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

Abstract

Synthetic nucleic acids that can be used for astrocyte directed expression of heterologous nucleotide sequences are described. Also described are methods of using the same for the targeted expression of astrocytes of such nucleotide sequences for the treatment of neurodegenerative diseases.

Description

[0001] Reference to electronically submitted sequence listings

[0002] The present disclosure is submitted together with a sequence listing in ST.26 XML format. The sequence listing is provided as a file named "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. Technical Field

[0003] The present disclosure relates generally to biology and medicine, and more particularly, it relates to synthetic nucleic acids that can be used as expression control elements and methods of using them for astrocyte-directed expression of heterologous nucleotide sequences, especially in the treatment of neurodegenerative diseases. Background Art

[0004] 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 abnormal protein deposits present throughout the brain that inhibit neuronal function, destroy connections between neurons, and ultimately lead to cell death. These deposits contain plaques of amyloid beta and tangles formed by phosphorylated tau protein. Individuals with mild AD experience memory loss, leading to distraction, difficulty managing finances, repeated questioning, and personality and behavioral changes. In addition, individuals with moderate AD show increased memory loss, leading to confusion and difficulty recognizing friends and family, inability to learn new things, hallucinations, delusions, and paranoia. In addition, individuals with severe AD are unable to communicate and are completely dependent on the care of others. Ultimately, protein plaques and tangles spread throughout the brain, leading to significant tissue atrophy.

[0005] Polymorphisms in the apolipoprotein E gene (APOE) are the major genetic risk determinants for late-onset AD. In the central nervous system (CNS), apolipoprotein E protein (ApoE) is primarily found 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.

[0006] In addition to AD, there are many other neurodegenerative diseases characterized by astrocyte dysfunction, including but not limited to AD-associated gliosis, amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD).

[0007] There is a need for synthetic nucleic acids that can be used as promoters to drive expression primarily in astrocytes, and methods of using them for astrocyte-directed expression of one or more heterologous nucleotide sequences, particularly in the treatment of neurodegenerative diseases. Summary of the Invention

[0008] In order to meet this need, the present disclosure first describes a synthetic nucleic acid that can be used as an expression control element (i.e., a promoter). In some cases, the expression control element (i.e., a promoter) is used for the astrocyte-directed expression of one or more operably connected heterologous nucleotide sequences (i.e., transgenic and / or inhibitory nucleic acids). In some cases, the expression control element (i.e., a promoter) includes a nucleotide sequence with 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., a promoter) is SEQ ID NO: 1.

[0009] In some cases, the synthetic nucleic acid is an expression construct comprising a first nucleotide sequence operably linked to a second nucleotide sequence, wherein the first nucleotide sequence has 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, 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 some cases, the first nucleotide sequence is SEQ ID NO: 1.

[0010] Alternatively, the synthetic nucleic acid is an expression construct comprising a first nucleotide sequence operably linked to a second nucleotide sequence, wherein the first nucleotide sequence has 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, 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 some cases, the first nucleotide sequence is SEQ ID NO: 1.

[0011] In some cases, the synthetic nucleic acid is a vector comprising a first nucleotide sequence operably linked to a second nucleotide sequence, the 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, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes a transgene, and wherein 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 yet other cases, the vector is a viral vector, particularly a recombinant adeno-associated virus (rAAV) vector or a baculovirus vector. In some cases, the first nucleotide sequence is SEQ ID NO: 1.

[0012] Alternatively, the synthetic nucleic acid is a vector comprising a first nucleotide sequence operably linked to a second nucleotide sequence, the 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, wherein the first nucleotide sequence is an expression control element and the second nucleotide sequence encodes an inhibitory nucleic acid, and wherein the vector is a plasmid or viral vector. In other cases, the vector includes a third nucleotide sequence, wherein the third nucleotide sequence encodes a transgene. In yet other cases, the vector is a viral vector, particularly an rAAV vector or a baculovirus vector. In some cases, the first nucleotide sequence is SEQ ID NO: 1.

[0013] Under any of the above, the transgene can be an astrocyte-associated gene, such as APOE2 or another gene associated with gliosis in AD, ALS, or HD.

[0014] Under any of the above, the inhibitory nucleic acid can be directed against an astrocyte-related gene, such as, for example, APOE4 or another gene associated with gliosis in AD, ALS, or HD.

[0015] Secondly, the present disclosure describes compositions comprising synthetic nucleic acids as described herein. In some cases, the composition is an rAAV comprising capsid proteins and synthetic nucleic acids as described herein, including capsid proteins that can cross the blood-brain barrier (BBB). In other cases, the composition is a host cell comprising synthetic nucleic acids or rAAV as described herein. In other cases, the composition is a pharmaceutical composition comprising synthetic nucleic acids or rAAV as described herein and a pharmaceutically acceptable carrier.

