Synthetic nucleic acids containing astrocyte-directed promoter constructs and methods of use thereof

Abstract

Synthetic nucleic acids are described that can be used for astrocyte-directed expression of heterologous nucleotide sequences, as well as methods of using same for astrocyte-directed expression of such nucleotide sequences for the treatment of neurodegenerative diseases.

Description

[Technical Field]

[0001] Reference to an electronically submitted sequence listing

[0001] The present disclosure is filed with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as file "30357," created on November 7, 2022, and is 42 kilobytes (kb) in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to biology and medicine, and more particularly to synthetic nucleic acids that can be used as expression control elements and methods for astrocyte-directed expression of heterologous nucleotide sequences, particularly in the treatment of neurodegenerative diseases. [Background technology]

[0003] Alzheimer's disease (AD) is the most common form of dementia, affecting more than 5 million people in the United States alone. AD is an irreversible, progressive brain disorder characterized by the presence of abnormal protein deposits throughout the brain, which impair nerve cell function, destroy connections between nerve cells, and ultimately lead to cell death. These deposits consist of amyloid-beta (β) plaques and tangles formed by phosphorylated tau protein. Individuals with mild AD experience memory loss, leading to wandering, difficulty handling money, repetitive questioning, and personality and behavioral changes. Furthermore, individuals with moderate AD exhibit worsening memory loss, difficulty confusing and recognizing friends and family, an inability to learn new things, and hallucinations, delusions, and delusional disorder. Furthermore, individuals with severe AD are unable to communicate and are completely dependent on others for their care. Eventually, protein plaques and tangles spread throughout the brain, leading to significant tissue shrinkage.

[0004] Polymorphisms in the apolipoprotein E gene (APOE) are the major genetic risk determinant for late-onset AD. In the central nervous system (CNS), apolipoprotein E protein (ApoE) is present primarily in astrocytes (although some is found in microglia and stressed neurons), is the major cholesterol carrier in the brain, and is required for cholesterol transport from astrocytes to neurons.

[0005] In addition to AD, there are numerous other neurodegenerative diseases characterized by astrocyte dysfunction, including, but not limited to, AD-associated gliosis, Amyotrophic Lateral Sclerosis (ALS), and Huntington's Disease (HD).

[0006] There is a need for synthetic nucleic acids that can be used as promoters to drive expression primarily in astrocytes, as well as methods for using same for astrocyte-directed expression of one or more heterologous nucleotide sequences, particularly in the treatment of neurodegenerative diseases. Summary of the Invention

[0007] To address this need, the present disclosure first describes synthetic nucleic acids that can be used as expression control elements (i.e., promoters). In some cases, the expression control element (i.e., promoter) is for astrocyte-directed expression of one or more operably linked heterologous nucleotide sequences (i.e., transgenes and / or inhibitory nucleic acids). In some cases, the expression control element (i.e., promoter) comprises a nucleotide sequence having at least about 90% (i.e., or about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO:1. In other cases, the expression control element (i.e., promoter) is SEQ ID NO:1.

[0008] In some cases, the synthetic nucleic acid is an expression construct comprising a first nucleotide sequence having at least about 90% (i.e., or about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO:1 operably linked to a second nucleotide sequence, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes a transgene. In other cases, the expression construct comprises a third nucleotide sequence, wherein the third nucleotide sequence encodes an inhibitory nucleic acid. In certain cases, the first nucleotide sequence is SEQ ID NO:1.

[0009] Alternatively, the synthetic nucleic acid is an expression construct comprising a first nucleotide sequence having at least about 90% (i.e., or about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO:1 operably linked to a second nucleotide sequence, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes an inhibitory nucleic acid. In other cases, the expression construct comprises a third nucleotide sequence, wherein the third nucleotide sequence encodes a transgene. In certain cases, the first nucleotide sequence is SEQ ID NO:1.

[0010] In some cases, the synthetic nucleic acid is a vector comprising a first nucleotide sequence having at least 90% (i.e., or about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO:1 operably linked to a second nucleotide sequence, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes a transgene, and the vector is a plasmid or viral vector. In other cases, the vector comprises a third nucleotide sequence, wherein the third nucleotide sequence encodes an inhibitory nucleic acid. In still other cases, the vector is a viral vector, particularly a recombinant adeno-associated virus (rAAV) vector or a baculovirus vector. In certain cases, the first nucleotide sequence is SEQ ID NO:1.

[0011] Alternatively, the synthetic nucleic acid is a vector comprising a first nucleotide sequence having at least about 90% (i.e., or about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) sequence identity to SEQ ID NO:1 operably linked to a second nucleotide sequence, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes an inhibitory nucleic acid, and the vector is a plasmid or viral vector. In other cases, the vector comprises a third nucleotide sequence, which encodes a transgene. In still other cases, the vector is a viral vector, particularly a rAAV vector or a baculovirus vector. In certain cases, the first nucleotide sequence is SEQ ID NO:1.

[0012] In any of the above, the transgene can be, for example, an astrocyte-associated gene such as APOE2 or another gene associated with gliosis in AD, ALS, or HD.

[0013] In any of the above, the inhibitory nucleic acid may target an astrocyte-associated gene, such as, for example, APOE4 or another gene associated with gliosis in AD, ALS, or HD.

[0014] Second, this disclosure describes compositions comprising synthetic nucleic acids as described herein. In some cases, the composition is an rAAV comprising a capsid protein as described herein and a synthetic nucleic acid, the capsid protein comprising a capsid protein capable of crossing the blood-brain barrier (BBB). In other cases, the composition is a host cell comprising a synthetic nucleic acid or rAAV as described herein. In yet other cases, the composition is a pharmaceutical composition comprising a synthetic nucleic acid or rAAV as described herein and a pharmaceutically acceptable carrier.

[0015] Third, this disclosure describes a method for preferentially expressing a nucleotide sequence in astrocytes, which may include providing to a cell, tissue, organ, or individual an effective amount of a synthetic nucleic acid, vector, rAAV, or composition as described herein.

[0016] Fourth, the present disclosure describes methods of treating neurodegenerative diseases, particularly neurodegenerative diseases in which astrocyte-directed expression is desired, in an individual in need thereof. The methods can include administering to the individual an effective amount of an expression construct, vector, rAAV, or composition as described herein. In some cases, administration is via direct injection into the CNS of the individual, which can be intracerebroventricular (ICV) injection, intracisternal (ICM) injection, intraparenchymal injection, intrathecal injection, or a combination thereof. In certain cases, the direct injection is convection-enhanced delivery (CED). In still other cases, administration is a peripheral injection. In certain cases, the peripheral injection is via intravenous (IV) injection or subcutaneous (SC) injection.

