Nucleic acid regulation of APOE

Abstract

The present invention relates to nucleic acids comprising two RNA coding sequences, each of the RNAs comprising a hairpin and a guide sequence substantially complementary to a portion of the APOE gene, and related AAVs, compositions, pharmaceutical compositions and therapeutic uses thereof.

Description

[Technical field]

[0001] The present invention relates to the fields of biotechnology, medicine and gene therapy. The present invention relates to nucleic acids comprising two or more RNA coding sequences that include guide sequences substantially complementary to portions of the APOE gene, related compositions, pharmaceutical compositions and their therapeutic uses. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is believed to be the leading cause of dementia in adults (Van Cauwenberghe et al. 2016, Genet Med.;18:421:30). The disease is characterized by the accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles that cause neuronal cell death and glial cell activation, ultimately resulting in a decline in brain function. The formation of amyloid plaques is due to the cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The formation of neurofibrillary tangles is instead due to the misfolding of tau protein (Pospich&Raunser 2017, Science;358:45-46).

[0003] One of the genetic factors that increases the risk of developing AD concerns the human apolipoprotein E (APOE) gene. The human APOE gene has single nucleotide polymorphisms (SNPs) that generate three major allelic variants: ε2, ε3, and ε4 (i.e., APOE isoforms E2, E3, and E4) (Belbin et al. 2007, Hum Mol Genet.;16:2199-208). The ε4 variant is involved in late-onset AD (LOAD), and carriers of APOE ε4 have an increased risk of developing AD in an allele-number-dependent manner, i.e., having one APOE ε4 allele leads to an average age at onset 2-5 years earlier, whereas the presence of two APOE ε4 alleles leads to an onset 5-10 years earlier. Importantly, 40-65% of AD patients carry at least one APOE ε4 allele. Furthermore, APOE4 has also been observed to be associated with adverse outcomes in atherosclerosis, traumatic brain injury (TBI), and other diseases. In contrast, APOEε2 and / or APOEε3 alleles exert protective or neutral effects against the development of AD (Yamazaki et al. 2016, CNS Drugs.;30:773-89).

[0004] The APOE protein, encoded by the APOE gene, is a secreted lipid transport protein found in peripheral and central body systems, such as the central nervous system (CNS). In the periphery, APOE is primarily secreted by hepatocytes, whereas in the CNS, astrocytes are the primary source of APOE (Chernick et al. 2019, Neurosci Lett.;708:134306).

[0005] SNPs within the APOE gene induce differences in amino acid residues located at positions 130 and 176, also called positions 112 and 158, respectively, when the signal peptide of the protein is excluded (APOEε2, Cys112 / Cys158; APOEε3, Cys112 / Arg158; APOEε4, Arg112 / Arg158). These single amino acid polymorphisms are believed to have profound effects on the structure and function of APOE, thereby affecting Aβ metabolism, aggregation, deposition, and tau phosphorylation. (Liu et al. 2013, Nat Rev Neurol.; 9:106-18). Summary of the Invention [Problem to be solved by the invention]

[0006] Given the complex pathophysiology of AD and the associated gene regulatory networks, there are currently no promising therapies for treating AD, and therefore there is an urgent need to find such therapies. [Means for solving the problem]

[0007] The present invention solves the problem by applying nucleic acid technology, where the nucleic acid comprises two or more RNA coding sequences, one of which contains a guide sequence substantially complementary to a portion of the APOE gene, and the other supports expression of the first, possibly through clustering, thus increasing the silencing of the APOE gene.

[0008] In one aspect there is provided an expression cassette comprising a nucleic acid according to the invention, wherein the expression cassette is a DNA molecule.

[0009] In one aspect, an adeno-associated virus (AAV) vector is provided comprising an expression cassette according to the invention.

[0010] In one aspect, there is provided a pharmaceutical composition comprising a nucleic acid according to the invention, an expression cassette according to the invention, or an AAV vector according to the invention and at least one pharma- ceutically acceptable excipient.

[0011] In one aspect there is provided a pharmaceutical composition according to the invention, a nucleic acid according to the invention, or an expression cassette according to the invention, or an AAV vector according to the invention for use as a medicament.

[0012] In one aspect there is provided a kit comprising a nucleic acid according to the invention, or an expression cassette according to the invention, or an AAV vector according to the invention, or a pharmaceutical composition according to the invention, the kit further comprising an immunosuppressant.