[0016] Third, the present disclosure describes a method for preferentially expressing a nucleotide sequence in astrocytes. The method may include providing an effective amount of a synthetic nucleic acid, vector, rAAV or composition as described herein to a cell, tissue, organ or individual.

[0017] Fourth, the present disclosure describes a method for treating a neurodegenerative disease in an individual in need thereof, especially a neurodegenerative disease in which astrocyte directional expression is required. The method can include administering an effective amount of an expression construct, a carrier, rAAV or a composition as described herein to an individual. In some cases, administration is via direct injection into the CNS of an individual, which can be intraventricular (ICV) injection, intracerebellar medullary cisterna (intracisterna magna) (ICM) injection, intraparenchymal injection, intrathecal injection or a combination thereof. In some cases, direct injection is convection enhanced delivery (CED). In other cases, administration is peripheral injection. In some cases, peripheral injection is via intravenous (IV) injection or subcutaneous (SC) injection.

[0018] 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 treating a neurodegenerative disease, particularly a neurodegenerative disease in which astrocyte-directed expression is desired.

[0019] Sixth, the present disclosure describes compositions comprising synthetic nucleic acids or rAAVs as described herein for use in treating neurodegenerative diseases, particularly neurodegenerative diseases in which astrocyte-directed expression is desired.

[0020] An advantage of the expression control element herein is that it is specific for astrocytes and, therefore, can drive expression of heterologous nucleotide sequences, such as transgenes and / or inhibitory nucleic acids, to certain levels in astrocytes, but not other cells in the CNS.

[0021] Another advantage of the expression control element herein is that it can be used to match the endogenous expression of a target gene, such as APOE, in neurodegenerative diseases where there is abnormal astrocyte-associated gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Advantages, effects, features and objects other than those set forth above will become more apparent upon consideration of the following detailed description. Such detailed description refers to the following drawings, in which:

[0023] Figure 1Shown is the expression of codon-optimized ApoE driven by three different expression control elements, i.e., a known promoter (SEQ ID NO: 9 (known promoter)) and one of two potential astrocyte-specific promoters (SEQ ID NO: 1 (first promoter) or 10 (second promoter)) in three different cell lines.

[0024] Figure 2 A-2C shows the expression of green fluorescent protein (GFP) driven by one of three different expression control elements, i.e., a known promoter (SEQ ID NO: 9; known promoter) and two potential astrocyte-specific promoters (SEQ ID NO: 1 (first promoter) or 10 (second promoter)), in three different cell lines. Figure 2 A shows the normalized GFP RNA level in one cell line, U87; Figure 2 B shows normalized GFP RNA levels in a second cell line, HEK293; and Figure 3 C shows the normalized GFP RNA levels in the third cell line SH-SY5Y.

[0025] Figure 3 A and 3B are images from mouse studies using promoters of SEQ ID NOs: 1 and 9, where Figure 3 A shows that SEQ ID NO: 9 weakly drives in vivo expression of enhanced green fluorescent protein (EGFP) in astrocytes of mouse brain, and wherein Figure 3 B shows that SEQ ID NO: 1 drives the in vivo expression of EGFP in astrocytes of mouse brain.

[0026] Figure 4 A and 4B are images from mouse studies using promoters of SEQ ID NOs: 1 and 9, where Figure 4 A shows that SEQ ID NO: 9 drives the in vivo expression of EGFP in neurons of the mouse brain, and wherein Figure 4 B shows that SEQ ID NO: 1 weakly drives in vivo expression of EGFP in neurons of mouse brain. DETAILED DESCRIPTION

[0027] Overview

[0028] APOE is implicated in the development of late-onset AD. APOE has several isoforms. One isoform, APOE2, protects against AD; however, another isoform, APOE4, is associated with an increased risk of developing late-onset AD relative to the more common isoform, APOE3. Homozygous individuals carry two copies of APOE4 (i.e., APOE4 + / +) and compared with heterozygous individuals carrying one copy of APOE4 and one copy of APOE2 or APOE3 (APOE4 + / APOE2 + or APOE4 + / APOE3 + ) are at even greater risk of developing late-onset AD.

[0029] 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)).

[0030] Accumulating evidence suggests that ApoE influences tau pathology, tau-mediated neurodegeneration, and microglial responses to AD-related pathology. Furthermore, ApoE4 is either pathogenic or exhibits reduced efficiency in multiple brain homeostatic pathways, including lipid transport, synaptic integrity and plasticity, glucose metabolism, and cerebrovascular function.

[0031] Astrocyte-directed expression of heterologous nucleotide sequences is therefore of interest in the treatment of neurodegenerative diseases such as AD as well as diseases caused by other astrocyte-related genes.

[0032] Abbreviations and definitions

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods herein, preferred methods and materials are described herein.

[0034] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. The indefinite article "a" or "an" therefore usually means "at least one."