[0017] Fifth, the present disclosure describes the use of a composition comprising a synthetic nucleic acid or rAAV as described herein in the manufacture of a medicament for the treatment of a neurodegenerative disease, particularly a neurodegenerative disease in which astrocyte-directed expression is desired.

[0018] Sixth, the present disclosure describes compositions comprising synthetic nucleic acids or rAAV as described herein for use in the treatment of neurodegenerative diseases, particularly neurodegenerative diseases in which astrocyte-directed expression is desired.

[0019] An advantage of the expression control element herein is that it is specific to astrocytes and therefore can drive expression of heterologous nucleotide sequences (e.g., transgenes and / or inhibitory nucleic acids) in astrocytes to levels not seen in other cells in the CNS.

[0020] Another advantage of the expression control elements herein is that they can be used to match endogenous expression of target genes, such as APOE, in neurodegenerative diseases where there is aberrant astrocyte-associated gene expression.

[0021] Further advantages, benefits, features and objects will become more readily apparent from a consideration of the following detailed description, which refers to the following drawings. [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1 shows the expression of codon-optimized ApoE driven by three different expression control elements: one known promoter (SEQ ID NO: 9 (known Prom)) and one of two potential astrocyte-specific promoters (SEQ ID NO: 1 (first Prom) or SEQ ID NO: 10 (second Prom)) in three different cell lines. [Figure 2A]Figure 2 shows the expression of Green Fluorescent Protein (GFP) driven by three different expression control elements (i.e., one known promoter (SEQ ID NO: 9 (known Prom)) and one of two potential astrocyte-specific promoters (SEQ ID NO: 1 (first Prom) or SEQ ID NO: 10 (second Prom)) in three different cell lines. Figure 2A shows normalized GFP RNA levels in one cell line—U87. Figure 2B shows normalized GFP RNA levels in a second cell line—HEK293, and Figure 3C shows normalized GFP RNA levels in a third cell line—SH-SY5Y. [Figure 2B] Figure 2 shows the expression of Green Fluorescent Protein (GFP) driven by three different expression control elements (i.e., one known promoter (SEQ ID NO: 9 (known Prom)) and one of two potential astrocyte-specific promoters (SEQ ID NO: 1 (first Prom) or SEQ ID NO: 10 (second Prom)) in three different cell lines. Figure 2A shows normalized GFP RNA levels in one cell line—U87. Figure 2B shows normalized GFP RNA levels in a second cell line—HEK293, and Figure 3C shows normalized GFP RNA levels in a third cell line—SH-SY5Y. [Figure 2C] Figure 2 shows the expression of Green Fluorescent Protein (GFP) driven by three different expression control elements (i.e., one known promoter (SEQ ID NO: 9 (known Prom)) and one of two potential astrocyte-specific promoters (SEQ ID NO: 1 (first Prom) or SEQ ID NO: 10 (second Prom)) in three different cell lines. Figure 2A shows normalized GFP RNA levels in one cell line—U87. Figure 2B shows normalized GFP RNA levels in a second cell line—HEK293, and Figure 3C shows normalized GFP RNA levels in a third cell line—SH-SY5Y. [Figure 3A]Images from mouse studies using the promoters of SEQ ID NO: 1 and SEQ ID NO: 9: Figure 3A shows that SEQ ID NO: 9 weakly drives in vivo expression of Enhanced Green Fluorescent Protein (EGFP) in astrocytes of mouse brain; Figure 3B shows that SEQ ID NO: 1 drives in vivo expression of EGFP in astrocytes of mouse brain. [Figure 3B] Images from mouse studies using the promoters of SEQ ID NO: 1 and SEQ ID NO: 9: Figure 3A shows that SEQ ID NO: 9 weakly drives in vivo expression of Enhanced Green Fluorescent Protein (EGFP) in astrocytes of mouse brain; Figure 3B shows that SEQ ID NO: 1 drives in vivo expression of EGFP in astrocytes of mouse brain. [Figure 4A] Images from mouse studies using the promoters of sequence number 1 and sequence number 9, where FIG. 4A shows that sequence number 9 drives in vivo expression of EGFP in neurons of the mouse brain, and FIG. 4B shows that sequence number 1 weakly drives in vivo expression of EGFP in neurons of the mouse brain. [Figure 4B] Images from mouse studies using the promoters of sequence number 1 and sequence number 9, where FIG. 4A shows that sequence number 9 drives in vivo expression of EGFP in neurons of the mouse brain, and FIG. 4B shows that sequence number 1 weakly drives in vivo expression of EGFP in neurons of the mouse brain. DETAILED DESCRIPTION OF THE INVENTION

[0023] overview

[0024] APOE is involved in the development of late-onset AD. APOE has several isoforms. One isoform, APOE2, is protective against AD; however, another isoform, APOE4, is associated with an increased risk of developing late-onset AD compared to the more common isoform, APOE3. Homozygous individuals carry two copies of APOE4 (i.e., APOE4 + / + heterozygous individuals who carry one copy of APOE4 and one copy of either APOE2 or APOE3 (APOE4 + / APOE2 + or APOE4 + / APOE3 + ) have a higher risk of developing late-onset AD compared to non-Hodgkin's disease.

[0025] Human ApoE is a 34 kDa glycoprotein with 299 amino acids after cleavage of an 18 amino acid signal peptide. ApoE isoforms differ from each other only at positions 130 and 176 (i.e., ApoE2-Cys130 and Cys176 (see SEQ ID NO: 4), ApoE3-Cys130 and Arg176 (see SEQ ID NO: 6), and ApoE4-Arg130 and Arg176 (see SEQ ID NO: 8)).

[0026] Accumulating evidence suggests that ApoE4 influences tau pathology, tau-mediated neurodegeneration, and microglial responses to AD-related pathologies. Furthermore, ApoE4 is pathogenic or exhibits reduced efficiency in multiple brain homeostatic pathways, including lipid transport, synaptic integrity and plasticity, glucose metabolism, and cerebrovascular function.

[0027] Astrocyte-directed expression of heterologous nucleotide sequences is therefore of interest in treating neurodegenerative diseases such as AD, as well as diseases caused by other astrocyte-associated genes.

[0028] Abbreviations and Definitions

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, the preferred methods and materials are described herein.

[0030] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0031] Furthermore, the use of "including" and other forms such as "including but not limited to," "include," "includes," and "included" is not limiting.