[0013] In one aspect, there is provided an expression cassette comprising a nucleic acid encoding one or more APOE2 and APOE3 proteins selected from SEQ ID NOs:249-254 for use in gene therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Description of the Invention definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. Indeed, the present invention is in no way limited to this method.

[0015] In this specification and the claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of the elements is present, unless the context clearly requires that one and only one of the element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0016] For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtained". Hereinafter, if for example an antibody is defined as being obtainable from a particular source, this should be understood to also disclose the antibody obtained from this source.

[0017] As used herein, the term "and / or" indicates that one or more of the stated instances may occur alone or in combination with at least one of the stated instances, and up to all of the stated instances.

[0018] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15... etc.

[0019] The terms "about" or "approximately" when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be a given value, more or less than 0.1% of the value (10).

[0020] As used herein, "effective amount" refers to the amount of drug required to improve the symptoms of a disease compared to untreated patients. The effective amount of an active agent used to practice the present invention, for example for the therapeutic treatment of cancer, will vary depending on the mode of administration, the age, weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount and may be determined as genome copies per kilogram (GC / kg). Thus, in the context of this disclosure, in the context of administering a drug that is "effective against" a disease or condition indicates that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant proportion of patients, such as amelioration of symptoms, cure, reduction of at least one disease sign or symptom, prolongation of life, improvement in quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of a particular type of disease or condition.

[0021] The use of a substance as a medicament as described herein can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. The products for use as medicaments described herein can be used in a method of treatment, such a method of treatment comprising the administration of the product for use.

[0022] The terms "homology", "sequence identity" and the like are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as may be determined by the match between strings of such sequences. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

[0023] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptides or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences can be called "substantially identical" or "essentially similar" when they share at least a certain minimum percentage of sequence identity (defined below) (e.g., when optimally aligned by the programs GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, a global alignment is appropriately used to determine sequence identity. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and score for percent sequence identity can be determined using computer programs such as GCG Wisconsin Package, Version 10.3 available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open source software such as the programs "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP described above, or using default settings (for both "needle" and "water", and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5, and the default scoring matrix is ​​Blossum62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments such as those using the Smith Waterman algorithm are preferred.

[0024] Alternatively, the percentage of similarity or identity can be determined by searching against public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTn) can be used. See the National Center for Biotechnology Information homepage (http: / / www.ncbi.nlm.nih.gov / ).

[0025] As used herein, the terms "selectively hybridizing", "selectively hybridizing" and similar terms are intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other, i.e., such hybridizing sequences may share at least 45%, at least 50%, at least 55%, at least 60%, at least 65, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% sequence identity.

[0026] A preferred, non-limiting example of such hybridization conditions is hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 1x SSC, 0.1% SDS at about 50°C, preferably about 55°C, preferably about 60°C, and even more preferably about 65°C.

[0027] Highly stringent conditions include, for example, hybridization in 5xSSC / 5xDenhardt's solution / 1.0% SDS at about 68°C and washing in 0.2xSSC / 0.1% SDS at room temperature. Alternatively, washing may be performed at 42°C.

[0028] Those of skill in the art will know what conditions to apply for stringent and highly stringent hybridization conditions. Further guidance regarding such conditions can be found, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 rd Current Protocols in Molecular Biology, (John Wiley & Sons, NY), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, are readily available in the art.

[0029] Of course, polynucleotides used to specifically hybridize to portions of the nucleic acids of the invention would not include polynucleotides that hybridize only to poly-A sequences (such as the 3' terminal poly(A) region of an mRNA) or only to complementary stretches of T (or U), since such polynucleotides will hybridize to any nucleic acid molecule containing a poly(A) stretch or its complement (e.g., virtually any double-stranded cDNA clone).