[0035] Furthermore, the use of "include" as well as other forms such as "including but not limited to," "include," "includes," and "included" is not limiting.

[0036] Some abbreviations used in this article are as follows:

[0037] “AAV” refers to 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-carboximidazole (C 16 H 15 N5); “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; “ICM” refers to cisterna magna; “ICV” refers to intracerebroventricular; “IRES” refers to internal ribosome entry site; “ITR” refers to inverted terminal repeat; “IV” refers to intravenous; “kDa” refers to kilodaltons; “min” refers to minute; “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.

[0038] Certain definitions used in this article are as follows:

[0039] As used herein, "about" means within a statistically meaningful range of one or more values, such as a stated concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges may typically be on the order of 20%, more typically 10%, and even more typically 5% of a given value or range. The permissible variations encompassed by "about" will depend on the specific system under study and will be readily apparent to those skilled in the art.

[0040] As used herein, "administer," "administering," "administration," and the like mean providing a substance (e.g., an oligonucleotide herein or a composition herein, such as an rAAV as described herein) to an individual in a pharmacologically useful manner (e.g., to treat a disease, disorder, condition, or symptom in the individual).

[0041] As used herein, "astrocyte-associated gene" means a gene encoding a peptide, polypeptide, or protein that is genetically, biochemically, or functionally equivalent to a gene predominantly expressed in astrocytes. Exemplary astrocyte-associated genes include, but are not limited to, APOE2 and APOE4, and GFAP.

[0042] As used herein, "astrocyte-directed expression" means the expression of a nucleotide sequence of interest encoding a peptide, polypeptide or protein that is predominant in astrocytes compared to other cells in the CNS, such as neurons, including dorsal root ganglia (DRG).

[0043] As used herein, "astrocyte-specific promoter" means a promoter that drives expression of an operably linked nucleotide sequence predominantly in astrocytes compared to other cells in the CNS (such as, for example, neurons, including DRGs).

[0044] As used herein, "codon optimized" means, with respect to a nucleotide sequence such as a gene of interest (e.g., an AD-related gene), a change in the codons or sequences in a gene or coding region thereof to reflect the typical codon usage of a host organism (e.g., a mammal, such as a human) or its cell, without changing the polypeptide encoded by the nucleotide sequence. Codon optimized transgenes are therefore optimized for expression in specific organisms, organs, tissues, or cell types, especially mammals or mammalian organs, tissues, or cell types. Alternatively, "codon optimized" means a change in the codons or sequences in a gene to improve protein expression compared to a sequence lacking a change in a site that may be a potential splice site, a stop codon, a miRNA recognition sequence, or the like, for example, by eliminating or changing the site. The entire nucleotide sequence may be codon optimized, or only one or more parts, parts, or regions of the nucleotide sequence may be codon optimized.

[0045] As used herein, "comparison window" means a contiguous and specified region of a nucleotide sequence or amino acid sequence in which the sequence in the comparison window may include additions and / or deletions (i.e., gaps) compared to a reference sequence (which does not include additions and / or deletions) for optimal alignment of the two sequences. Typically, the comparison window is at least 10 contiguous nucleotides / amino acids in length, and optionally can be 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides / amino acids or longer.

[0046] As used herein, " complementary " means the structural relationship between two nucleotides (for example, on two relative nucleic acids or on the relative region of a single nucleic acid chain), which allows two nucleotides to form base pairs (bp) with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide in a relative nucleic acid in a nucleic acid may be base-paired together by forming hydrogen bonds with each other. Complementary nucleotides can carry out base pairing in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. Similarly, two nucleic acids may have a region of multiple nucleotides that complement each other to form a region of complementarity, as described herein.

[0047] As used herein, "effective amount" means the amount, concentration, or dosage of a therapeutic agent (e.g., a nucleic acid, vector, or rAAV as described herein), or a pharmaceutical composition thereof, which, following single or multiple dose administration to an individual in need thereof, provides the desired effect in such 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 skilled in the art using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, a variety of factors are taken into account, including, but not limited to, the species of mammal, its size, age, and overall health, the specific disease, disorder, condition, or symptom involved, the extent or severity of the disease, disorder, condition, or symptom, the response of the individual, the therapeutic agent administered, the mode of administration, the bioavailability characteristics of the administered formulation, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances.

[0048] As used herein, "expression construct" means a nucleotide sequence that can replicate and express a nucleotide sequence of interest (e.g., a transgenic 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, especially AAV vectors or baculovirus vectors. Here, the expression construct can include at least one expression control element operably linked to a nucleotide sequence of interest, such as a transgenic (and / or inhibitory nucleic acid). In this way, the expression construct can be an expression control element that can interact operably with a transgenic (and / or inhibitory nucleic acid), such as a promoter, which can direct the expression of a transgenic (and / or inhibitory nucleic acid) in a cell, tissue, organ, or individual, especially astrocytes.