[0032] Certain abbreviations used herein are as follows:

[0033] "AAV" refers to recombinant adeno-associated virus. "AD" refers to Alzheimer's disease. "ALS" refers to amyotrophic lateral sclerosis. "APOE" refers to apolipoprotein E gene. "ApoE" refers to apolipoprotein E protein. "BBB" refers to blood-brain-barrier. "bp" refers to base pair. "CED" refers to convection-enhanced delivery. "DAPI" refers to 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (C 16 H 15N5)(2-(4-amidinophenyl)-1H-indole-6-carboxamidine (C 16 H 15N5). "DNA" refers to deoxyribonucleic acid. "DRG" ​​refers to dorsal root ganglion. "ds" refers to double-stranded. "EGFP" refers to enhanced green fluorescent protein. "GAPDH" refers to glyceraldehyde 3-phosphate dehydrogenase. "GFAP" refers to glial fibrillary acidic protein gene. "Gfap" refers to glial fibrillary acidic protein. "HD" refers to Huntington's disease. "hr" refers to hour(s). "ICM" refers to intracisterna magna. "ICV" refers to intracerebroventricular. "IRES" refers to Internal Ribosome Entry Site(s). "ITR" refers to Inverted Terminal Repeat. "IV" refers to intravenous. "kDa" refers to kilodalton(s). "min" refers to minute(s). "PNM" refers to Pan Neuronal Marker. "qPCR" refers to quantitative reverse transcription Polymerase Chain Reaction. "rAAV" refers to recombinant Adeno-Associated Virus. "RNA" refers to ribonucleic acid."RT-qPCR" refers to Real-Time quantitative reverse transcription Polymerase Chain Reaction. "SC" refers to subcutaneous. "ss" refers to single-stranded, and "vg" refers to vector genome(s).

[0034] Certain definitions used herein are defined as follows:

[0035] As used herein, "about" means within a statistically significant range of a value or values, e.g., a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0036] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., a composition described herein, such as an oligonucleotide described herein or an rAAV as described herein) to an individual in a manner that is pharmacologically useful (e.g., to treat a disease, disorder, condition, or symptom of the individual).

[0037] As used herein, "astrocyte-associated gene" means a gene that encodes a peptide, polypeptide, or protein that is genetically, biochemically, or functionally equivalent to a gene that is primarily expressed in astrocytes. Exemplary astrocyte-associated genes include, but are not limited to, APOE2 and APOE4, and GFAP.

[0038] As used herein, "astrocyte-directed expression" refers to the predominant expression of a nucleotide sequence of interest encoding a peptide, polypeptide, or protein in astrocytes relative to other cells in the CNS (e.g., neurons, including the dorsal root ganglion (DRG)).

[0039] As used herein, "astrocyte-specific promoter" means a promoter that drives expression of an operably linked nucleotide sequence preferentially in astrocytes relative to other cells in the CNS (e.g., neuronal cells, including DRGs).

[0040] As used herein, "codon optimization," with respect to a nucleotide sequence of a gene of interest, e.g., an AD-associated gene, refers to altering the codons or sequences in the gene or coding region therein to reflect the typical codon usage of a host organism (e.g., a mammal, such as a human) or cells thereof, without altering the polypeptide encoded by the nucleotide sequence. Thus, a codon-optimized transgene is optimized for expression in a particular organism, organ, tissue, or cell type, particularly a mammal, or mammalian organ, tissue, or cell type. Alternatively, "codon-optimized" refers to altering the codons or sequences in a gene to improve protein expression compared to a sequence lacking the alteration, for example, by eliminating or altering sites that may be cryptic splice sites, stop codons, miRNA recognition sequences, etc. The entire nucleotide sequence may be codon-optimized, or only one or more portions, sections, or regions of the nucleotide sequence may be codon-optimized.

[0041] As used herein, a "comparison window" refers to a specific contiguous segment of a nucleotide or amino acid sequence, where the sequence in the comparison window may contain additions and / or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions and / or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 10 contiguous nucleotides / amino acids in length, and optionally may be 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides / amino acids in length, or longer.

[0042] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to form base pairs (bp) with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid may base pair with each other by forming hydrogen bonds. Complementary nucleotides may base pair in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids may have regions of multiple nucleotides that are complementary to each other and form a region of complementarity, as described herein.

[0043] As used herein, "effective amount" refers to an amount, concentration, or dosage of a therapeutic agent (e.g., a nucleic acid, vector, or rAAV as described herein), or a pharmaceutical composition thereof, that, when administered in single or multiple doses to an individual in need thereof, provides the desired effect in such an individual under diagnosis or treatment (i.e., is likely to produce a clinically measurable difference in the individual's condition). An effective amount can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, numerous factors are taken into consideration, including, but not limited to, the species of mammal, its size, age, and general health; the particular disease, disorder, condition, or symptom involved; the extent, involvement, or severity of the disease, disorder, condition, or symptom; the individual's response; the therapeutic agent administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosage regimen; the use of concomitant medications; and other relevant circumstances.

[0044] As used herein, "expression construct" refers to a nucleotide sequence that is capable of replicating and expressing a nucleotide sequence of interest (e.g., a transgene or inhibitory nucleic acid) when transformed, transfected, or transduced into a target cell, tissue, organ, or individual. Exemplary expression constructs are vectors, such as viral vectors, particularly AAV vectors or baculovirus vectors. Here, an expression construct can include at least one expression control element operably linked to a nucleotide sequence of interest, such as a transgene (and / or inhibitory nucleic acid). Thus, an expression construct can be an expression control element, such as a promoter, operably interacting with a transgene (and / or inhibitory nucleic acid) that can direct expression of the transgene (and / or inhibitory nucleic acid) in a cell, tissue, organ, or individual, particularly astrocytes.

[0045] As used herein, "expression control element" means nucleotide sequences such as promoters, polyadenylation signals, transcriptional or translational termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, and the like, which collectively provide for the replication, transcription, and / or translation of a desired nucleic acid (e.g., a transgene or inhibitory nucleic acid) in a cell, tissue, organ, or individual. Not all of these control sequences need always be present so long as the desired nucleotide sequence can be replicated, transcribed, and translated in the appropriate cell, tissue, organ, or individual.

[0046] As used herein, "in combination with" means that a therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition as described herein) is administered either simultaneously, sequentially, or in a single combined formulation with one or more additional therapeutic agents.

[0047] As used herein, "individual" means any mammal, including cats, dogs, mice, rats, and primates, particularly humans. Furthermore, the terms "subject" or "patient" may be used interchangeably with "individual."

[0048] As used herein, "individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein. Specifically, the preferred individual to be treated is a human.

