered (e.g., implanted) to the patient; (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 implant remain in situ; and (7) the therapeutic agent production rate of the genetically modified cells. Considering the above factors and the clinical spectrum of patients, the selection and optimization of these factors for delivering a specific therapeutic agent at an effective dose is considered to be within the scope of those skilled in the art without requiring excessive experimentation.

[0131] 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 (e.g., a neomycin resistance gene or a fluorescent protein gene) to facilitate selection of cells transfected or transduced with the expression vector. Alternatively, cells may be transfected with two or more expression vectors, at least one vector containing a gene encoding a therapeutic agent, and another vector containing a selection gene. The selection of suitable promoters, enhancers, selection genes, and / or signal sequences is considered to be within the scope of those skilled in the art without requiring excessive experimentation.

[0132] The coding sequences disclosed herein can be inserted into any type of target cell or host cell. In the case of expression vectors, the vectors can be readily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0133] 4. Delivery of carriers / polynucleotides

[0134] As disclosed herein, the aforementioned polynucleotides or nucleic acid molecules can be used to treat a subject's condition. Therefore, this disclosure provides systems and methods for delivering polynucleotides to target cells or a subject.

[0135] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for generating 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, ColdSpring Harbor Laboratory, New York).

[0136] Biological methods for introducing polynucleotides of interest 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 (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0137] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems 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 used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0138] The polynucleotides or nucleic acids described herein (e.g., repressive nucleic acids, polynucleotides or nucleic acids encoding CRISPR-Cas systems, expression cassettes, and expression vectors) can be added directly, or conjugated with cationic lipids, packaged in liposomes, or used as recombinant plasmids or viral vectors, or otherwise delivered to target cells or tissues. Methods for delivering nucleic acid molecules are known in the art. See, for example, U.S. Patent Nos. 6,395,713 and WO 94 / 02595; Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, edited by 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 to deliver virtually any nucleic acid molecule. Nucleic acid molecules can be applied to cells by a variety of methods known to those skilled in the art, including but not limited to encapsulation in liposomes, via iontophoresis, or by incorporation into other media such as biodegradable polymers, hydrogels, cyclodextrins (see, for example, Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; WO 03 / 47518 and WO 03 / 46185), poly(lactic-co-glycolic acid) (PLGA) and PLCA microspheres (see, for example, U.S. Patent Nos. 6,447,796 and 2002130430), biodegradable nanocapsules, and bioadhesive microspheres) or via protein carriers (see, for example, WO 00 / 53722).

[0139] On one hand, this application provides a carrier system containing the nucleic acid molecules described herein. In some embodiments, the carrier system is a lipid-based carrier system, a cationic lipid or liposomal nucleic acid complex, liposomes, micelles, virions, lipid nanoparticles, or mixtures thereof. In other embodiments, the carrier system is a polymer-based carrier system, such as a cationic polymer-nucleic acid complex. In still other embodiments, the carrier system is a cyclodextrin-based carrier system, such as a cyclodextrin polymer-nucleic acid complex. In yet another embodiment, 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”) formulation described herein can be applied to any nucleic acid molecule (e.g., RNA molecule) or combination of nucleic acid molecules described herein.

[0140] In some embodiments, the nucleic acid molecules described herein are formulated into lipid nanoparticle compositions, as described in U.S. Patent Nos. 7,514,099 and 7,404,969. In some embodiments, this application is characterized by compositions comprising nucleic acid molecules formulated as any of the formulations described in US 20120029054, 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.

[0141] In other embodiments, this disclosure is characterized by conjugates and / or complexes of nucleic acid molecules described herein. Such conjugates and / or complexes can be used to facilitate the delivery of nucleic acid molecules into biological systems, such as cells. The conjugates and complexes thus provided can confer 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. Patent Nos. 7,833,992; 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136; and 5,138,045.

[0142] In various embodiments, polyethylene glycol (PEG) may be covalently linked to the nucleic acid molecule described herein. The linked PEG may be of any molecular weight, preferably from about 100 Daltons (Da) to about 50,000 Daltons (Da). Therefore, this disclosure is characterized by compositions or formulations comprising surface-modified liposomes and the nucleic acid molecule described herein, said surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, long-circulating, or stealthy liposomes). See, for example, WO 96 / 10391, WO 96 / 10390, and WO 96 / 10392.

[0143] In some embodiments, the nucleic acid molecule may also be formulated or compounded with polyethyleneimine and its 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, the nucleic acid molecule may be formulated in the manner described in US20030077829.

[0144] In other embodiments, the nucleic acid molecules described herein may be complexed with membrane disruptors (such as those described in US20010007666). In still other embodiments, one or more membrane disruptors and molecules may be complexed with cationic lipids or cofactor lipid molecules (such as those described in US Patent No. 6,235,310).

[0145] In some embodiments, the nucleic acid molecules described herein may be combined with delivery systems as described in U.S. Patent Application Publications Nos. 2003077829, 20050287551, 20050164220, 20050191627, 20050118594, 20050153919, 20050085486, and 20030158133; as well as WO 00 / 03683 and WO 02 / 087541.

[0146] In some embodiments, the liposome formulations described herein may comprise the same components as those in 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 Publication No. 2006 / 0019912; 2006 The compounds and compositions described in Nos. 0019258, 2006 / 0008909, 2005 / 0255153, 2005 / 0079212, 2005 / 0008689, 2003 / 0077829, 2005 / 0064595, 2005 / 0175682, 2005 / 0118253, 2004 / 0071654, 2005 / 0244504, 2005 / 0265961, and 2003 / 0077829 are formulated or compounded with nucleic acid molecules (e.g., inhibitory nucleic acids) as described herein.

[0147] As disclosed herein, the nucleic acid molecules described above can be used to treat a subject's condition. Vectors discussed above (such as recombinant plasmids and viral vectors) can be used to deliver therapeutic agents, such as the inhibitory nucleic acids or CRISPR-Cas systems described herein. Delivery of the vector can be systemic, such as by intravenous or intramuscular administration; by administration to target cells transplanted from the subject, followed by reintroduction into the subject; or by any other means allowing introduction into desired target cells. Such recombinant vectors can also be administered directly or in combination with suitable delivery agents, including, for example, the lipophilic agent Mirus Transit LT1; lipofectin; lipofectamine; cellfectin; polycationic (e.g., polylysine) or liposomal lipid-based carrier systems, cationic lipids or liposomal nucleic acid complexes, micelles, virions, lipid nanoparticles.

[0148] A. Viral vector

[0149] In some embodiments, the polynucleotide encoding a therapeutic agent (e.g., an RNA molecule) may be inserted into or encoded by a vector (such as a plasmid or 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, adenovirus vector, AAV vector, lentiviral vector, etc. 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. Patent Nos. 5,399,346; 5,124,263; 4,650,764 and 4,980,289; the contents of each of these U.S. Patents are incorporated herein by reference in their entirety).

[0150] Lentiviral vector

[0151] Lentiviruses (such as HIV) are “slow viruses.” Vectors derived from lentiviruses can be expressed long-term in host cells, for example, through in vitro transduced stem cells or progenitor cells, after several administrations to a patient. Long-term expression is crucial for successful treatment of most diseases and conditions, including genetic disorders, cancer, and neurological disorders. Regarding the safety of lentiviral vectors, many strategies for eliminating the replication capacity of lentiviral vectors are currently known in the art. See, for example, US 20210401868 and 20210403517, each of which is incorporated herein by reference in its entirety. For example, deletion of promoter and enhancer elements from the U3 region of a long terminal repeat (LTR) is considered to eliminate LTR-directed transcription. The resulting vector is referred to as “self-inactivation” (SIN).

[0152] Lentiviral vectors are particularly well-suited for long-term gene transfer because they allow transgenes to integrate stably and persistently and multiply in daughter cells. Lentiviral vectors offer additional advantages over vectors derived from oncogenic retroviruses (such as murine leukemia virus) because they can transduce non-proliferating cells (such as CNS cells). They also have the added advantage of low immunogenicity. Typically, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectivity markers (e.g., WO01 / 96584 and WO01 / 29058; and U.S. Patent No. 6,326,193). Several vector promoter sequences can be used to express transgenes. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any operatively linked polynucleotide sequence. Another example of a suitable promoter is EF1a. However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Raoult sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0153] This disclosure provides recombinant lentiviruses capable of infecting both dividing and non-dividing cells, such as oligodendrocytes, astrocytes, or glial progenitor cells. The viruses can be used for in vivo and in vitro transfer and expression of nucleic acid sequences. The lentiviral vectors of this disclosure can be lentiviral transfer plasmids or infectious lentiviral particles. The construction of lentiviral vectors, helper constructs, envelope constructs, etc., for lentiviral transfer systems has been described, for example, in US 20210401868 and 20210403517, each of which is incorporated herein by reference in its entirety.