[0049] As used herein, "expression control elements" refers to nucleotide sequences such as promoters, polyadenylation signals, transcription or translation termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites (IRES), enhancers, etc., which together 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 to be present at all times, as long as the desired nucleotide sequence can be replicated, transcribed, and translated in the appropriate cell, tissue, organ, or individual.

[0050] As used herein, "in combination with" means administering a therapeutic agent (eg, a nucleic acid, vector, rAAV, or composition as described herein) simultaneously, sequentially, or in a single combined formulation with one or more additional therapeutic agents.

[0051] As used herein, "individual" means any mammal, including cats, dogs, mice, rats and primates, especially humans. In addition, "subject" or "patient" may be used interchangeably with "individual".

[0052] As used herein, "a subject in need thereof" means a mammal, such as a human, having a condition, disease, disorder or symptom for which treatment or therapy is required, including, for example, those listed herein. In particular, the preferred subject to be treated is a human.

[0053] 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, and the like. Here, the inhibitory nucleic acid may be 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).

[0054] As used herein, "nucleoside" means a nucleobase-sugar combination, wherein the nucleobase portion is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The sugar is typically a pentose such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0055] As used herein, "nucleotide" means an organic molecule having a nucleoside (a nucleobase such as adenine, cytosine, guanine, thymine, or uracil; and a pentose such as ribose or 2'-deoxyribose) and a phosphate group, which can serve as the monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0056] As used herein, "oligonucleotide" means a short nucleic acid molecule (eg, less than about 100 nucleotides in length). An oligonucleotide may be single-stranded (ss) or double-stranded (ds).

[0057] As used herein, "operably linked" and the like means that the elements of an expression construct (or other nucleic acid construct) are configured so as to perform their usual functions (i.e., under the influence of an expression control element). 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. The control element need not be contiguous with the desired nucleotide sequence, as long as it functions to direct its expression (i.e., maintains the proper reading frame). Thus, for example, there can be untranslated but transcribed intervening sequences between a promoter and the desired nucleotide sequence, and the promoter can still be considered "operably linked" to the desired nucleotide sequence.

[0058] 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 the therapeutic agent (e.g., a nucleic acid, vector, rAAV, or composition as described herein) and is relatively nontoxic (i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any component of the composition in which it is contained).

[0059] As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transportation of a therapeutic agent in or to an individual, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, so that it may perform its intended function. Additional ingredients that may be included in the 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, 21st edition, University of the Sciences in Philadelphia, PA (2006).

[0060] 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, dispersant, suspending agent, thickener, excipient, and the like.

[0061] As used herein, " polynucleotide " means the polymer of nucleotide. Although it may comprise any type of nucleotide unit, the term is generally applicable to the nucleotide polymer of RNA or DNA. Polynucleotide is used to include ss nucleic acid, ds nucleic acid and RNA and DNA prepared by nucleotide or nucleoside analogs that may be identified by its sequence, and it is generally presented (as coding strand) in the direction of 5' to 3', wherein 5' and 3' indicate the bonding formed between the 5' hydroxyl group of a nucleotide and the 3' hydroxyl group of the next nucleotide. For the coding strand presented in the 5'-3' direction, its complement (or non-coding strand) is the chain that hybridizes with this sequence according to Watson-Crick base pairing. Therefore, as used herein, the complement of nucleic acid such as polynucleotide is identical with " reverse complement ", and describes the nucleic acid that is paired with the nucleic acid base in its native form in question.

[0062] As used herein, "recombinant adeno-associated virus," "recombinant AAV," and "rAAV" mean viral particles comprising a rAAV vector encapsidated by AAV capsid proteins.

[0063] As used herein, "recombinant adeno-associated virus vector," "recombinant AAV vector," and "rAAV vector" refer to polynucleotide vectors comprising one or more heterologous sequences (i.e., nucleic acid sequences that are not of AAV origin) flanked by at least one AAV inverted terminal repeat (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in host cells that have been infected with a suitable helper virus (or a helper virus expressing suitable helper functions) that expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0064] As used herein, "sequence identity" in the context of two nucleotide sequences or two amino acid sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0065] As used herein, "synthetic" means a nucleic acid or other molecule or compound that is artificially modified (i.e., recombinantly produced), or synthesized using a machine such as a solid phase nucleic acid synthesizer, or otherwise not derived from a natural source that normally produces nucleic acids or other compounds (i.e., non-naturally occurring). As used herein, "transgenic" means a nucleotide sequence that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. The transgene confers desired properties to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome. Here, the transgene may be a nucleotide sequence encoding a polypeptide of interest, such as an AD-related gene (e.g., a gene encoding ApoE2).