[0049] As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can attenuate, reduce, or prevent the expression of a gene or mRNA. Exemplary inhibitory nucleic acids include, but are not limited to, shRNA, siRNA, miRNA, amiRNA, etc. Here, the inhibitory nucleic acid may be, for example, a nucleotide sequence encoding an antisense sequence to a nucleotide sequence of interest, such as an AD-related gene (e.g., a gene encoding ApoE4).

[0050] As used herein, "nucleoside" refers to a nucleobase-sugar combination, where the nucleobase moiety is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The sugar is typically a pentose sugar, such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0051] As used herein, "nucleotide" means an organic molecule having a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a five-carbon sugar such as ribose or 2'-deoxyribose) and a phosphate group, which can function as a monomer unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0052] As used herein, "oligonucleotide" refers to a short nucleic acid molecule (e.g., less than about 100 nucleotides in length). Oligonucleotides may be single-stranded (ss) or double-stranded (ds).

[0053] As used herein, "operably linked" and the like means that the elements of an expression construct (or other nucleic acid construct) are configured to perform their normal function (i.e., under the influence of expression control elements). Thus, an expression control element (e.g., a promoter) operably linked to a desired nucleotide sequence (e.g., a transgene or inhibitory nucleic acid) is capable of effecting expression of the desired nucleic acid. Control elements need not be contiguous with the desired nucleotide sequence, so long as they function to direct its expression (i.e., maintain the proper reading frame). Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter and the desired nucleotide sequence, and the promoter would still be considered "operably linked" to the desired nucleotide sequence.

[0054] As used herein, "pharmaceutically acceptable," when referring to a material such as a carrier or diluent, means that it does not abrogate the biological activity or properties of a therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition as described herein) and is relatively non-toxic (i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is included).

[0055] As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which is involved in carrying or transporting a therapeutic agent within or to an individual so that the therapeutic agent may perform its intended function. Additional ingredients that may be included in pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 21 stEdition, University of the Sciences in Philadelphia, PA (2006).

[0056] As used herein, "pharmaceutical composition" means a composition or therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition as described herein) mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersing agent, suspending agent, thickener, excipient, etc.

[0057] As used herein, "polynucleotide" refers to a polymer of nucleotides. The term can contain any type of nucleotide unit but generally applies to RNA or DNA nucleotide polymers. Polynucleotide is used to include ss nucleic acids, ds nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogs that can be identified by their sequence, which are generally presented in a 5' to 3' direction (as the coding strand), with 5' and 3' indicating the bond formed between the 5' hydroxyl group of one nucleotide and the 3' hydroxyl group of the next nucleotide. For a coding strand presented in a 5'-3' direction, its complement (or non-coding strand) is the strand that hybridizes to that sequence according to Watson-Crick base pairing. Thus, as used herein, the complement of a nucleic acid, e.g., a polynucleotide, is the same as a "reverse complement" and describes a nucleic acid that, in its natural form, base-pairs with the nucleic acid in question.

[0058] As used herein, "recombinant adeno-associated virus," "recombinant AAV," and "rAAV" refer to a viral particle containing a rAAV vector encapsidated by AAV capsid proteins.

[0059] As used herein, "recombinant adeno-associated virus vector," "recombinant AAV vector," and "rAAV vector" refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one AAV inverted terminal repeat (ITR) sequence. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with (or expressing suitable helper functions for) a suitable helper virus that expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0060] As used herein, "sequence identity," in the context of two nucleotide sequences or two amino acid sequences, means the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0061] As used herein, "synthetic" refers to a nucleic acid or other molecule or compound that has been artificially engineered (i.e., recombinantly produced) or synthesized using a machine, such as a solid-phase nucleic acid synthesizer, or that is otherwise not derived from a natural source that normally produces that nucleic acid or other compound (i.e., non-naturally occurring). As used herein, "transgene" refers to a nucleotide sequence that can be introduced into a cell, transcribed into RNA, optionally translated, and / or expressed under appropriate conditions. A transgene confers a desired characteristic on a cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result. Here, a transgene may be, for example, a nucleotide sequence encoding a polypeptide of interest, such as an AD-associated gene (e.g., a gene encoding ApoE2).

[0062] As used herein, "treat," "treatment," or "to treat" refers to a process that may slow, control, retard, or halt the progression of a disease or disorder disclosed herein, or may ameliorate disease or disorder symptoms, but does not necessarily indicate the complete disappearance of all disease or disorder symptoms. Treatment, etc., includes the administration of a nucleic acid, expression construct, vector, rAAV, or composition as described herein to treat a disease or disorder in an individual, particularly a human.

[0063] As used herein, "vector" refers to a nucleic acid construct, such as a plasmid, cosmid, or phage, for introducing / transferring one or more heterologous nucleotide sequences, such as the expression constructs herein, into a target cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0064] As used herein, "viral vector" refers to a vector derived from a naturally occurring or modified virus, particularly an rAAV vector or a baculovirus vector (e.g., an Autographa californica Nuclear Polyhedrosis Vector (AcNPV) vector).

[0065] composition

[0066] synthetic nucleic acid

[0067] Expression control elements for use as astrocyte-specific promoters: A synthetic nucleic acid can be an expression control element, for example, an astrocyte-specific promoter (i.e., can be used for astrocyte-directed expression of heterologous nucleic acid sequences, such as transgenes and / or inhibitory nucleic acids). In some cases, a synthetic nucleic acid for use as an astrocyte-specific promoter comprises a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 1. Alternatively, the nucleotide sequence has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1. In certain cases, the nucleotide sequence is SEQ ID NO: 1.

[0068] In other cases, the synthetic nucleic acid is complementary to a nucleotide sequence having at least about 90% sequence identity to SEQ ID NO: 2. Alternatively, the nucleotide sequence is complementary to a nucleotide sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2.

[0069] Synthetic nucleic acids as described herein can exist by themselves or can be present as part of an expression construct, vector, or rAAV as further described herein.

[0070] Expression Constructs: As described above, synthetic nucleic acids can be incorporated into expression constructs for astrocyte-directed expression of heterologous nucleotide sequences. In some cases, synthetic nucleic acids for use as expression constructs include at least SEQ ID NO: 1 (or a nucleotide sequence having at least about 90% to about 100% sequence identity thereto) as an expression element and the nucleotide sequence of a transgene. In other cases, synthetic nucleic acids for use as expression constructs include at least SEQ ID NO: 1 (or a nucleotide sequence having at least about 90% to about 100% sequence identity thereto) as an expression element and the nucleotide sequence of an inhibitory nucleic acid. In still other cases, synthetic nucleic acids for use as expression constructs include at least SEQ ID NO: 1 (or a nucleotide sequence having at least about 90% to about 100% sequence identity thereto) as an expression control element, the nucleic acid sequence of a transgene, and the nucleic acid sequence of an inhibitory nucleic acid. In certain cases, the expression element is SEQ ID NO: 1.