[0154] adenovirus

[0155] Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver nucleic acids to a variety of cell types in the body and have been widely used in gene therapy protocols, including for targeting genes to nerve cells and glial cells. Various replication-defective adenoviruses and minimal adenovirus vectors for nucleic acid therapy have been described (see, for example, PCT patent publications WO199426914, WO199502697, WO199428152, WO199412649, WO199502697 and WO199622378; the contents of each of these PCT patent publications are incorporated herein by reference in their entirety). Such adenovirus vectors can also be used to deliver the therapeutic molecules of this disclosure to cells.

[0156] 4.AAV

[0157] Adeno-associated virus (AAV) is a widely used gene therapy vector due to its proven clinical safety, non-pathogenicity, ability to infect non-dividing cells (such as neurons), and ability to provide long-term gene expression after a single administration (Hocquemiller et al., 2016). Currently, multiple human and non-human primate AAV serotypes have been identified (Gao et al., 2004). AAV vectors have demonstrated safety in hundreds of clinical trials worldwide and clinical efficacy in hemophilia B, spinal muscular atrophy, α1-antitrypsin, and Leber congenital amaurosis (Keeler et al., 2017). Three AAV-based gene therapies have been approved. The first drug, Glybera, was approved by the European Medicines Agency (EMA) in 2012 (but was withdrawn in 2017, primarily due to commercial failure). Luxturna was approved by the FDA in 2017 for the treatment of rare, inherited retinal dystrophy, and Zolgensma was approved by the FDA in 2019 for the treatment of spinal muscular atrophy.

[0158] AAVs (such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9) are commonly used gene therapy vectors in CNS applications due to their safety, non-pathogenicity, and ability to infect neurons. However, after direct CNS infusion, these serotypes exhibit significant neuronal tropism and low expression in glial cells, especially when gene expression is driven by constitutive promoters. AAV1 / 2, AAV2, and AAV8 have been shown to transduce oligodendrocytes, but only when using oligodendrocyte-specific promoters (Chen et al., 1998; Lawlor et al., 2009; Li et al., 2019). Cell-specific promoters are required to achieve expression specificity, enabling non-selective cellular uptake and leaky transgene expression through cryptic promoter activity in non-oligodendrocyte lineages.

[0159] The methods described in this article for mitigating these problems include the use of AAV serotypes with high tropism for glial cells. Recently, a chimeric AAV capsid with strong selectivity for oligodendrocytes, namely AAV / Olig001, has been described using DNA shuffling and directed evolution (Powell et al., 2016). Subsequently, AAV / Olig001 was shown to transduce new oligodendrocytes in a mouse model of Canavan disease (Francis et al., 2021). Other methods, such as random mutagenesis and peptide library insertion, can be used to generate capsid libraries with tropism and selectivity that can screen for glial progenitor cells, astrocytes, or oligodendrocytes.

[0160] As discussed above, the terms “adeno-associated virus” and / or “AAV” refer to parvoviruses and their variants having a linear single-stranded DNA genome. Unless otherwise required, the terms encompass all subtypes and both naturally occurring and recombinant forms. Parvoviruses (including AAVs) can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids (e.g., transgenes) into the cell nucleus. In some embodiments, the introduced nucleic acids (e.g., rAAV vector genome) form circular polymers that persist as episomes in the nucleus of the transduced cell. In some embodiments, the transgene is inserted into a specific site in the host cell genome, such as a site on human chromosome 19. Site-specific integration, in contrast to random integration, is considered likely to produce a predictable long-term expression profile. The insertion site for AAV into the human genome is called AAVS1. Once introduced into a cell, RNA or polypeptide encoded by the nucleic acid can be expressed by the cell. Because AAVs are not associated with any pathogenic disease in humans, nucleic acids delivered by AAVs can be used to express therapeutic RNAs or polypeptides to treat diseases, conditions, and / or symptoms in human subjects.

[0161] AAV exists in many serotypes naturally, and at least fifteen wild-type serotypes (i.e., AAV1-AAV15) have been identified in humans to date. The difference between naturally occurring serotypes and variant serotypes lies in the presence of a protein capsid that is serologically different from other AAV serotypes. Examples include AAV1, AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74 (see WO 2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV, as well as recombinant variants (e.g., capsid variants with insertions, deletions, and substitutions), such as variants called AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, etc. "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, etc.

[0162] Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies against one AAV and antibodies against another. Such differences in cross-reactivity are typically due to differences in capsid protein sequences and antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAVs or artificial AAV mutants (e.g., recombinant AAVs) do not exhibit serological differences from any currently known serotype. These viruses can then be considered subgroups of the corresponding type, or more simply, variant AAVs. Therefore, as used herein, the term "serotype" refers to serologically distinct viruses, as well as serologically distinct viruses that may belong to a subgroup or variant of a given serotype.

[0163] A comprehensive list and alignment of the 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 AAV serotypes, as well as sequences of the native ITR, rep protein, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank login 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); the public information thereof is incorporated herein by reference. See also, 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. (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. USA99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publication WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; WO 2013 / 063379; WO 2014 / 194132; WO 2015 / 121501, and U.S. Patent Nos. 6,156,303 and 7,906,111.

[0164] 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 a non-natural sequence. The incorporation of a non-natural sequence into a virus defines a viral vector as a “recombinant” vector, and is therefore defined as an “rAAV vector.” An rAAV vector may comprise a heteropolynucleotide encoding a desired RNA, protein, or polypeptide (e.g., the RNA molecule disclosed herein). The recombinant vector sequence may be capsidated or packaged into an AAV capsid and is referred to as an “rAAV vector,” “rAAV vector particle,” “rAAV viral particle,” or simply “rAAV.”

[0165] For the generation of the rAAV carrier, the required VP1:VP2:VP3 ratio 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. Although the required VP1:VP2 ratio can be 1:1, the VP1:VP2 ratio can vary from 1:50 to 50:1.

[0166] This disclosure provides rAAV vectors comprising polynucleotide sequences not derived from AAV (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and sometimes two, AAV terminal repeat sequences (e.g., inverted terminal repeat sequences). The heterologous polynucleotide flanked by an ITR (also referred to herein as the “vector genome”) typically encodes an RNA or polypeptide of interest or a gene of interest, such as a target for therapeutic treatment. Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the subject with the encoded RNA / protein / peptide. Therefore, rAAV vectors can be used to transfer / deliver heterologous polynucleotides for expression, for example, for the treatment of a variety of diseases, conditions, and symptoms.

[0167] The rAAV vector genome typically retains a 145-base ITR, cis-linked with heterologous nucleic acid sequences of alternative viral rep and cap genes. Such ITRs can be used to generate recombinant AAV vectors; however, modified AAV ITRs and non-AAV terminal repeat sequences (including partially or fully synthesized sequences) can also be used for this purpose. ITRs form hairpin structures and, for example, serve as primers for host cell-mediated complementary DNA strand synthesis after infection. ITRs also play a role in viral packaging, integration, etc. The ITR is the only AAV viral element cis-required for AAV genome replication and packaging into the rAAV vector. The rAAV vector genome optionally contains two ITRs, typically located 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, CRISPR molecules, etc.). Both the 5' and 3' ITRs may contain the same sequence or may each contain different sequences. AAV ITRs can come from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or any other AAV.

[0168] The rAAV vectors disclosed herein may contain an ITR from an AAV serotype (e.g., wild-type AAV2, fragments thereof, or variants thereof), said AAV serotype being different from the capsid serotype (e.g., AAV8, Oligo001). Such rAAV vectors containing at least one ITR from one serotype but containing a capsid from a different serotype may be referred to as hybrid viral vectors (see U.S. Patent No. 7,172,893). The AAV ITR may comprise the complete wild-type ITR sequence, or its variants, fragments, or modifications, but will retain functionality.