[0030] A "nucleic acid construct" or "nucleic acid vector" is understood herein to mean an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may include (parts of) naturally occurring nucleic acid molecules. A "vector" is a nucleic acid construct (typically DNA or RNA) that serves to transfer an exogenous nucleic acid sequence (i.e. DNA or RNA) into a host cell. The vector is preferably maintained in the host by at least one of autonomous replication and integration into the genome of the host cell. The term "expression vector" or "expression construct" refers to a nucleotide sequence that can affect the expression of a gene in a host cell or host organism that is compatible with such a sequence. These expression vectors typically contain at least one "expression cassette", which is a functional unit that can affect the expression of a sequence that codes for an expressed product, the coding sequence being operably linked to at least a suitable expression control sequence that includes a suitable transcription control sequence and optionally a 3' transcription termination signal. Further factors necessary or useful for affecting expression may also be present, such as expression enhancer elements. The expression vector can be introduced into a suitable host cell to affect expression of the coding sequence in an in vitro cell culture of the host cell. Preferred expression vectors are suitable for expressing viral proteins and / or nucleic acids, in particular recombinant AAV proteins and / or nucleic acids.

[0031] As used herein, the term "promoter" or "transcriptional control sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream in the direction of transcription of the transcription start site of the coding sequence, and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequence of nucleotides known to those of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer or biological entity.

[0032] The term "reporter" can be used interchangeably with marker, but is primarily used to refer to a visible marker such as green fluorescent protein (GFP) or luciferase.

[0033] The terms "protein" and "polypeptide" are used interchangeably and refer to a molecule made up of a chain of amino acids, without reference to a particular mode of action, size, three-dimensional structure, or origin.

[0034] The term "gene" refers to a DNA fragment that contains a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell and is operably linked to appropriate control regions (e.g., a promoter). A gene will usually contain several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) that contains a polyadenylation site. "Expression of a gene" refers to the process by which a DNA region operably linked to appropriate control regions, particularly a promoter, is transcribed into RNA that is biologically active, i.e., can be translated into a biologically active protein or peptide.

[0035] The term "homologous", when used to indicate the relationship of a given (recombinant) nucleic acid or polypeptide molecule to a given host organism or host cell, is understood to mean that in nature, the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same type or strain. When homologous to a host cell, the nucleic acid sequence encoding the polypeptide is typically (but not necessarily) operably linked to another (heterologous) promoter sequence than in its natural environment, and, if applicable, another (heterologous) secretion signal sequence and / or terminator sequence. It is understood that control sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloning" genetically modified organisms (GMOs) (self-cloning is defined herein as in Annex II of the European Directive 98 / 81 / EC). When used to indicate the relationship of two nucleic acid sequences, the term "homologous" means that one single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization will depend on many factors, including the amount of identity between the sequences and the hybridization conditions, such as temperature and salt concentration, as discussed below.

[0036] The terms "heterologous" and "exogenous", when used in reference to a nucleic acid (DNA or RNA) or protein, refer to a nucleic acid or protein that is not naturally occurring as part of the organism, cell, genome or DNA or RNA sequence in which it is found, or that is found in a location in a cell or genome or DNA or RNA sequence different from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell in which they are introduced, but are obtained from another cell or are synthetically or recombinantly produced. Generally, although not necessarily, such nucleic acids code for proteins that are not normally produced by the cell in which the DNA is transcribed or expressed, i.e., exogenous proteins. Similarly, exogenous RNA codes for proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous / exogenous nucleic acids and proteins are sometimes referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is encompassed herein by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the combined sequences are heterologous to each other.

[0037] As used herein, the term "non-naturally occurring" when used with respect to an organism means that the organism has at least one genetic change that is not normally found in naturally occurring strains of the referenced species, including wild-type strains of the referenced species. Genetic changes include, for example, modifications that introduce expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruptions of the organism's genetic material. Such modifications include, for example, coding regions of heterologous or homologous polypeptides of the referenced species and functional fragments thereof. Further modifications include, for example, non-coding control regions where the modifications alter the expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes or functional fragments thereof can confer biochemical reaction capabilities or metabolic pathway capabilities to a non-naturally occurring organism that has been altered from its naturally occurring state.

[0038] As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0039] An expression control sequence is "operably linked" to a nucleotide sequence if the expression control sequence controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, expression control sequences can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons.

[0040] The term "expression control sequence" is intended to include at least a sequence whose presence is designed to affect expression, and may also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term may also include the design of a nucleic acid sequence such that undesirable potential initiation codons in or out of frame are removed from the sequence. It may also include the design of a nucleic acid sequence such that undesirable potential splice sites are removed. This includes sequences that direct the addition of a series of adenine residues at the 3' end of the mRNA, or polyadenylation sequences (pA), sequences called polyA sequences. They may also be designed to increase mRNA stability. Expression control sequences that affect transcriptional and translational stability, such as promoters, as well as sequences that affect translation, such as Kozak sequences, are known in insect cells. Expression control sequences may be of such a nature that they regulate the nucleotide sequence to which they are operably linked, so that lower or higher levels of expression are achieved.