[0071] In some cases, the transgene encodes an astrocyte-associated gene. In some cases, the inhibitory nucleic acid targets an astrocyte-associated gene. Examples of astrocyte-associated genes include, but are not limited to, APOE2, APOE4, or another gene associated with gliosis in AD, ALS, or HD.

[0072] An expression construct as described herein can exist by itself or can exist as part of a vector or rAAV as described herein.

[0073] Vectors: As noted above, synthetic nucleic acids or expression constructs as described herein can also be incorporated into vectors, particularly viral vectors such as rAAV vectors. The rAAV vector may comprise either the "plus strand" or "minus strand" of the rAAV vector. In some cases, the rAAV vector is single-stranded (ss) (e.g., ss DNA or ss RNA). In other cases, the rAAV vector is double-stranded (ds) (e.g., ds DNA or ds RNA).

[0074] In other cases, the vector is a baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis virus (AcNPV) vector).

[0075] A vector, such as an rAAV vector, may include not only an expression control element having the nucleotide sequence of SEQ ID NO: 1 (or a nucleotide sequence having at least about 90% to about 100% sequence identity thereto) and a transgene and / or inhibitory nucleic acid, but also other expression control elements, such as at least one or more nucleotide sequences selected from the group consisting of promoters, enhancers, transcription factor binding sites, repressor binding sites, intron splice sites, post-transcriptional regulatory elements, polyadenylation signals, and combinations thereof. See, e.g., PCT Publication No. WO 2020 / 112802.

[0076] In still other cases, the vector comprises at least an expression control element having the nucleotide sequence of SEQ ID NO: 1 (or a nucleotide sequence having at least about 90% to about 100% sequence identity thereto), a nucleic acid sequence of a transgene, and a nucleic acid sequence of an inhibitory nucleic acid.

[0077] In some cases, the transgene encodes an astrocyte-associated gene. In some cases, the inhibitory nucleic acid targets an astrocyte-associated gene. Examples of astrocyte-associated genes include, but are not limited to, APOE2, APOE4, or another gene associated with gliosis in AD, ALS, or HD.

[0078] A vector as described herein can exist by itself or can exist as part of a rAAV as described herein.

[0079] rAAV

[0080] As described above, a synthetic nucleic acid, expression construct, or vector as described herein can be incorporated into a rAAV. In some cases, the rAAV may have a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In other cases, the rAAV may have a capsid protein derived from a non-human host, such as a rhesus AAV capsid protein, e.g., AAVrh.10, AAVrh.39, etc.

[0081] In some cases, the rAAV comprises a capsid protein that is a variant of a wild-type capsid protein, where such a capsid protein variant has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) compared to the wild-type AAV capsid protein from which it is derived.

[0082] In some embodiments, rAAVs contain capsid proteins that allow them to spread easily through the CNS, particularly when introduced directly into the CSF space or into the brain parenchyma. In this way, such rAAVs are able to cross the BBB. Examples of capsid proteins that can cross the BBB include, but are not limited to, capsid proteins having the AAV9 or AAVrh.10 serotypes.

[0083] Methods for producing rAAV are described, for example, in Samulski et al. (1989) J. Virol. 63:3822-3828 and Wright (2009) Hum. Gene Ther. 20:698-706. In some cases, rAAV can be produced in a baculovirus vector expression system (BEVS). Production of rAAV using BEVS is described, for example, in Urabe et al. (2002) Hum. Gene Ther. 13:1935-1943, Smith et al. (2009) Mol. Ther. 17:1888-1896, and U.S. Patent Nos. 8,945,918 and 9,879,282, and PCT Publication Nos. 2017 / 184879 and 2022 / 082017. Alternatively, rAAV can be produced in human embryonic kidney (e.g., HEK293) cells (see, e.g., PCT Publication Nos. WO 2020 / 210689 and WO 2022 / 035900). However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

[0084] Pharmaceutical Composition

[0085] The synthetic nucleic acids described herein (i.e., expression constructs or vectors) or the rAAV described herein can be formulated as pharmaceutical compositions comprising the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier.

[0086] The pharmaceutical compositions may be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, such as by topical powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual, by intratracheal instillation, bronchial instillation, and / or inhalation, and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, IV administration (e.g., systemic intravenous injection), topical administration via the blood and / or lymphatic supply, and / or direct administration to the affected site.

[0087] Generally, the most appropriate route of administration will depend on various factors, including, but not limited to, the properties of the agent (e.g., its administration and / or its stability in the intended target environment) and / or the condition of the individual (e.g., whether the subject can tolerate oral administration). In some cases, the synthetic nucleic acid, rAAV, or pharmaceutical composition is suitable for administration to the CNS of an individual.

[0088] kit

[0089] In some cases, a synthetic nucleic acid (i.e., an expression construct or vector) described herein, a rAAV described herein, or even other therapeutic oligonucleotides comprising expression control elements as described herein, can be included in a kit containing the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide and instructions for its use. In other cases, the kit includes the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide, and a package insert containing instructions for use of the kit and / or its components. In still other cases, the kit includes a suitable container or other means for containing the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art. In some cases, the container includes at least one vial, well, test tube, flask, bottle, syringe, or other container means into which the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide is placed, and, in some cases, suitably aliquoted. If an additional component is provided, the kit includes an additional container into which this component is placed. The kits may also include a means for enclosing the synthetic nucleic acids, rAAV or other therapeutic oligonucleotides and other reagents in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained. The containers and / or kits may include instructions and / or warning labels.

[0090] In some cases, the kit includes a synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide and a pharmaceutically acceptable carrier, or a pharmaceutical composition including a synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide and instructions for treating or slowing the progression of a neurodegenerative disease in an individual in need thereof.

[0091] In some cases, the kit includes a synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide and a pharmaceutically acceptable carrier or a pharmaceutical composition comprising the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide, and instructions for administering the synthetic nucleic acid, rAAV, or other therapeutic oligonucleotide or pharmaceutical composition. method

[0092] How to use

[0093] The synthetic nucleic acids, rAAV, other therapeutic oligonucleotides or pharmaceutical compositions may be used in methods of treating neurodegenerative diseases, such methods comprising at least the step of administering to an individual in need of such treatment an effective amount of the synthetic nucleic acid, rAAV, other therapeutic oligonucleotide or pharmaceutical composition comprising the same.

[0094] In some cases, the synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition is administered by intravenous or SC injection. In other cases, the synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition is administered directly to the CNS of an individual, for example, by direct injection into the brain and / or spinal cord. Examples of direct CNS administration modes include, but are not limited to, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing.