[0169] In some implementations, the rAAV vector genome is linear, single-stranded, and flanked by AAV ITRs. Before heterologous gene transcription and translation, second-strand synthesis must be initiated by a DNA polymerase (e.g., a transducing intracellular DNA polymerase) using the free 3'-OH of one of the self-initiating ITRs, converting the approximately 4700 nucleotide single-stranded DNA genome into a double-stranded form. In some implementations, the full-length single-stranded vector genome (i.e., sense and antisense) is annealed 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 simultaneously transduced into the same cell. Regardless of how they are generated, once the double-stranded vector genome is formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0170] Transgenic expression efficiency of rAAV vectors may be hampered by the need to convert single-stranded rAAV genomes (ssAAV) into double-stranded DNA prior to expression. This step can be circumvented by using self-complementary AAV genomes (scAAV), which can package inverted repeat genomes that fold into double-stranded DNA without the need for 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.

[0171] The viral capsid of the rAAV vector can be derived from wild-type AAV or variant AAV, such as 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 WO 2015 / 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, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV (avian), AAV (cattle), AAV (dog), AAV (horse), AAV (primate), AAV (non-primate), AAV (snake), AAV (goat), AAV (shrimp), AAV (sheep), and their variants (see, for example, Fields et al., VIROLOGY, Vol. 2, Chapter 69 (4th edition), Lippincott-Raven Publishers). The capsid can be derived from various AAV serotypes disclosed in the following documents: U.S. Patent No. 7,906,111; Gao et al. (2004) J. Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO 2013 / 063379; WO 2014 / 194132; and includes the AAV (AAV-TT) variant disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 to RHM15-6 and their variants disclosed in WO 2015 / 013313. Complete complements of the AAV cap protein include VP1, VP2, and VP3. An ORF containing the nucleotide sequence encoding the AAV VP capsid protein may contain fewer complete complement sequences than the AAV cap protein, or may provide complete complement sequences of the AAV cap protein.

[0172] In some embodiments, an rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises a capsid sequence derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or variants thereof, to produce a chimeric capsid protein comprising 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 comprise a mixture of VP1 from one serotype, VP2 from a different serotype, VP3 from yet another different serotype, and combinations thereof. For example, the chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. Chimeric capsids may, for example, comprise AAV capsids having one or more B19 cap subunits, whereby the AAV cap protein or subunits may be replaced by B19 cap protein or subunits. For example, in one embodiment, the VP3 subunit of the AAV capsid may be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is the Olig001 capsid as described in WO 2021221995 and WO 2014052789, which are incorporated herein by reference.

[0173] In some implementations, chimeric vectors have been engineered to exhibit altered tropism or tropism for specific tissue or cell types. The term "tropism" refers to the preferential entry of a virus into certain cell types (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) and / or preferential interaction with cell surfaces, which facilitates entry into certain cell or tissue types. AAV tropism 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 the cell, a sequence carried by the vector genome (e.g., rAAV vector genome) (e.g., a heterologous sequence, such as a transgene) is expressed.

[0174] "Tendency profile" refers to the transduction pattern of one or more target cells in various tissues and / or organs. For example, the tendency profile of a chimeric AAV capsid is characterized by highly efficient transduction of astrocytes, while only low transduction of neurons, oligodendrocytes, and other CNS cells.

[0175] If, when applied directly to the CNS, such a chimeric capsid preferentially transduces astrocytes rather than neurons, oligodendrocytes, and other CNS cell types, then such a chimeric capsid can be considered "astrocytogenic," exhibiting a tropism towards astrocytes. 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.

[0176] 5. Virus particles and their production

[0177] Viral vectors carrying transgenes (e.g., transgenes encoding RNA disclosed herein) (e.g., rAAV vectors) can be assembled from polynucleotides encoding transgenes, suitable regulatory elements, and elements necessary for the production of viral proteins mediating cell transduction. Examples of viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, herpesviruses, and AAV vectors, and particularly rAAV vectors.

[0178] The vector genome components of the rAAV vector generated according to the method of this disclosure include at least one transgene (e.g., a polynucleotide encoding an RNA molecule) and an associated expression control sequence for controlling RNA expression. In a preferred embodiment, the vector genome includes a portion of the parvovirus genome, such as an AAV genome with rep and cap deletions and / or replacements by the transgene and its associated expression control sequence. The transgene is typically inserted near one or two AAVITR or ITR elements sufficient for viral replication (i.e., flanked by said AAV ITR or ITR elements), replacing the nucleic acids encoding the viral rep and cap proteins. Additional regulatory sequences suitable for promoting tissue-specific expression of the transgene in target cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes) may also be included.

[0179] A. Packaging cells

[0180] Those skilled in the art will understand that an rAAV vector containing transgenes but lacking viral proteins (e.g., cap and rep) required for viral replication cannot replicate, as such proteins are essential for viral replication and packaging. The cap and rep genes can be provided as part of a plasmid (e.g., a host cell, such as a packaging cell), and this plasmid is separate from the plasmid that provides the vector genome containing the transgenes.

[0181] Packaging cells or production cells are cells or cell lines that can be transfected with vectors, plasmids, or DNA constructs and trans-provide all the missing functions required for the complete replication and packaging of a viral vector. Genes required for rAAV vector assembly include the vector genome (e.g., transgenes encoding RNA, regulatory elements, and ITRs), the AAV rep gene, the AAV cap gene, and certain accessory genes from other viruses (e.g., adenoviruses). Those skilled in the art will understand that the genes required for AAV production can be introduced into packaging cells in a variety of ways, including, for example, by transfecting one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, accessory genes) may already be present in the packaging cells, integrated into the genome, or carried on episomes. In some embodiments, the packaging cells express one or more missing viral functions constitutively or inducibly.

[0182] Any suitable packaging cell known in the art can be used to generate the packaged viral vector. Mammalian or insect cells are preferred. Examples of cells that can be used to generate packaging cells in the practice of this disclosure include, for example, human cell lines such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cell line, 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.

[0183] In some embodiments, the packaging cells are capable of growth in suspension cultures. In some embodiments, the packaging cells are capable of growth in serum-free media. For example, HEK293 cells are grown in suspension in serum-free media. In another embodiment, the packaging cells are HEK293 cells as described in U.S. Patent No. 9,441,206 and deposited at the American Type Culture Collection (ATCC) under PTA 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002 / 46359.

[0184] Cell lines used as packaging cells include insect cell lines. According to this disclosure, any insect cell that allows AAV replication and can be maintained in culture can be used. Examples include fall armyworm (Spodoptera frugiperda) cell lines such as Sf9 or Sf21, Drosophila spp. cell lines, or mosquito cell lines, such as Aedes albopictus-derived cell lines. A preferred cell line is the fall armyworm Sf9 cell line. The following references are incorporated in this paper for their teachings on the use of insect cells for the expression of heterologous peptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in cultures: Methods in Molecular Biology, edited by 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. (2000) Virol. 272:382-393; and U.S. Patent No. 6,204,059.

[0185] As an alternative, the viral vector of this disclosure can be generated in insect cells using a baculovirus vector to deliver the rep / cap gene and rAAV template, as described, for example, in Urabe et al. (2002) Human Gene Therapy 13:1935-1943. When AAV is generated using baculovirus, 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 auxiliary functions, thereby facilitating the generation of the viral capsid.

[0186] Packaging cells typically include one or more viral vector functions, as well as auxiliary and packaging functions sufficient to induce viral vector replication and packaging. These various functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicones, and they can be present extrachromosomally within the cell line or integrated into the host cell's chromosome.

[0187] B. Accessibility Functions

[0188] AAV cannot replicate in cells without helper virus co-infection. Helper functions include helper viral elements required to establish active infection of packaging cells, which is necessary to initiate the packaging of the viral vector. Helper viruses typically include adenovirus or herpes simplex virus. Adenoviral helper functions typically include adenoviral components adenoviral early region 1A (E1a), E1b, E2a, E4, and virus-associated (VA) RNA. Helper functions (e.g., E1a, E1b, E2a, E4, and VA RNA) can be provided to packaging cells by transfecting cells with one or more nucleic acids encoding various helper elements. Alternatively, host cells (e.g., packaging cells) may contain nucleic acids encoding helper proteins. For example, HEK293 cells are generated by transforming human cells with adenovirus 5 DNA and now express many adenoviral genes, including but not limited to E1 and E3 (see, for example, Graham et al. (1977) J. Gen. Virol. 36:59-72). Therefore, these auxiliary functions can be provided by HEK 293 packaging cells without the need to provide them to the cells via, for example, plasmids that encode them.