[0066] As used herein, "treat," "to treat," "treatment," or "treating" means a process in which there may be a slowing, control, delay, or cessation of the progression of a disease or condition disclosed herein, or an amelioration of disease or condition symptoms, but does not necessarily indicate a complete elimination of all disease or condition symptoms. Treatment, among other things, includes the administration of a nucleic acid, expression construct, vector, rAAV, or composition as described herein for the treatment of a disease or condition in an individual, particularly a human.

[0067] As used herein, "vector" means a nucleic acid construct, such as a plasmid, clay or phage, for example, an expression construct herein that is used to introduce / transfer one or more heterologous nucleotide sequences to a target cell. Some vectors can be autonomously replicated (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after introducing the host cell, and thereby replicate together with the host genome.

[0068] As used herein, "viral vector" means a vector derived from a naturally occurring or modified virus, particularly a rAAV vector or a baculovirus vector (eg, Autographa californica nuclear polyhedra (AcNPV) vector).

[0069] Composition

[0070] synthetic nucleic acids

[0071] Expression control elements used as astrocyte-specific promoters: The synthetic nucleic acid can be an expression control element, such as 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, the synthetic nucleic acid used 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 some cases, the nucleotide sequence is SEQ ID NO: 1.

[0072] In other cases, the synthetic nucleic acid is complementary to a nucleotide sequence that has at least about 90% sequence identity to SEQ ID NO: 2. Alternatively, the nucleotide sequence is complementary to a nucleotide sequence that 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: 2.

[0073] The synthetic nucleic acids as described herein may exist alone, or may be present as part of an expression construct, vector, or even rAAV as described herein.

[0074] Expression Constructs: As noted above, synthetic nucleic acids can be incorporated into expression constructs for astrocyte-directed expression of heterologous nucleotide sequences. In some cases, the synthetic nucleic acid used as an expression construct includes 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 a nucleotide sequence for a transgene. In other cases, the synthetic nucleic acid used as an expression construct includes 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 a nucleotide sequence for an inhibitory nucleic acid. In still other cases, the synthetic nucleic acid used as an expression construct includes 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, a nucleic acid sequence for a transgene, and a nucleic acid sequence for an inhibitory nucleic acid. In some cases, the expression element is SEQ ID NO: 1.

[0075] In some cases, the transgene encodes an astrocyte-associated gene. In some cases, the inhibitory nucleic acid is directed against 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.

[0076] The expression constructs as described herein may exist alone, or may be present as part of a vector as described herein or even rAAV.

[0077] Vector: As noted above, the synthetic nucleic acids or expression constructs described herein can be further incorporated into a vector, particularly a viral vector such as an rAAV vector. The rAAV vector may comprise a "positive strand" or a "negative strand" of the rAAV vector. In some cases, the rAAV vector is single-stranded (ss) (e.g., ssDNA or ssRNA). In other cases, the rAAV vector is double-stranded (ds) (e.g., dsDNA or dsRNA).

[0078] In other cases, the vector is a baculovirus vector (eg, an Autographa californica nuclear polyhedrosis (AcNPV) vector).

[0079] A vector, such as an rAAV vector, may include not only an expression control element having a 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 may also include other expression control elements such as nucleotide sequences of at least one or more of a promoter, an enhancer, a transcription factor binding site, a repressor binding site, an intron splice site, a post-transcriptional regulatory element, a polyadenylation signal, and combinations thereof. See, for example, International Patent Application Publication No. WO 2020 / 112802.

[0080] In still other cases, the vector includes at least an expression control element having a 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 for a transgene, and a nucleic acid sequence for an inhibitory nucleic acid.

[0081] In some cases, the transgene encodes an astrocyte-associated gene. In some cases, the inhibitory nucleic acid is directed against 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.

[0082] The vectors as described herein may exist alone, or may exist as part of a rAAV as described herein.

[0083] rAAV

[0084] As noted above, the synthetic nucleic acids, expression constructs, or vectors described herein can be incorporated into rAAV. In some cases, the rAAV may have a capsid protein of 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 from a non-human host, such as, for example, a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39, and the like.

[0085] In some cases, rAAV includes a capsid protein that is a variant of a wild-type capsid protein, wherein such 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) relative to the wild-type AAV capsid protein from which it is derived.

[0086] In some embodiments, rAAV comprises a capsid protein that readily diffuses through the CNS, particularly when introduced into the CSF space or directly into the brain parenchyma. In this manner, such rAAV is 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.

[0087] Methods for producing rAAV are described, for example, in Samulski et al. (1989) J. Viral. 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). rAAV production 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 International Patent Application Publication Nos. WO 2017 / 184879 and WO 2022 / 082017. Alternatively, rAAV can be produced in human embryonic kidney (e.g., HEK293) cells (see, e.g., International Patent Application 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).

[0088] Pharmaceutical composition

[0089] The synthetic nucleic acids described herein (ie, expression constructs or vectors) or the rAAV described herein can be formulated as a pharmaceutical composition comprising the synthetic nucleic acid or rAAV and a pharmaceutically acceptable carrier.