[0095] In some cases, direct CNS administration is by convection-enhanced delivery (CED), which involves surgically exposing the brain, placing a small-diameter catheter directly into the target region of the brain, and then injecting the therapeutic agent (e.g., a synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition as described herein) directly into the brain. CED is described in Debinski et al. (2009) Expert Rev. Neurother. 9:1519-1527.

[0096] In some cases, the neurodegenerative disease is an AD-related disease. In other cases, the neurodegenerative disease is AD. In still other cases, the individual is characterized by an APOE4 allele. The individual may be homozygous (e.g., APOE4 + / + ) or heterozygous for APOE4 (e.g., APOE4 + / - Optionally, the individual may be heterozygous for APOE4, and the individual's second APOE allele may be APOE2 or APOE3.

[0097] In some cases, the effective amount is about 10 9 Genome copies (GC) / kg ~ approx. 10 14 In other cases, the titer is in the range of about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 12 GC / kg, or approximately 10 14 In still other cases, the titer is >10 GC / kg by injection into the CSF space or by intraparenchymal injection. 12 GC / kg.

[0098] In other cases, the effective amount is about 1 x 10 12 vg~approx. 1×10 15 vg or approximately 1 x 10 13 vg~approx. 7×10 14 In other cases, the dose is in the range of about 3.5 x 10 13 vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 In still other cases, the dose is about 1 x 10 14 vg, approx. 2.0×10 14 vg, or approximately 4.0 × 10 14 vg. Alternatively, the dose is about 2 x 10 13 vg, approx. 3×10 13 vg, approx. 4×10 13 vg, approx. 5×10 13 vg, approx. 6×10 13 vg, approx. 7×10 13vg, approx. 8×10 13 vg, approx. 9×10 13 vg, approx. 1×10 14 vg, or approximately 2 × 10 14 vg. In certain cases, the dose is 7.0 x 10 13 vg or 1.4 x 10 14 vg.

[0099] In some cases, the individual is between about 1 month and about 10 years old (e.g., about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or any age in between). In other cases, the individual is between about 10 years old and about 20 years old (e.g., about 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, 17 years old, 18 years old, 19 years old, 20 years old, or any age in between). In other cases, the individual is over 20 years old (e.g., about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any age in between), over 30 years old (e.g., about 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or any age in between), over 40 years old (e.g., about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or any age in between), or even over 50 years old (e.g., about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, or any age in between).

[0100] use

[0101] rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein, or pharmaceutical compositions comprising same, can be used or adapted for use to treat individuals (e.g., humans) having or suspected of having an AD-related disease. Accordingly, rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein, or pharmaceutical compositions comprising same, are provided for or adapted for use in treating individuals having or suspected of having an AD-related disease. Also provided is the use of rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein, or pharmaceutical compositions comprising same, for use in, or adapted for use in, the manufacture of a medicament or pharmaceutical composition for treating an AD-related disease. [Example]

[0102] The following non-limiting examples are offered by way of illustration and not limitation.

[0103] In vitro function

[0104] Example 1: Astrocyte-specific expression control elements

[0105] Objective: To develop expression control elements (eg, promoters) that can drive astrocyte-specific expression of heterologous nucleotide sequences (eg, transgenes or inhibitory nucleic acids).

[0106] Methods: Two potential astrocyte-specific expression control elements were generated and compared to a known promoter (SEQ ID NO: 9). The first potential expression control element has the nucleotide sequence of SEQ ID NO: 1, and the second potential expression control element has the nucleotide sequence of SEQ ID NO: 10.

[0107] A plasmid expressing the codon-optimized human ApoE2 nucleotide sequence (SEQ ID NO: 11) under the control of one of three expression control elements was synthesized and cloned by Vigene Biosciences.

[0108] HEK293T (human embryonic kidney cell line), U87 (human glioblastoma cell line), and SH-SY5Y (human neuroblastoma cell line) cell lines were transfected with these three constructs to test in vitro expression. Briefly, cells were transfected into 96-well plates using Lipofectamine® 2000 (Invitrogen) at a volume:mass ratio of 3:1 and plated at a density of 30,000 cells / well. 72 hours after transfection, RNA was harvested and gene expression was analyzed by RT-qPCR. Expression of codon-optimized ApoE was measured using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which served as a loading control.

[0109] Results: In U87 cells (astrocytoma line), all three expression control elements drove approximately equal expression, with the second expression control element (SEQ ID NO: 10) having slightly lower expression (Figure 1). In HEK293 cells, the second expression control element (SEQ ID NO: 10) had lower expression than either the known expression control element (SEQ ID NO: 9) or the first expression control element (SEQ ID NO: 1) (Figure 1). In SH-SY5Y cells (neuroblastoma line), the three expression control elements again drove approximately equal expression, with the second expression control element (SEQ ID NO: 10) having potentially slightly higher expression (Figure 1). In vivo function

[0110] Example 2: In vivo studies of enhanced astrocyte-specific expression of GFP

[0111] Objective: To evaluate the ability of the astrocyte-specific expression control element of Example 1 in an in vivo environment.

[0112] Methods: rAAV9 expressing enhanced GFP (EGFP, SEQ ID NO: 12) under the control of three expression control elements was generated by Virovek, Inc. (expression control elements SEQ ID NO: 1 and SEQ ID NO: 10) or Prevail Therapeutics (expression control element SEQ ID NO: 9). The rAAV had the following nucleotide sequences: SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. rAAV was delivered to neonatal (P2) C57BL / 6 mice at Psychogenics via unilateral ICV injection (4.84 x 10 in 4 uL per mouse). 10 vg, 6 animals for each of the three expression control elements. The animals were euthanized 4 weeks after injection, and tissues were harvested for molecular biology and imaging analysis.

[0113] Brains, spinal cords, and livers were mounted for imaging, and GFP fluorescence was visualized with DAPI nuclear counterstain.

[0114] Following the study, DNA and mRNA were extracted from cortex, spinal cord, and liver samples and analyzed by qPCR and RT-qPCR, respectively, to determine viral biodistribution and GFP mRNA expression.

[0115] Results: AAVs using the known expression control element of SEQ ID NO: 9 conferred widespread expression throughout the brain, almost exclusively in neurons, whereas AAVs using the first expression control element of SEQ ID NO: 1 conferred nearly equal expression. However, expression was largely localized to astrocytes (confirmed by preliminary GFAP counterstaining). In contrast, AAVs using the second expression control element of SEQ ID NO: 10 conferred low levels of expression in only a few cells, mostly neurons.