[0189] In some embodiments, the packaging cells are transfected with at least the following: (i) a plasmid containing a vector genome, the vector genome containing transgenes and AAV ITR and further containing at least one of the following regulatory elements: enhancer, promoter, exon, intron and poly A; (ii) a plasmid containing a rep gene (e.g., AAV2 rep) and a cap gene (e.g., Olig001 cap); and (iii) a plasmid containing helper functions.

[0190] Any method can be used to introduce a nucleotide sequence carrying an accessory function into the cell host for replication and packaging, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and combinations of liposomes with nuclear localization signals. In some embodiments, standard methods for generating viral infection can be used to provide the accessory function by using viral vector transfection or by using helper viral infection.

[0191] The vector genome can be any suitable recombinant nucleic acid, such as a DNA or RNA construct, and can be single-stranded, double-stranded, or bistranded (i.e., self-complementary as described in WO 2001 / 92551).

[0192] C. Production of viral vectors in packaging

[0193] Viral vectors can be prepared by several methods known to those skilled in the art (see, for example, WO 2013 / 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. In short, efficient transfection of HEK293 cells serves as a starting point, where adherent HEK293 cell lines from qualified clinical master cell banks are used for growth in shake flasks and WAVE bioreactors under animal-free suspension conditions, thereby achieving rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), HEK293 cell line suspensions collected 48 hours post-transfection can produce greater than 1 × 10⁻⁶ cells / mL. 5 One particle (vg) / cell containing the vector genome, or greater than 1 × 10⁻⁶ 14 Each vg / L cell culture. More specifically, triple transfection refers to a method of transfecting packaged cells with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various auxiliary functions (e.g., adenovirus or HSV proteins such as E1a, E1b, E2a, E4, and VA RNA), and a third plasmid encodes the transgene (e.g., the RNA described herein) and various elements controlling the expression of said transgene.

[0194] The single-stranded vector genome is packaged into the capsid in approximately equal proportions as either the positive or negative strand. In some embodiments of the rAAV vector, the vector genome is positively polar (i.e., the sense or coding sequence of the DNA strand). In some embodiments of the rAAV vector, the vector is negatively polar (i.e., the antisense or template DNA strand). Where a positive strand nucleotide sequence in the 5' to 3' direction is given, the negative strand nucleotide sequence in the 5' to 3' direction can be identified as the inverse complementary sequence of the positive strand nucleotide sequence.

[0195] To achieve the desired yield, many variables were optimized, such as selecting a compatible serum-free suspension medium that supports growth and transfection, and choosing transfection reagents, transfection conditions, and cell density.

[0196] The rAAV vector can be purified using standard methods in the art, such as column chromatography or a cesium chloride gradient. Methods for purifying the rAAV vector 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 Nos. 6,566,118 and WO 98 / 09657.

[0197] A general purification strategy based on ion-exchange chromatography can be used to produce high-purity carrier preparations of AAV serotypes 1-6, 8, 9, and various chimeric capsids. In some embodiments, this process can be completed within one week, producing a high ratio of intact to empty capsids (>90% intact capsids), providing a purified yield suitable for clinical applications (>1 × 10⁻⁶). 13 (vg / L) and purity. In some implementations, such methods are universal for all serotypes and chimeric capsids. Scalable manufacturing technologies can be used to manufacture GMP clinical and commercial-grade rAAV vectors (e.g., for the treatment of hereditary or acquired neurodegenerative diseases).

[0198] After generating and purifying the rAAV vector disclosed herein, it can be titrated (e.g., the amount of rAAV vector in a sample can be quantified) to prepare a composition for administration to a subject (such as a human subject suffering from a hereditary or acquired neurodegenerative disease). The rAAV vector titration can be performed using methods known in the art.

[0199] In some implementations, the number of viral particles (including particles containing the vector genome and “empty” capsids without the vector genome) can be determined by electron microscopy (e.g., 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.

[0200] In some implementations, the rAAV vector genome can be titrated using quantitative PCR (qPCR) using primers targeting sequences in the vector genome (e.g., ITR sequences, and / or sequences in transgenes or regulatory elements). A standard curve can be generated by performing qPCR in parallel with dilutions of standards of known concentrations (e.g., plasmids containing vector genome sequences), allowing the concentration of the rAAV vector to be calculated as the number of vector genomes (vg) per unit volume (e.g., μL or mL). The number of empty capsids can be determined by comparing the number of vector particles, measured via, for example, electron microscopy, with the number of vector genomes in the sample. Because the vector genome contains therapeutic transgenes, the vg / kg or vg / ml of the vector sample may be a better indicator of the therapeutic dose of the vector that the subject will receive than the number of vector particles, 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 or dialyzed against a suitable buffer for preparation of a composition to be administered to a subject (e.g., a subject with a hereditary or acquired neurodegenerative disease).

[0201] 6. Uses and Treatments

[0202] Nucleic acids (such as RNA molecules or polynucleotides encoding said RNA molecules) disclosed herein can be used for gene therapy treatment and / or prevention of diseases, conditions, or symptoms. Specifically, they can be used to treat or prevent diseases, conditions, or symptoms associated with deficiency or dysregulation by targeting specific target genes (e.g., FXYD1), and any other symptom and / or ailment for which reducing the expression of related target genes can produce therapeutic benefit or improvement, such as diseases, conditions, or symptoms mediated or associated with increased levels or function of related proteins (e.g., FXYD1) compared to the levels or function of said proteins in healthy individuals. The vector genomes and / or rAAV vectors described herein can be used for gene therapy treatment and / or prevention of the same diseases, conditions, or symptoms.

[0203] In some embodiments, the methods of this disclosure include treating a subject's disease, condition, or symptom with an rAAV vector or a pharmaceutical composition thereof. In some embodiments, the methods of this disclosure include using an rAAV vector or a pharmaceutical composition thereof to reduce the level of a gene of interest (e.g., FXYD1) in a subject in need.

[0204] The nucleic acids, vector genomes, and / or rAAV vectors described above can be used to prepare drugs for the treatment and / or prevention of diseases, conditions, or symptoms (e.g., neurodegenerative diseases) associated with or caused by deficiency or dysregulation of one or more related proteins, as well as any other condition or ailment for which downregulation of one or more related proteins may produce therapeutic benefit or improvement.

[0205] In some embodiments, the compositions described herein may be administered in combination with cognitive-enhancing (nootropic) agents. Exemplary agents include any medicine, supplement, or other substance that improves cognitive function in healthy individuals, particularly executive function, memory, creativity, or motivation. Non-limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), nutritional foods (e.g., bacopa monnieri, panax ginseng, ginkgo biloba, and GABA), stimulants (e.g., amphetamine drugs, methylphenidate, eugeroic, xanthine, and nicotine), L-theanine, tolcapone, levodopa, atomoxetine, and desipramine.

[0206] The total dose of the therapeutic agent (e.g., an RNA molecule, a polynucleotide encoding said RNA molecule, a vector genome or vector (such as an rAAV vector) or cell) will be the therapeutically effective dose, which depends on several factors, including the subject's overall health condition, the subject's disease state, the severity of the symptoms, the observed improvement, and the formulation and route of administration of one or more of the chosen agents. The determination of the therapeutically effective dose is within the competence of those skilled in the art. The exact formulation, route of administration, and dose can be selected by an individual physician considering the subject's condition.

[0207] In other embodiments, the cellular or nucleotide composition can be applied in an amount that effectively promotes the survival of CNS neurons in a subject, increasing the number of surviving 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%.

[0208] In some embodiments, a second agent is also administered to the subject to treat or prevent neurological diseases or conditions. 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 during a period when they produce overlapping therapeutic effects in the patient. When the first and second agents are administered simultaneously or during a period when they produce overlapping therapeutic effects, the agents may be administered via the same or different routes of administration (e.g., oral versus infusion).

[0209] 7. Pharmaceutical Composition

[0210] This disclosure provides a pharmaceutical composition or medicament for the prevention or treatment of hereditary or acquired neurodegenerative diseases. In some embodiments, the pharmaceutical composition comprises one or more of the RNA molecule, polynucleotide, expression cassette, expression vector (e.g., viral vector genome, expression vector, rAAV vector), and host cell.

[0211] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and / or other agent. A pharmaceutically acceptable carrier, adjuvant, diluent, excipient, or other agent is an agent that has no adverse effects biologically or otherwise; for example, the material can be administered to a subject without causing adverse biological effects exceeding the beneficial biological effects of said material. Any suitable pharmaceutically acceptable carrier or excipient can be used to prepare the pharmaceutical composition according to the invention (see, for example, Remington, The Science and Practice of Pharmacy, AdeboyeAdejare (ed.), Academic Press, November 2020).