[0090] The pharmaceutical composition can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by 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), administration to a region supplied by the blood and / or lymph, and / or direct administration to the site of the lesion.

[0091] In general, the most appropriate route of administration depends on various factors, including, but not limited to, the nature of the agent (e.g., its stability in the environment in which it is administered and / or its intended target) 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.

[0092] Reagent test kit

[0093] In some cases, the synthetic nucleic acids described herein (i.e., expression constructs or vectors), rAAV described herein, or even other therapeutic oligonucleotides including expression control elements as described herein, may be included in a kit comprising synthetic nucleic acids, rAAV or other therapeutic oligonucleotides and instructions for use thereof. In other cases, the kit includes synthetic nucleic acids, rAAV or other therapeutic oligonucleotides and a package insert containing instructions for use of the kit and / or any of its components. In other cases, the kit includes synthetic nucleic acids, rAAV or other therapeutic oligonucleotides, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art in a suitable container or other means for holding. In some cases, the container contains synthetic nucleic acids, rAAV or other therapeutic oligonucleotides placed therein, and in some cases at least one vial, well, test tube, flask, bottle, syringe, or other container means that is appropriately subpackaged. In those cases where additional components are provided, the kit includes additional containers in which such components are placed. The kit may also include means for accommodating synthetic nucleic acids, rAAV or other therapeutic oligonucleotides and any other reagents in a confined space for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are retained. The container and / or kit may include a label with instructions for use and / or warnings.

[0094] 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 delaying progression of a neurodegenerative disease in an individual in need thereof.

[0095] 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 a synthetic nucleic acid, rAAV or other therapeutic oligonucleotide, and instructions for administering the synthetic nucleic acid, rAAV or other therapeutic oligonucleotide or pharmaceutical composition.

[0096] method

[0097] How to use

[0098] Synthetic nucleic acids, rAAV, other therapeutic oligonucleotides, or pharmaceutical compositions may be used in methods for treating neurodegenerative diseases, wherein such methods comprise at least the step of administering to an individual in need of such treatment an effective amount of a synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition comprising the same.

[0099] In some cases, the synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition is administered via IV injection or SC injection. In other cases, the synthetic nucleic acid, rAAV, other therapeutic oligonucleotide, or pharmaceutical composition is administered directly to the CNS of the individual, for example, by direct injection into the brain and / or spinal cord. Examples of direct CNS modes of administration include, but are not limited to, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing.

[0100] In some cases, direct CNS administration is by convection-enhanced delivery (CED), which involves surgical exposure of the brain and placement of a small diameter catheter directly into a target area of ​​the brain, followed by direct infusion of the therapeutic agent (e.g., a synthetic nucleic acid, rAAV, other therapeutic oligonucleotides, or a pharmaceutical composition as described herein) into the brain. CED is described in Debinski et al. (2009) Expert Rev. Neurother. 9: 1519-1527.

[0101] 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 the APOE4 allele. The individual may be homozygous for APOE4 (e.g., APOE4 + / + ) or heterozygous (e.g., APOE4 + / - ). In some cases, the individual is heterozygous for APOE4, and the individual's second APOE allele may be APOE2 or APOE3.

[0102] In some cases, an effective amount is in the range of about 10 9 genome copies (GC) / kg to about 10 14 GC / kg. In other cases, the titer is about 10 9 GC / kg, about 10 10 GC / kg, about 10 11 GC / kg, about 10 12 GC / kg, about 10 12 GC / kg or about 10 14 GC / kg. In other cases, by injection into the CSF space or by intraparenchymal injection, the titer is >10 12GC / kg.

[0103] In other cases, an effective amount is in the range of about 1 x 10 12 vg to about 1x10 15 vg or about 1x10 13 vg to about 7x10 14 In other cases, the dose is about 3.5 x 10 13 vg, about 7.0x10 13 vg or about 1.4x10 14 In other cases, the dose is about 1x10 14 vg, about 2.0x10 14 vg or about 4.0x10 14 Alternatively, the dose is about 2x10 13 vg, about 3x10 13 vg, about 4x10 13 vg, about 5x10 13 vg, about 6x10 13 vg, about 7x10 13 vg, about 8x10 13 vg, about 9x10 13 vg, about 1x10 14 vg or about 2x10 14 In some cases, the dose is 7.0x10 13 vg or 1.4x10 14 vg.

[0104] In some cases, the subject is between about 1 month and about 10 years of age (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 subject is between about 10 and about 20 years of age (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years, or any age in between). In other cases, the subject is older than 20 years old (e.g., about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any age in between), older than 30 years old (e.g., about 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or any age in between), older than 40 years old (e.g., about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or any age in between), or even older than 50 years old (e.g., about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, or any age in between).