[0041] As used herein, the term "codon optimization" refers to an experimental approach designed to improve the codon composition of a recombinant gene based on various criteria without changing the amino acid sequence. This is possible because most amino acids are coded for by more than one codon. Most codon optimization approaches avoid the use of rare codons. However, different approaches vary in the extent to which other features are considered, including mRNA elements that can inhibit expression, the nucleotide context of the start codon, mRNA secondary structure, sequence repeats, nucleotide composition, internal ribosome entry sites, promoter sequences, and putative splice donor and acceptor sites. In addition, some programs take into account protein structural information, intragenic poly(A) sites, stop codons in alternative reading frames, and dinucleotides that are targets for RNase cleavage, mutation, and methylation-dependent gene silencing. Those skilled in the art understand the requirements necessary to design such codon-optimized nucleic acids.

[0042] The present inventors set out to develop nucleic acids comprising RNA coding sequences for modifying APOE expression in cells. The human APOE gene is associated with an increased risk of developing AD. In particular, the ε4 variant is involved in late-onset AD (LOAD), and APOE ε4 carriers increase the risk of developing AD in an allele-number-dependent manner, i.e., having one APOE ε4 allele leads to an average age of onset 2-5 years earlier, whereas the presence of two APOE ε4 alleles leads to an onset 5-10 years earlier. Importantly, 40-65% of AD patients carry at least one APOE ε4 allele. In contrast, APOE ε2 and / or APOE ε3 alleles exert a protective or neutral effect on AD development. Thus, reducing RNA expression levels aims at reducing at least the neuropathology associated with APOE4 expression. Use of the gene therapy approaches outlined herein will significantly benefit affected human patients by thereby reversing, preventing, slowing the progression, or completely halting neuropathology.

[0043] In a first aspect of the invention, there is provided a nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, the second RNA comprising a guide sequence of at least 19 nucleotides substantially complementary to a portion of the APOE gene, and wherein the first RNA and the second RNA each comprise a hairpin.

[0044] RNA The term "nucleic acid" as used herein takes its ordinary meaning in the art. Thus, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" as used herein refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30 or more ribonucleotides), and the term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" as used herein refers to a polymer of deoxyribonucleotides. DNA and RNA can be naturally synthesized (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis, where ribosomes bind to the mRNA.

[0045] As used herein, the term "small interfering RNA" ("siRNA") refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. Preferably, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating RNA interference. Preferably, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term refers to siRNAs that contain about 21 nucleotides (or nucleotide analogs), such as 19, 20, 21, or 22 nucleotides. The term "long" siRNAs refers to siRNAs that contain about 24-25 nucleotides, such as 23, 24, 25, or 26 nucleotides. Short siRNAs may contain fewer than 19 nucleotides, such as 16, 17, or 18 nucleotides, in some cases, so long as the shorter siRNA retains the ability to mediate RNAi. Similarly, long siRNAs may contain more than 26 nucleotides in some cases, so long as the longer siRNA retains the ability to mediate RNAi without further processing, such as enzymatic processing, to short siRNAs.

[0046] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (e.g., viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA that direct the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be induced, for example, to silence the expression of a target gene. Double-stranded RNA structures suitable for inducing RNAi are well known in the art. For example, small interfering RNA (siRNA) can induce RNAi. siRNA comprises two separate RNA strands, one strand comprising a first RNA sequence and the other strand comprising a second RNA sequence, thus comprising a first and a second strand. Frequently used siRNA designs comprise consecutive base pairs with a 3' overhang. The first and / or second strand may comprise a 3'-overhang. The 3'-overhang is preferably a dinucleotide overhang on both strands of the siRNA. Such designs are based on observed Dicer processing of larger double-stranded RNAs resulting in siRNAs with these characteristics. A 3'-overhang may be included in the first strand. A 3'-overhang may be added to the first strand. The length of the duplex from which the siRNA is composed may be 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides or more.