[0212] Pharmaceutical compositions are typically sterile, pyrogen-free, and stable under manufacturing and storage conditions. Pharmaceutical compositions can be formulated as solutions (e.g., water, saline, glucose solutions, buffer solutions, or other sterile pharmaceutical fluids), microemulsions, liposomes, or other ordered structures suitable for accommodating high concentrations of products (e.g., viral vector particles, microparticles, or nanoparticles).

[0213] In some embodiments, pharmaceutical compositions comprising the RNA molecules, polynucleotides, expression cassettes, expression vectors, vector genomes, host cells, or rAAV vectors of the present disclosure described above are formulated in water or buffered saline solutions. The carrier can 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. For example, a coating (e.g., lecithin) can be used to maintain the desired particle size in the case of a dispersion, and appropriate flowability can be maintained by using a surfactant. In some embodiments, it is preferable to include an isotonic agent, such as sugar, polyol (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including a delayed-absorption agent (e.g., monostearate and gelatin) in the composition. In some embodiments, the nucleic acids, vectors, and / or host cells of the present disclosure can be administered in controlled-release formulations, for example, in compositions comprising sustained-release polymers or other carriers that protect the product from rapid release, said compositions including implants and microencapsulated delivery systems.

[0214] In some embodiments, the pharmaceutical compositions of this disclosure are parenteral pharmaceutical compositions, comprising compositions suitable for intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, intraparenchymal (IP), intrathecal (IT), intraventricular (ICV), and / or intracerebellomedullary cistern (ICM) administration. In some embodiments, the pharmaceutical compositions of this disclosure are formulated for administration via ICV injection. In some embodiments, the rAAV carrier is formulated in PBS containing 350 mM NaCl and 5% D-sorbitol.

[0215] 8. Application method

[0216] The RNA molecules or polynucleotides or vectors (e.g., vector genomes, rAAV vectors) described above can be administered to a subject (e.g., a patient) to treat the subject. The vectors can be administered to human subjects or animals in need by any method known in the art for administering vectors. Target cells for the vectors disclosed herein include cells of the CNS, preferably glial progenitor cells, astrocytes, or oligodendrocytes.

[0217] The carrier can be administered in addition to and as an adjunct to standard care treatment. That is, the carrier can be administered simultaneously, concurrently, or at defined dosing intervals with another agent, compound, drug, treatment, or treatment regimen, said dosing intervals being determined by those skilled in the art using conventional methods. Uses disclosed herein include concurrent administration of the rAAV carrier of this disclosure in addition to standards of care known in the art, and / or administration of the rAAV carrier of this disclosure according to dosing regimens concurrent with standards of care known in the art.

[0218] In some embodiments, the combination composition includes one or more immunosuppressants. In some embodiments, the combination composition includes an rAAV vector and one or more immunosuppressants, said rAAV vector containing a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein). In some embodiments, the method includes administering or delivering the transgene-containing rAAV vector to a subject and administering an immunosuppressant to the subject prophylactically before administering the vector, or administering the immunosuppressant to the subject after administering the vector (i.e., before or after symptoms of a response to the vector and / or the protein provided therein become apparent).

[0219] In one embodiment, the vector of this 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 tissue or organ injection. Those skilled in the art will understand that systemic administration can deliver nucleic acids to all tissues. In some embodiments, direct tissue or organ administration includes administration to areas directly affected by deficiency (e.g., the brain and / or the central nervous system). In some embodiments, the vector of this disclosure and its pharmaceutical composition are administered to the brain parenchyma (i.e., intraparenchymal administration), to the spinal canal or subarachnoid space such that they reach the cerebrospinal fluid (CSF) (i.e., intrathecal administration), to the ventricles (i.e., intraventricular administration), and / or to the cerebellomedullary cistern of the brain (i.e., intracerebellomedullary administration).

[0220] Therefore, in some embodiments, the carriers of this disclosure are administered to treat neurodegenerative diseases by direct injection into the brain (e.g., into the brain parenchyma, ventricles, cerebellomedullary cistern, etc.) and / or into the CSF (e.g., into the spinal canal or subarachnoid space). Target cells of the carriers of this 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 carriers of this disclosure are glial cells (glial progenitor cells, astrocytes, or oligodendrocytes). Other routes of administration may include local application of the carrier under direct visualization, such as superficial cortical application or other stereotactic applications.

[0221] In some embodiments, the vector of this disclosure is administered via at least two routes. For example, the vector can be administered systemically and directly to the brain. If administered via at least two routes, the administration of the vector may, but does not have to, be simultaneous or concurrent. Instead, administration via different routes can be performed separately, with a certain time interval between each administration.

[0222] The RNA molecules described above, or the polynucleotides encoding said RNA molecules, or the vector genomes or rAAV vectors containing said polynucleotides, can be used for in vitro transduction of cells or for direct administration to a subject (e.g., direct administration to the CNS of a patient with a disease). In some embodiments, transduced cells (e.g., host cells) are administered to a subject to treat or prevent a disease, symptom, or disease (e.g., cell therapy for a disease). rAAV vectors containing therapeutic nucleic acids (e.g., encoding RNA molecules) are preferably administered to cells in a biologically effective amount. In some embodiments, the biologically effective amount of the vector is an amount sufficient to cause a reduction in the expression of the relevant gene in the target cells.

[0223] In some embodiments, this disclosure includes methods for reducing the level and / or activity of genes in cells by administering a polynucleotide encoding an RNA molecule described herein to cells (in vivo, in vitro, or ex vivo), said polynucleotide being administered alone or in a vector (including plasmids, viral vectors, nanoparticles, liposomes, or any known method for delivering nucleic acids to cells).

[0224] The dosage of the rAAV vector depends on factors such as the administration method, the disease or condition to be treated, the stage and / or invasiveness of the disease, the individual subject's condition (age, sex, weight, etc.), the specific viral vector, the stability of the protein to be expressed, the host's immune response to the vector, and / or the gene to be delivered. Typically, the dosage range is at least 1 × 10⁻⁶. 8 or more, for example, 1 × 10 9 1 × 10 10 1 × 10 11 1×10 12 1 × 10 13 1 × 10 14 1 × 10 15 More vector genomes (vg) / kg subject weight to achieve therapeutic effects.

[0225] In some embodiments, the polynucleotide encoding the RNA molecule described herein may be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having regulatory elements (e.g., promoters) suitable for expression in target cells (e.g., glial progenitor cells, astrocytes, or oligodendrocytes). The polynucleotide may be administered as a component of a plasmid or viral vector (e.g., an rAAV vector). The rAAV vector may be administered in vivo by delivering the vector directly to a patient requiring treatment (e.g., direct delivery to the CNS). The rAAV vector may be administered ex vivo to a patient by in vitro administration of the vector to cells from a donor patient requiring treatment, followed by returning the transduced cells to the donor (e.g., cell therapy).

[0226] 9. Reagent kit

[0227] This disclosure provides a kit having packaging material and one or more components therein. The kit typically includes a label or packaging insert containing a description of the components or instructions for use of the components in vitro, in vivo, or ex vivo. The kit may contain a collection of such components, such as RNA molecules, polynucleotides, nucleic acids, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors), and host cells, and optionally a second active agent, such as a compound, therapeutic agent, drug, or composition.

[0228] A kit is a physical structure containing one or more components. Packaging materials can retain the components aseptically and can be made of materials commonly used for such purposes (e.g., paper, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0229] Labels or inserts may include identification information, dosage, clinical pharmacology (including mechanism of action), pharmacokinetics, and pharmacodynamics of one or more of the components. Labels or inserts may include information identifying the manufacturer, batch number, place and date of manufacture, and expiration date. Labels or inserts may include information on the diseases for which the kit components can be used (e.g., hereditary or acquired neurodegenerative diseases). Labels or inserts may include instructions for clinicians or subjects regarding the use of one or more of the kit components in methods, uses, treatment protocols, or therapeutic regimens. Instructions for use may include dosage, duration of use, frequency, and instructions for implementing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0230] Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians about situations where the particular composition is not suitable for use.