[0105] use

[0106] rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein or pharmaceutical compositions comprising the same can be used for or applicable to treating individuals (e.g., humans) suffering from or suspected of having AD-related diseases. Therefore, rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein or pharmaceutical compositions comprising the same are provided for or applicable to treating individuals suffering from or suspected of having AD-related diseases. Also provided are rAAV or other therapeutic oligonucleotides comprising expression control elements as described herein or pharmaceutical compositions comprising the same for or applicable to the manufacture of a medicament or pharmaceutical composition for treating AD-related diseases.

[0107] Example

[0108] The following non-limiting examples are offered for illustrative and non-limiting purposes.

[0109] In vitro function

[0110] Example 1: Astrocyte-specific expression control element

[0111] Objective: To develop expression control elements (eg, promoters) that can drive astrocyte-specific expression of heterologous nucleotide sequences (eg, transgenes or inhibitory nucleic acids).

[0112] Methods: Two potential astrocyte-specific expression control elements were generated and compared with a known promoter (SEQ ID NO: 9). The first potential expression control element had the nucleotide sequence of SEQ ID NO: 1, while the second potential expression control element had the nucleotide sequence of SEQ ID NO: 10.

[0113] Plasmids expressing the codon-optimized human ApoE2 nucleotide sequence (SEQ ID NO: 11) under the control of one of three expression control elements were synthesized and cloned by Vigene Biosciences.

[0114] 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 the expression in vitro. Cells were transfected in 96-well plates using Invitrogen 2000 (Invitrogen) at a volume to mass ratio of 3:1, with cells 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) as a loading control.

[0115] 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) being slightly lower ( Figure 1 In HEK293 cells, the second expression control element (SEQ ID NO: 10) has lower expression than 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 (a neuroblastoma line), the three expression control elements again drive approximately equal expression, with the second expression control element (SEQ ID NO: 10) having potentially slightly higher expression ( Figure 1 ).

[0116] Function in the body

[0117] Example 2: In vivo study on astrocyte-specific expression of enhanced GFP

[0118] Objective: To evaluate the ability of the astrocyte-specific expression control element of Example 1 in an in vivo environment.

[0119] 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 of SEQ ID NO: 1 and SEQ ID NO: 10) or Prevail Therapeutics (expression control element of SEQ ID: 9). The rAAV had the following nucleotide sequences: SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. The rAAV was injected unilaterally ICV (4.84 x 10 in 4 uL per animal). 10 vg; 6 animals for each of the 3 expression control elements) were administered to newborn (P2) C57BL / 6 mice at Psychogenics, Inc. Animals were euthanized 4 weeks after injection, and tissues were collected for molecular biology and imaging analyses.

[0120] Brain, spinal cord, and liver were fixed for imaging, and GFP fluorescence was visualized with DAPI nuclear counterstain.

[0121] 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.

[0122] Results: AAV using the known expression control element of SEQ ID NO: 9 resulted in widespread expression throughout the brain, almost exclusively in neurons, while AAV using the first expression control element of SEQ ID NO: 1 resulted in roughly equivalent expression; however, expression was largely localized to astrocytes (confirmed by preliminary GFAP counterstaining). In contrast, AAV using the second expression control element of SEQ ID NO: 10 resulted in low-level expression in only a few cells, primarily neurons.

[0123] Expression in the spinal cord was qualitatively different between the first expression control element of SEQ ID NO: 1 and the known expression control element of SEQ ID NO: 9, but was still generally equivalent.

[0124] Expression in the liver appears to be almost identical between the first expression control element of SEQ ID NO: 1 and the known expression control element of SEQ ID NO: 9.

[0125] Regarding the second expression control element of SEQ ID NO: 10, fluorescence was hardly detectable in the liver or spinal cord.

[0126] There were no significant differences in the biodistribution of AAV across all tissues (i.e., brain, liver, and spinal cord). In fact, 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 were up to 10-fold lower than the second expression control element of SEQ ID NO: 10.

[0127] 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, and the identical viral capsid used for all constructs, the differences in expression patterns may be due to differences in the effectiveness of the expression control elements to act as promoters. Expression from the first expression control element of SEQ ID NO: 1 was predominantly in astrocytes, which was not achieved using the second expression control element of SEQ ID NO: 10 ( Figure 2 A-2C). Surprisingly, these in vivo data could not be predicted based on the in vitro data, in which expression in a neuronal-like cell line (SH-SY5Y) and an astrocyte-like cell line (U87) was similar across all three expression control elements.

[0128] Example 3: In vivo study on astrocyte-specific expression of GFP

[0129] Objective: To further evaluate the ability of the astrocyte-specific expression control element of Example 1 in an in vivo environment.