[0116] Expression in the spinal cord differs qualitatively between the first expression control element of SEQ ID NO: 1 and the known expression control element of SEQ ID NO: 9, but remains approximately equal overall.

[0117] Expression in the liver appears to be nearly identical between the first expression control element of SEQ ID NO:1 and the known expression control element of SEQ ID NO:9.

[0118] For the second expression regulatory element of SEQ ID NO: 10, almost no fluorescence was detectable in either the liver or spinal cord.

[0119] There were no significant differences in biodistribution across all tissues (i.e., brain, liver, and spinal cord) between the AAVs. Indeed, EGFP mRNA levels were similar or identical between the known expression control element of SEQ ID NO:9 and the first expression control element of SEQ ID NO:1, but up to 10-fold lower with the second expression control element of SEQ ID NO:10.

[0120] Given the similar biodistribution and overall expression levels between the known expression control element of SEQ ID NO:9 and the first expression control element of SEQ ID NO:1, as well as the same viral capsid used in all constructs, differences in expression patterns may be due to differences in the effectiveness of the expression control elements as promoters. Expression from the first expression control element of SEQ ID NO:1 was predominant in astrocytes, which was not achieved with the second expression control element of SEQ ID NO:10 (Figures 2A-2C). Surprisingly, these in vivo data could not be predicted from in vitro data, in which expression in neuronal cell lines (SH-SY5Y) and astrocyte-like cell lines (U87) was similar across all three expression control elements.

[0121] Example 3: In vivo studies on astrocyte-specific expression of GFP

[0122] Objective: To further evaluate the ability of the astrocyte-specific expression control element of Example 1 in an in vivo environment.

[0123] Methods: In vivo validation was performed at PsychoGenics, Inc. (Paramus, NJ). Briefly, rAAV9 encoding EGFP (as in Example 2) under the control of an expression construct having SEQ ID NO:1, SEQ ID NO:9, or SEQ ID NO:10 was administered by ICV injection to mixed-sex C57BL / 6 mice at P2. rAAV was administered at a concentration of 1.21 x 10 in a 4 μL volume. 13 At a concentration of 4.84 x 10 vg / mL 10 The total dose was administered at 100 mg / animal. Thirty days after ICV administration, animals were euthanized, and tissues were collected and fixed for immunohistochemistry or flash-frozen for molecular biology analysis. Brains, spinal cords, and livers were stained with antibodies against GFAP to identify astrocytes and PNM to identify neurons, and co-imaged with native GFP fluorescence to determine cellular expression. The cortex, spinal cord, and liver were also analyzed for biodistribution and GFP mRNA expression by qPCR at Prevail Therapeutics (New York, NY).

[0124] Results: The expression construct under the control of SEQ ID NO: 9 weakly drives the in vivo expression of EGFP in astrocytes of the mouse brain (Figure 3A). In contrast, the expression construct under the control of SEQ ID NO: 1 drives the in vivo expression of EGFP in astrocytes of the mouse brain (Figure 3B). However, the expression construct under the control of SEQ ID NO: 9 drives the in vivo expression of EGFP in neurons of the mouse brain (Figure 4A). In contrast, the expression construct under the control of SEQ ID NO: 1 weakly drives the in vivo expression of EGFP in neurons of the mouse brain (Figure 4B). Sequence Listing

[0125] The following nucleotide and / or amino acid sequences are mentioned in the above disclosure and are provided below for reference.

[0126] SEQ ID NO: 1 - Synthetic nucleic acid 1 (603 nt) attcggtacctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgggggtgtctgtattactgggcgaggtgtcctcccttcctggggactgtggggggtggtcaaaagacctctatgccccacctccttcctccctctgccctgctgtgcctggggcagggggagaacagcccacctcgtgactgggggctggcccagcccgccctatccctgggggagggggcgggacagggggagccctataattggacaagtctgggatccttgagtcct

[0127] SEQ ID No. 2 - Synthetic nucleic acid 2 (603 nt, complementary sequence to SEQ ID No. 1) aggactcaaggatcccagacttgtccaattatagggctccccctgtcccgccccctcccccagggatagggcgggctgggccagcccccagtcacgaggtgggctgttctccccctgccccaggcacagcagggcagagggaggaaggaggtggggcatagaggtcttttgaccaccccccacagtccccaggaagggaggacacctcgcccagtaatacagacacccccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaataactaggtaccgaat

[0128] SEQ ID NO: 3 - Human APOE2 mRNA (1234 nt, NCBI Reference Sequence No. NM_000041.3)

[0129] SEQ ID NO:4 - Human ApoE2 protein (317 aa) MKVLWAALLVTLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASH LRKLRKRLLRDADDLQKCLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0130] SEQ ID NO:5 - Human APOE3 mRNA (1144 nt, NCBI reference sequence NM_001302689.2)

[0131] SEQ ID NO:6 - Human ApoE3 protein (317 aa) MKVLWAALLVTLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQTAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASH LRKLRKRLLRDPDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0132] SEQ ID NO:7 - Human APOE4 mRNA (1265 nt, NCBI reference sequence NM_001302690.1)

[0133] SEQ ID NO:8 - Human ApoE4 protein (317 aa) MKVLWAALLVTLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQTAQARLGADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLASH LRKLRKRLLRDPDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0134] SEQ ID NO: 9 - Synthetic nucleic acid 3 (658 nt) gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcg

[0135] SEQ ID NO: 10-S Synthetic Nucleic Acid 4 (228 nt) gggtgtctgtattactgggcgaggtgtcctcccttcctggggactgtggggggtggtcaaaagacctctatgccccacctccttcctccctctgccctgctgtgcctggggcagggggagaacagcccacctcgtgactgggggctggcccagcccgccctatccctgggggagggggcgggacagggggagccctataattggacaagtctgggatccttgagtcct

[0136] Array No. 11-S Synthetic Nucleic Acid 5 (954 nt) atgaaggtgctgtgggccgccctgctggtgaccttcctggccggctgccaggccaaagtcgaacaggccgtcgagaccgagcccgagcccgagctgcgccagcagaccgagtggcagagcggccagcgctgggagctggccctgggccgcttctgggactacctgcgctgggtgcagaccctgagcgagcaggtgcaggaggagctgctgagcagccaggtgacccaggagctgcgcgccctgatggacgagaccatgaaagaactcaaagcttataagagcgagctggaggagcagctgacccccgtggccgaggagacccgcgcccgcctgagcaaggagctgcaggccgcccaggcccgcctgggcgccgacatggaggacgtgtgcggccgcctggtgcagtaccgcggcgaggtgcaggccatgctgggccagagcaccgaggagctgcgcgtgcgcctggccagccacctgcgcaagctgcgcaagcgcctgctgcgcgacgccgacgacctgcagaagtgcctggccgtgtaccaggccggcgcccgcgagggcgccgagcgcggcctgagcgccatccgcgagcgcctgggccccctggtggagcagggccgcgtgcgcgccgccaccgtgggcagcctggccggccagcccctgcaggagcgcgcccaggcctggggcgagcgcctgcgcgcccgcatggaggagatgggcagccgcacccgcgaccgcctggacgaggtgaaggagcaggtggccgaggtgcgcgccaagctggaggagcaggcccagcagatccgcctgcaggccgaggccttccaggcccgcctgaagagctggttcgagcccctggtggaggacatgcagcgccagtgggccggcctggtggagaaggtgcaggccgccgtgggcaccagcgccgcccccgtgcccagcgacaaccactaa