[0231] 10. Definition

[0232] As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” interchangeably refer to any molecule consisting of or containing monomeric nucleotides linked by phosphodiester bonds. Nucleic acids can be oligonucleotides or polynucleotides. Nucleic acid sequences are presented herein in a 5' to 3' orientation. The nucleic acid sequences (i.e., polynucleotides) disclosed herein can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules, and refer to all forms of nucleic acids, such as double-stranded molecules, single-stranded molecules, small or short hairpin RNA (shRNA), micro-interfering RNA or microRNA (miRNA), small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, and ribosomal RNA. When the polynucleotide is a DNA molecule, the molecule can be a gene, cDNA, an antisense molecule, or a fragment of any of the foregoing. Nucleotides are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Nucleotide sequences can be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threononucleotides (TNA). Each of these sequences differs from naturally occurring DNA or RNA in the variation of the molecular backbone. Additionally, phosphate-thioester nucleotides can be used. Other deoxynucleotide analogs include methylphosphonates, aminophosphates, dithiophosphates, N3'-P5'-aminophosphates, and oligonucleotide phosphate-thioesters, as well as their 2'-O-allyl analogs, and 2'-O-methylribonucleotide methylphosphonates, which can be used in the nucleotide sequences disclosed herein.

[0233] In some embodiments, the nucleic acid may comprise one or more non-standard nucleotides, which may be naturally occurring or non-natural (i.e., artificial; not found in nature) and / or may contain modified sugars or modified backbone bonds. Nucleic acid modifications (e.g., base, sugar, and / or backbone modifications), non-standard nucleotides, or nucleosides, such as those known in the art for use in RNA interference (RNAi), aptamers, CRISPR technology, peptide generation, reprogramming, or in the context of antisense molecules for research or therapeutic purposes, may be incorporated into the embodiments. For example, such modifications may improve stability (e.g., by reducing sensitivity to nuclease cleavage), reduce in vivo clearance, increase cellular uptake, or confer other properties to improve translation, potency, efficacy, specificity, or otherwise make the nucleic acid more suitable for its intended use. Various non-limiting examples of nucleic acid modifications are described, for example, in the following literature: Deleavey GF, et al., Chemical modification of siRNA. Curr. Protoc. NucleicAcid Chem. 2009; 39:16.3.1-16.3.22; Crooke, ST (edited) Antisense drugtechnology: principles, strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J. (edited) Therapeutic oligonucleotides, RSC biomolecularsciences. Cambridge: Royal Society of 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 Application Publications WO 00 / 56746 and WO 01 / 14398. Different modifications can be used on the two strands of a double-stranded nucleic acid. Nucleic acids can be modified uniformly or only on a portion thereof and / or can contain a variety of 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, the number refers to the number of nucleotides in each strand of a single-stranded or double-stranded nucleic acid. An "oligonucleotide" is a relatively short nucleic acid, typically between approximately 5 nt and approximately 100 nt in length.

[0234] In some implementations, the protein or nucleic acid is 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 by, for example, polymerase chain reaction (PCR); (ii) produced in a recombinant manner by cloning; (iii) purified, for example, by cutting and gel separation; or (iv) synthesized by, for example, chemical synthesis. Isolated nucleic acids are nucleic acids that can be readily manipulated using recombinant DNA techniques well known in the art. Thus, a nucleotide sequence containing known 5' and 3' restriction sites or publicly disclosed polymerase chain reaction (PCR) primer sequences in a vector is considered isolated, but a nucleic acid sequence present in its natural state in its natural host is not. Isolated nucleic acids may be substantially purified, but not necessarily so. For example, nucleic acids isolated within a cloning or expression vector are not pure because they may constitute only a very small percentage of the material in the cell in which they reside. However, such nucleic acids are isolated because the term is used herein because they can be readily manipulated using standard techniques known to those skilled in the art. As used herein with respect to proteins or peptides, the term "isolated" means a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by 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 nucleotide suitable for base pairing with adenosine nucleotides (e.g., by Watson-Crick base pairing). For example, T may be replaced with U, and U may be replaced with T.

[0235] "Heterologous" means originating from an entity that is genotyped differently from the remainder of an entity being compared with it, or from the remainder of an entity into which it is introduced or incorporated. For example, polynucleotides introduced into different cell types through genetic engineering are heterologous polynucleotides (and, when expressed, can encode heterologous polypeptides). Similarly, cellular sequences (e.g., genes or portions thereof) incorporated into viral vectors are heterologous nucleotide sequences relative to the vector.

[0236] The term "transgenic" refers to a heteropolynucleotide introduced into a cell that is capable of being transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In various ways, it confers desired properties on the cell into which it is introduced, or otherwise leads to desired therapeutic or diagnostic outcomes. Alternatively, it can be transcribed into molecules that mediate RNA interference, such as miRNA, siRNA, or shRNA.

[0237] As used herein, the term "recombinant" refers to a vector, polynucleotide (e.g., recombinant nucleic acid), polypeptide, or cell, which is the product of various combinations of procedures involving cloning, restriction, or ligation steps (e.g., related to the polynucleotide or polypeptide contained therein) and / or other procedures that produce a construct different from those found in nature. A recombinant virus or vector (e.g., an rAAV vector) contains a vector genome containing recombinant nucleic acids (e.g., nucleic acids containing transgenes and one or more regulatory elements). The terms respectively include copies of the original polynucleotide construct and progeny of the original viral construct.

[0238] As used herein, the term "operably linked" refers to nucleic acid sequence (or polypeptide) elements linked in a functional relationship. Nucleic acids are operably linked when one nucleic acid is in a functional relationship with another nucleic acid sequence. For example, if a promoter or other transcriptional regulatory sequence (e.g., an enhancer) affects the transcription of a coding sequence, then it is operably linked to said 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 contiguously linked, but rather that there are insert sequences between those linked nucleic acid sequences.

[0239] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences) flanked by at least one (and in embodiments two) AAV inverted terminal repeat sequences. Such rAAV vectors, when present in host cells already infected with a suitable helper virus (or expressing a suitable helper function) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), can be replicated and packaged into infectious viral particles. When the 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 can be referred to as a "pro-vector," which can be "rescued" by replication and capsidation in the presence of AAV packaging function and a suitable helper function. The rAAV vector can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, encapsulated in liposomes, and capsidated within viral particles (particularly AAV particles). The rAAV vector can be packaged into the capsid of an AAV virus to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0240] As used herein, the term "vector" refers to plasmids, viruses (e.g., rAAV), granules, or other agents that can be manipulated by inserting or incorporating nucleic acids (e.g., recombinant nucleic acids). Vectors can be used for a variety of purposes, including, for example, genetic manipulation (e.g., cloning vectors), to introduce / transfer nucleic acids into / to cells, thereby transcribing or translating the inserted nucleic acids in the cells. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for proliferation in the cells. In some embodiments, the vector nucleic acid includes a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and / or an ITR. In some embodiments, the nucleic acid sequence proliferates upon delivery to a host cell. In some embodiments, the cell expresses a polypeptide encoded by the heterologous nucleic acid sequence upon delivery to a host cell, either in vitro or in vivo. In some embodiments, the nucleic acid sequence or a portion thereof is packaged into a capsid upon delivery to a host cell. The host cell can be an isolated cell or a cell within a host organism. In addition to the nucleic acid sequence encoding RNA, polypeptide, or protein (e.g., transgene), other sequences (e.g., regulatory sequences) may exist within the same vector (i.e., in cis-regulation with the gene) and flanked by the gene. In some embodiments, the regulatory sequence may be present on a separate (e.g., a second) vector that acts trans-regulates to regulate gene expression. Plasmid vectors may be referred to herein as “expression vectors.”

[0241] As used herein, the term "vector genome" refers to the recombinant nucleic acid sequence that is packaged or capsidated to form an rAAV vector. Typically, a vector genome includes heterologous polynucleotide sequences, such as transgenes, regulatory elements, and ITRs not originally present in the capsid. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors (e.g., rAAV vectors), the vector genome does not include the entire plasmid, but only the sequences intended for delivery via a viral vector. This non-vector genome portion of a recombinant plasmid is often referred to as the "plasmid backbone," which is important for cloning. Plasmid selection and amplification are processes required for the proliferation of recombinant viral vectors, but plasmids themselves are not packaged or capsidated into the rAAV vector.

[0242] As used herein, the term "viral vector" generally refers to a viral particle that acts as a nucleic acid delivery medium, and said viral particle contains a vector genome (e.g., nucleic acids containing transgenes rather than encoding AAV rep and cap) packaged within the viral particle (i.e., capsid), and includes, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not contain a vector genome including rep and / or cap genes.