[0130] 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, 9, or 10 was administered by ICV injection to mixed-sex C57BL / 6 mice at P2. rAAV was expressed at 1.21 x 10 13 vg / mL was used for 4.84x10 10The total dose of vg / animal was 400 mg / day. After 30 days of ICV administration, animals were euthanized and tissues were collected and fixed for immunohistochemical analysis or quick-frozen for molecular biological analysis. The brain, spinal cord, and liver were stained with antibodies against GFAP to identify astrocytes, and stained with antibodies against PNM to identify neurons, and co-imaged with native GFP fluorescence to determine cell expression. Biodistribution and GFP mRNA expression in the cortex, spinal cord, and liver were analyzed by qPCR at Prevail Therapeutics (New York, NY).

[0131] Results: The expression construct under the control of SEQ ID NO: 9 weakly drives the expression of EGFP in vivo in astrocytes of mouse brain ( Figure 3 A). In contrast, an expression construct under the control of SEQ ID NO: 1 drives in vivo expression of EGFP in astrocytes of mouse brain ( Figure 3 B). However, the expression construct under the control of SEQ ID NO: 9 drives in vivo expression of EGFP in neurons of the mouse brain ( Figure 4 A). In contrast, an expression construct under the control of SEQ ID NO: 1 weakly drives in vivo expression of EGFP in neurons of the mouse brain ( Figure 4 B).

[0132] Sequence Listing

[0133] The following nucleotide and / or amino acid sequences are mentioned in the disclosure above and are provided below for reference.

[0134] SEQ ID NO: 1 - Synthetic nucleic acid 1 (603 nt)

[0135]

[0136] SEQ ID NO: 2—Synthetic nucleic acid 2 (603 nt; complementary sequence of SEQ ID NO: 1)

[0137]

[0138] SEQ ID NO:3-human APOE2mRNA (1234nt; NCBI Ref.Seq.No.NM_000041.3)

[0139]

[0140]

[0141] SEQ ID NO:4—Human ApoE2 protein (317aa)

[0142]

[0143] SEQ ID NO:5-human APOE3mRNA (1144nt; NCBI Ref.Seq.No.NM_001302689.2)

[0144]

[0145]

[0146] SEQ ID NO:6—Human ApoE3 protein (317aa)

[0147]

[0148] SEQ ID NO:7-human APOE4mRNA (1265nt; NCBI Ref.Seq.No.NM_001302690.1)

[0149]

[0150] SEQ ID NO:8—Human ApoE4 protein (317aa)

[0151]

[0152] SEQ ID NO:9—Synthetic nucleic acid 3 (658 nt)

[0153]

[0154] SEQ ID NO:10—Synthetic nucleic acid 4 (228 nt)

[0155]

[0156] SEQ ID NO:11—Synthetic nucleic acid 5 (954 nt)

[0157]

[0158]

[0159] SEQ ID NO:12—Enhanced green fluorescent protein (720 nt)

[0160]

[0161] SEQ ID NO:13 - CAPO-GFP AAV expression construct / vector (7351 nt)

[0162]

[0163]

[0164]

[0165]

[0166] SEQ ID NO:14—CBA-GFP expression construct / vector (7313 nt)

[0167]

[0168]

[0169]

[0170] SEQ ID NO:15—ApoP-GFP expression construct / vector (7047 nt)

[0171]

[0172]

[0173]

Claims

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

1. The promoter according to claim 1 , wherein the nucleotide sequence is SEQ ID NO:

1.

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

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

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

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

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

8. The synthetic nucleic acid of claim 4, wherein the second nucleotide sequence is an inhibitory nucleic acid, and wherein 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. The synthetic nucleic acid according to claim 9 or 10, wherein the first AD-related gene is APOE2.

12. The synthetic nucleic acid according to claim 10 or 11, wherein the second AD-related 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. The vector of claim 14, wherein the vector is an AAV vector, and wherein the vector further comprises a nucleotide sequence for 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 poly A tail, and a repressor binding site, and combinations thereof.

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

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

18. A pharmaceutical composition comprising: (i) the synthetic nucleic acid of any one of claims 3 to 12, the vector of any one of claims 13 to 15, or the rAAV of claim 16 or 17; and (ii) a pharmaceutically acceptable carrier.

19. A method of treating an individual suffering from or suspected of suffering from a neurodegenerative disease, the method comprising the steps of: An effective amount of the synthetic nucleic acid according to any one of claims 3 to 12, the vector according to any one of claims 13 to 15, the rAAV according to claim 16 or 17, or the pharmaceutical composition according to claim 18 is administered to the individual.

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

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

22. A method for expressing a nucleic acid of interest in astrocytes, the method comprising the steps of: A vector comprising a promoter comprising SEQ ID NO: 1 operably linked to a nucleic acid of interest is introduced into a cell, tissue, organ or individual.

23. The method of claim 22, wherein the nucleic acid of interest is a transgene for an Alzheimer's disease (AD)-associated gene. The method according to claim 23 , wherein the AD-related gene is APOE2.

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

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

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

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

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

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

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