[0137] Accession No. 12 - Enhanced Green Fluorescent Protein (720 nt) Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa

[0138] Accession No. 13 - CAPO-GFP AAV Expression Construct / Vector (7351 nt)

[0139] SEQ ID NO: 14 - CBA-GFP expression construct / vector (7313 nt)

[0140] SEQ ID NO: 15 - ApoP-GFP expression construct / vector (7047 nt)

Claims

1. A promoter comprising a nucleotide sequence having at least about 95% sequence identity to SEQ ID NO:

1.

2. The promoter of claim 1 , wherein the nucleotide sequence is SEQ ID NO:

1.

3. a first nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1 operably linked to a second nucleotide sequence; A synthetic nucleic acid, wherein said first nucleotide sequence is a promoter for astrocyte-directed expression and said second nucleotide sequence is not an expression control element.

4. 4. The synthetic nucleic acid of claim 3, further comprising a third nucleotide sequence operably linked to the first nucleotide sequence, the third nucleotide sequence being downstream of the second nucleotide sequence, and the third nucleotide sequence not being an expression control element.

5. 5. The synthetic nucleic acid of claim 4, wherein the second nucleotide sequence is a transgene and the third nucleotide sequence is a different transgene.

6. 5. The synthetic nucleic acid of claim 4, wherein the second nucleotide sequence is a transgene and the third nucleotide sequence is an inhibitory nucleic acid.

7. 5. The synthetic nucleic acid of claim 4, wherein the second nucleotide sequence is an inhibitory nucleic acid and the third nucleotide sequence is a different inhibitory nucleic acid.

8. 5. The synthetic nucleic acid of claim 4, wherein the second nucleotide sequence is an inhibitory nucleic acid and the third nucleotide sequence is a transgene.

9. The synthetic nucleic acid of any one of claims 5 to 8, wherein the transgene encodes a first Alzheimer's disease (AD)-associated gene.

10. The synthetic nucleic acid of any one of claims 6 to 8, wherein the inhibitory nucleic acid inhibits the expression or activity of a second AD-related gene.

11. 11. The synthetic nucleic acid of claim 9 or 10, wherein the first AD-associated gene is APOE2.

12. 12. The synthetic nucleic acid of claim 10 or 11, wherein the second AD-associated gene is APOE4.

13. A vector comprising the promoter according to claim 1 or 2 or the synthetic nucleic acid according to any one of claims 3 to 12.

14. The vector of claim 13 , wherein the vector is a baculovirus vector or an adeno-associated virus (AAV) vector.

15. 15. The vector of claim 14, wherein the vector is an AAV vector, and the vector further comprises a nucleotide sequence of at least one additional expression control element selected from the group consisting of an AAV ITR, an enhancer, a transcription factor binding site, an intron splice site, a post-transcriptional regulatory element, a polyA tail, and a repressor binding site, and combinations thereof.

16. (i) AAV capsid proteins; and (ii) a synthetic nucleic acid according to any one of claims 3 to 12 or a vector according to any one of claims 13 to 15. A recombinant adeno-associated virus (rAAV).

17. 17. The rAAV of claim 16, wherein the AAV capsid protein is an AAV9 capsid protein or an AAVrh.10 capsid protein.

18. (i) a synthetic nucleic acid according to any one of claims 3 to 12, a vector according to any one of claims 13 to 15, or an rAAV according to claim 16 or 17; (ii) a pharmaceutically acceptable carrier.

19. 1. A method of treating an individual having or suspected of having a neurodegenerative disease, said method comprising:

19. A method comprising administering to the individual an effective amount of the synthetic nucleic acid of any one of claims 3 to 12, the vector of any one of claims 13 to 15, the rAAV of claim 16 or 17, or the pharmaceutical composition of claim 18.

20. the administering step comprises: (i) direct injection into the central nervous system (CNS) of said individual, wherein said direct injection is selected from the group consisting of intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, or a combination thereof; and / or 20. The method of claim 19, comprising (ii) a peripheral injection, wherein the peripheral injection is an intravenous injection or a subcutaneous injection.

21. 21. The method of claim 19 or 20, wherein the individual has Alzheimer's disease (AD) and is homozygous for the APOE4 allele.

22. 1. A method for expressing a nucleic acid of interest in an astrocyte, said method comprising: A method comprising the step of introducing into a cell, tissue, organ, or individual a vector comprising a promoter comprising SEQ ID NO: 1 operably linked to the nucleic acid of interest.

23. 23. The method of claim 22, wherein the nucleic acid of interest is a transgene of an Alzheimer's disease (AD)-associated gene.

24. 24. The method of claim 23, wherein the AD-associated gene is APOE2.

25. 23. The method of claim 22, wherein the nucleic acid of interest is an inhibitory nucleic acid of an Alzheimer's disease (AD)-associated gene.

26. 26. The method of claim 25, wherein the AD-associated gene is APOE4.

27. Use of the promoter of claim 1 or 2, or the synthetic nucleic acid of any one of claims 3 to 12, the vector of any one of claims 13 to 15, the rAAV of claim 16 or 17, or the pharmaceutical composition of claim 18, in the manufacture of a medicament for treating the neurodegenerative disease.

28. 28. The use according to claim 27, wherein the neurodegenerative disease is Alzheimer's disease (AD) or an AD-related disease.

29. 19. A promoter according to claim 1 or 2, a synthetic nucleic acid according to any one of claims 3 to 12, a vector according to any one of claims 13 to 15, an rAAV according to claim 16 or 17, or a pharmaceutical composition according to claim 18, for use in therapy.

30. 19. The promoter of claim 1 or 2, the synthetic nucleic acid of any one of claims 3 to 12, the vector of any one of claims 13 to 15, the rAAV of claim 16 or 17, or the pharmaceutical composition of claim 18, for use in treating the neurodegenerative disease.

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