[0243] As used herein, a “miRNA scaffold” can refer to a polynucleotide containing: (i) a double-stranded sequence targeting a gene of interest to be knocked down via RNAi, and (ii) an additional sequence forming a stem-loop structure similar to that of endogenous miRNAs. When the polynucleotide assembles into a miRNA-like secondary structure, the sequence of the gene of interest targeting RNAi (e.g., a short sequence of about 20 nt) can be linked to the sequence that generates the miRNA-like stem-loop and the sequence that pairs with the bases of the sequence of interest to form a double strand. As described herein, this double strand may not be fully hybridized; for example, it may contain one or more unpaired or mismatched bases. After cleavage of this polynucleotide by Dicer, this double strand containing the sequence targeting the gene of interest can unwind and be incorporated into the RISC complex. A miRNA scaffold can refer to the miRNA itself or the DNA polynucleotide encoding the miRNA. An example of a miRNA scaffold is the miR-155 sequence (Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9). Commercially available kits for cloning sequences into miRNA scaffolds are known in the art (e.g., the INVITROGEN BLOCK-IT Pol II miRNAi expression vector kit from LifeTechnologies, Thermo Fisher Scientific, Waltham, Massachusetts).

[0244] A functional variant or equivalent of a reference peptide, polypeptide, or protein refers to a polypeptide derivative of the reference peptide, polypeptide, or protein, such as a protein, fusion protein, or combination thereof having one or more point mutations, insertions, deletions, truncations, or variations thereof. It substantially retains the activity of the reference peptide, polypeptide, or protein. Typically, the functional equivalent is at least 60% identical to the reference peptide, polypeptide, or protein (e.g., any number between 60% and 100%, including, for example, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). In some embodiments, the point mutation may be a conserved modification.

[0245] As used herein, the term "conservative modification" refers to amino acid modifications that do not significantly affect or alter the biological properties of a peptide or protein. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into peptides or proteins using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. 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 with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions would require exchanging members of one class with members of another.

[0246] As used herein, the terms “treat,” “treating,” or “treatment” refer to the application of a therapy that partially or completely relieves, improves, reduces, or inhibits one or more symptoms, features, and / or causes of a particular disease, condition, and / or symptom, delays its onset, reduces its severity, and / or reduces its incidence.

[0247] The terms “decrease,” “reduce,” “reduced,” “reduction,” “decrease,” and “inhibit” are generally used herein to mean a reduction in a statistically significant amount relative to a reference. However, for the avoidance of doubt, “reduce,” “reduction,” or “decrease” or “inhibit” generally means a reduction of at least 10% compared to a reference level and may include, for example, a reduction of 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%, and at most, include, for example, the complete absence of a given entity or parameter compared to a reference level, or any reduction between 10% and 99% compared to the absence of a given treatment.

[0248] As used herein, the term “improvement” means a detectable or measurable improvement in a subject’s disease, condition, or symptom, or its symptoms, or underlying cellular responses. Detectable or measurable improvement includes, subjectively or objectively, a reduction, decrease, inhibition, suppression, limitation, or control of the occurrence, frequency, severity, progression, or duration of a disease, condition, or symptom; complications arising from or related to a disease, condition, or symptom; improvement of symptoms of a disease, condition, or symptom; or reversal of a disease, condition, or symptom.

[0249] As used herein, the term "associated with" means that if the presence, level, and / or form of one entity is associated with the presence, level, and / or form of another, then they are related to each other. For example, if the presence, level, and / or form of a particular entity (e.g., polypeptide, genetic trait, metabolite, microorganism, etc.) is associated with the incidence and / or susceptibility to a disease, condition, or symptom (e.g., in a relevant population), then that particular entity is considered associated with that particular disease, condition, or symptom.

[0250] As used herein, the terms "prevent" or "prevention" refer to delaying the onset of one or more signs or symptoms of a particular disease, condition, or symptom, and / or reducing their frequency and / or severity. In some embodiments, prevention is assessed on a population basis, such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more signs or symptoms of said disease, condition, or symptom is observed in a population susceptible to the particular disease, condition, or symptom, then the agent is considered to "prevent" said disease, condition, or symptom. Prevention is considered complete when the onset of the disease, condition, or symptom has been delayed for a predetermined period.

[0251] As used herein, the term "pharmaceutical" means any compound or substance, such as, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, viruses, cells, etc. "Pharmaceutical" can be any chemical substance, entity, or part, including but not limited to synthetic and naturally occurring protein and non-protein entities. In some embodiments, the pharmaceutical is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including but not limited to proteins, oligonucleotides, ribozymes, DNases, glycoproteins, siRNA, lipoproteins, aptamers, and modifications and combinations thereof. In some embodiments, the pharmaceutical is selected from the group consisting of nucleic acids, small molecules, polypeptides, and peptides. In some embodiments, the pharmaceutical is an oligonucleotide, protein, or small molecule. In some embodiments, the pharmaceutical comprises one or more oligonucleotides. In some aspects, the oligonucleotide is a splice-switching oligonucleotide. In some aspects, the oligonucleotide is an antisense oligonucleotide (ASO). In some embodiments, the pharmaceutical is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and their associated natural products or analogues. The compound is known to possess the desired activity and / or properties, or may be selected from a library of various compounds. In some embodiments, the agent is a genome modification system (e.g., CRISPR / Cas, zinc finger nucleases, or the TALEN system). The CRISPR / Cas system may employ 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 type V system.

[0252] "Small molecules" are defined as molecules with a molecular weight of less than 10 kDa (typically less than 2 kDa, and preferably less than 1 kDa). Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules may penetrate cells more easily, are less prone to degradation, and are less likely to elicit an immune response than large molecules.

[0253] As used herein, the term "peptide" or "protein" is used to designate a series of amino acid residues linked together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The term "peptide" refers to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. The term "peptide" is generally used to refer to small polypeptides, but its use in the art overlaps with "protein" or "peptide." Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, as well as both naturally occurring and non-naturally occurring variants, fragments, and analogs of the foregoing.

[0254] As used herein, the term "subject" refers to an organism, such as 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 pediatric subject. In some embodiments, the subject has a disease, condition, or symptom, such as a disease, condition, or symptom that can be treated as provided herein. In some embodiments, the subject has a disease, condition, or symptom associated with neuronal overexcitation. In some embodiments, the subject is susceptible to a disease, condition, or symptom. In some embodiments, susceptible subjects are susceptible to a disease, condition, or symptom and / or exhibit an increased risk of developing a disease, condition, or symptom (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, condition, or symptom. In some embodiments, the subject does not exhibit a specific symptom (e.g., clinical manifestations of a disease) or characteristic of a disease, condition, or symptom. In some embodiments, the subject does not exhibit any symptom or characteristic of a disease, condition, or symptom. In some implementations, the subject is a human patient. In some implementations, the subject is an individual who is receiving and / or has received a diagnosis and / or therapy.

[0255] As used herein, the term “pharmaceutically acceptable” means those pharmaceutical preparations, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues to the extent of correct medical judgment without causing excessive toxicity, irritation, allergic response, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0256] As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulation material (involving the carrying or delivery of the subject pharmaceutical agent 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 other components of the formulation and harmless to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium dodecyl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes. (9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) Ethylene glycols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates, and / or polyanhydrides; (22) Fillers, such as peptides and amino acids; (23) Serum components, such as serum albumin, HDL, and LDL; (22) C2-C 12 Alcohols, such as ethanol; and (23) other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, aromatizers, preservatives, and antioxidants may also be present in the preparations. Terms such as “excipients,” “carriers,” and “pharmaceutically acceptable carriers” are used interchangeably in this document.

[0257] As used herein, the term "therapeuticly effective amount" refers to an amount administered to produce the desired therapeutic effect. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, or symptom when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such a disease, condition, or symptom. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition, or symptom, and / or delays the onset of one or more symptoms of such a disease, condition, and / or symptom. Those skilled in the art will understand that the term "therapeuticly effective amount" does not actually require successful treatment in a particular individual. Rather, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to patients requiring such treatment.

[0258] As used herein, the term "administration" means placing an agent or composition into the body of a subject (e.g., a subject in need) by a method or route that at least partially positions the agent or composition at a desired site to produce the desired effect. Administration routes suitable for the methods of the present invention include both local and systemic administration routes. Generally, local application delivers more of the administered agent to a specific location than systemic application, while systemic application delivers the agent substantially throughout the body of the subject.

[0259] The compositions and pharmaceutical agents disclosed herein may be administered via any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, percutaneous, airway (aerosol), pulmonary, nasal, rectal, and local (including buccal and sublingual) administration. Exemplary administration modes include, but are not limited to, injection, infusion, drip, inhalation, or ingestion. “Injection” includes, but is not limited to, intravenous, intramuscular, intraarticular, intrathecal, intracranial, intracapsular, intracapsular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, intracerebrospinal, and intrasternal injection and infusion. In preferred embodiments of the aspects described herein, the compositions are administered via intravenous infusion or injection.

[0260] As used herein, the term "glial cell" refers to a group of non-neuronal cells that provide support and nutrition, maintain homeostasis, form myelin or promote myelination, and participate in signal transduction in the nervous system. As used herein, "glial cell" encompasses fully differentiated cells of the glial lineage, such as oligodendrocytes or astrocytes, as well as glial progenitor cells. Glial progenitor cells are cells with the potential to differentiate into cells of the glial lineage, such as oligodendrocytes and astrocytes.

[0261] The glial progenitor cells described herein can 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 example, the glial progenitor cells can be cells regenerated from glial progenitor cells as described herein or their progeny.

[0262] In some implementations, in order to treat subjects in need, the glial progenitor cells or regenerating cells are young glial cells or glial progenitor cells, or younger than their counterparts in the subjects to be treated.

[0263] As used herein, the terms “about” or “approximately” refer to measurable values ​​such as the amount of biological activity, homology or length of polynucleotide or polypeptide sequences, dosage, time, temperature, etc., and are intended to cover specified quantity 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 any direction (greater than or less than).

[0264] As used herein, the terms “homologous” or “homology” 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, peptides are homologous at that position when the same amino acid occupies an amino acid position in two peptides. It is worth noting that homologous peptides will retain the activity or function associated with the unmodified peptide or reference peptide, and modified peptides will generally have an amino acid sequence that is “substantially homologous” to the amino acid sequence of the unmodified sequence. When 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 strand), or fragment thereof with appropriate insertions or deletions, sequence identity is present in 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 the percentage of sequence homology (or identity) typically take into account sequence gaps and mismatches over the comparison region or area.

[0265] Example

[0266] Example 1

[0267] Meta-analysis was performed to identify dysregulated astrocyte genes in neurodegenerative disease-derived astrocytes but not in other healthy senescent astrocytes. The analysis was conducted using a large database encompassing gene expression in both murine and human astrocytes (e.g., derived from both wild-type and senescent cells) and compared with age-matched cells derived from patients with various neurodegenerative diseases.

[0268] More specifically, six transcriptome datasets were collected from published studies investigating 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 effects of oligodendrocytes in male APP NL-GF and 5XFADmice. 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 perform comparisons of differential expression between disease symptom and wild-type samples. Particular emphasis was placed on astrocytes, and all included datasets were derived from mice. These datasets cover both batch and single-cell resolution RNA sequencing experiments. Using the given datasets, the inventors were able to obtain or perform six different symptom / wild-type hybrids.

[0269] When available, a published list of differentially expressed genes (DEGs) was utilized. In cases where such a list was unavailable, the inventors used STAR to align a published FastQ file with a GRCm39 ens106 reference and used desired-maximum RNA-Seq (RSEM) (Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMCBioinformatics. 2011 Aug 4;12:323. doi:10.1186 / 1471-2105-12-323) for bulk RNA and STARsolo (Dobin A, Davis CA, Schlesinger F et al. STAR: ultrafastuniversal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15-21. doi:10.1093 / bioinformatics / bts635) for cell-resolution datasets. The cell resolution dataset was then restricted to clusters expressing GFAP and AQP4 to enrich astrocytes. The DEG list was generated by comparing the symptom to the wild type using the Wilcoxon rank sum, with adjusted p-values ​​of 0.05 or lower.These DEG lists were then filtered to include only those compatible with the Enrichr database (Chen EY, Tan CM, Kou Y, et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013 Apr 15;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. 2016 Jul 8;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.). The list of 280 genes associated with any of the 61 potassium-related gene ontology (GO) terms or pathways found in March 2021; 1(3):e90.doi:10.1002 / cpz1.90) is included. Finally, the inventors identified genes appearing in at least three of the six datasets, thus deriving a concise list of genes that are prevalent and significantly dysregulated in astrocytes of mice with the neurodegenerative disease we targeted.

[0270] Cortical [K] across disease models + ] e Consistent changes suggest the possible existence of a common molecular pathway mediating dysregulation of potassium homeostasis. Based on this, the inventors mined six publicly available transcriptome databases covering astrocyte gene expression in the AD, ALS, and HD mouse models we studied. Figure 1-2 Using a publicly available list of differentially expressed genes (DEGs) (if available), or by creating its own list (if not available), the inventors identified the intersection of DEGs present in at least three of the six datasets.

[0271] It is noteworthy that while many DEGs are shared between disease states, astrocytes do not share DEGs during normal aging. Fxyd1 (Na...) was discovered... + / K +(A negative regulator of ATPase activity) was selectively upregulated in all three AD and ALS models, but remained unchanged in normal aging. In contrast, its expression was decreased in Huntington's disease (HD) mouse models, which may be a compensatory mechanism for the previously described (Osipovitch et al., 2019) downregulation of K channel expression in HD glial cells.

[0272] Fxyd1 has received particular attention due to its common positive expression. Fxyd1 (containing the FXYD domain of ion transport regulator 1) regulates the activity of sodium / potassium transporter ATPase (NKA), which transports Na+ to potassium ions. + Transported out of the cell and K + It is transported into the cell. Since Fxyd1 is typically used to downregulate Na+... + / K + -ATPase activity, and its relative overexpression, indicate that it disrupts astrocytes in AD and ALS [K] + ] e Intake and buffering of extracellular K+, leading to these symptoms + High. It is noteworthy that Fxyd1 has been reported to inhibit Na only in the unphosphorylated state. + / K + -ATPase activity; its activity in the CNS if phosphorylated is unclear. Therefore, overexpression of Fxyd1 would have the effect of increasing extracellular K+.

[0273] Astrocytes have traditionally been considered to be responsible for K + The main cell types of buffers (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)). Na + / K + -ATPase helps extracellular K+ after neuronal activity. +The clearance of [K] (Larsen, BR et al. Glia 62, 608-622 (2014)), therefore the negative regulation of it by Fxyd1 may increase [K] + ] e This results in cortical [K] in both the ALS and AD models. + ] e Elevation and fright-induced [K] + ] e Enhance both.

[0274] The foregoing description of the embodiments and preferred embodiments should be considered illustrative and not limiting of the disclosure as defined by the claims. As will be readily understood, many variations and combinations of the features set forth above may be utilized without departing from the disclosure set forth in the claims. Such variations are not considered to depart from the scope of this disclosure, and all such variations are intended to be included within the scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. A method, said method (i) reducing brain hyperexcitation in a subject in need, or (ii) treating symptoms mediated by brain hyperexcitation, said method comprising increasing Na+ in the glial cells of said subject. + ,K + The level or activity of ATPase.

2. A method for reducing interstitial potassium levels in the brain of a subject in need, the method comprising increasing Na+ levels in the glial cells of the subject. + ,K + The level or activity of ATPase.

3. The method according to claim 1 or 2, wherein the subject suffers from symptoms mediated by neuronal hyperexcitability.

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

5. The method according to any one of the preceding claims, wherein the enhancement comprises reducing the expression level of the FXYD1 gene in the glial cells.

6. The method of claim 5, wherein the reduction comprises administering to the subject an agent that reduces the expression level of the FXYD1 gene in the glial cells.

7. The method of claim 6, wherein the agent comprises or encodes an inhibitory nucleic acid or a CRISPR / Cas system.

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

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

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

12. The method of claim 11, wherein the promoter is a glial cell-specific promoter or a regulated 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 preceding claims, wherein the glial cells are astrocytes.

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

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

17. The method according to any one of claims 1 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 repressive nucleic acid or siRNA molecule comprises or encodes a sequence that is at least 75% complementary to a segment of the FXYD1 gene or RNA.

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

20. A repressive nucleic acid or siRNA molecule, said repressive nucleic acid or siRNA molecule comprising or encoding a sequence that is at least 75% complementary to a segment of the FXYD1 gene or RNA.

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

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