[0047] siRNA can also function as a dicer substrate. For example, the dicer substrate can be a 27-mer consisting of a double strand of RNA with 27 consecutive base pairs. The first strand is located at the 3' end of the 27-mer duplex. As with siRNA, at the 3' end, each or one of the strands can include a two nucleotide overhang. The 3'-overhang can be included in the first strand. The 3' overhang can be added to the first strand. 5' from the first strand, additional sequences can be included that are adjacent or non-adjacent to the target RNA sequence that are complementary. The other end of the siRNA dicer substrate is blunt-ended. This dicer substrate design can favor processing by dicer so that siRNA can be formed like the above siRNA design with 19 consecutive base pairs and two nucleotide overhangs at both 3' ends. In either case, the siRNA etc. is composed of two separate RNA strands (Fire et al. 1998, Nature. Feb 19;391(6669):806-11), each RNA strand comprising or consisting of a first and second RNA strand according to the invention. Thus, the nucleic acid of the invention may be said to induce a first and second RNA strand, which is a first RNA, and a third and fourth RNA strand, which is a second RNA. Alternative naming conventions for each of the first, second, third and fourth RNA strands are within the scope of the invention, and may be complementary, substantially complementary or unique to each other, or in any other required arrangement as discussed herein.

[0048] The loop sequence may be a stem-loop sequence, which extends the double-stranded region of the shRNA.Similar to the above siRNA dicer substrate, the shRNA can be processed, for example, by dicer to provide an siRNA with the above siRNA design, for example, with 19 consecutive base pairs and 2 nucleotide overhangs at both 3' ends.When the shRNA is processed by dicer, it is preferred to have a first and second strand at the end of the shRNA, i.e., the putative strands of the siRNA are linked via a stem-loop sequence: 5'-first strand-apical loop sequence-second strand-optional 2nt overhang sequence-3'.Or conversely, 5'-second strand-apical loop sequence-first strand-optional 2nt overhang sequence-3'. Another shRNA design can be a shRNA structure that is processed by the RNAi machinery to provide an activated RISC complex that does not require Dicer processing (Liu et al., Nucleic Acids Res. 2013, Apr 1, 41(6):3723-33, incorporated herein by reference), the so-called Ago shRNA or Ago2 processed shRNA, which is based on a structure very similar to the miR-451 scaffold described below. Such shRNA structures contain a portion of the first RNA sequence in its loop sequence. Such shRNA structures can also consist of a first strand followed by a second strand.

[0049] In one embodiment, a nucleic acid is provided in which the sequence encoding the first RNA is followed by a spacer and a sequence encoding the second RNA, the spacer being at least 50 nucleotides.Thus, preferably, in the 5' to 3' direction, the sequence encoding the first RNA is followed by a first spacer comprising at least 50 nucleotides, which is followed by the sequence encoding the second RNA.The 5' to 3' direction is understood to refer to the coding strand in the case of a ds nucleic acid.It has been found by chance that the use of a spacer as described herein allows downstream processing of the sequence encoding the first RNA and the second RNA, allowing their combined effect. As mentioned above, the nucleic acid may be said to induce a first and second RNA strand, which is a first RNA, and a third and fourth RNA strand, which is a second RNA, where the sequences encoding the first and second RNA strands are followed by a spacer, and a sequence encoding the third and fourth RNA strands, where the spacer is at least 50 nucleotides, such as at least 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 or 130 nucleotides. In some embodiments, the spacer is 60-130 nucleotides. In a preferred embodiment, the spacer is 90-105 nucleotides. In a more preferred embodiment, the spacer is 92 nucleotides. In some embodiments of the invention, the spacer comprises SEQ ID NO: 237. Thus, the first and second RNAs may also be known as an RNA cluster when transcribed from physically adjacent genes. In further embodiments, the relevant above-mentioned shRNA structures are also applicable when one or both RNAs are shRNAs processed by Dicer. In an alternative embodiment, when one or both RNAs are AgoshRNAs, the related shRNA structures described above are also applicable. Thus, one or both RNAs can be processed by the same or different RNAi machinery, such as Dicer and / or Ago2 of the RNAi machinery.In a preferred embodiment, the first RNA is processed by Dicer, so that the putative strand of the subsequent siRNA is linked via a stem-loop sequence: 5'-first strand-apical loop sequence-second strand-optional 2nt overhang sequence-3' or vice versa: 5'-second strand-apical loop sequence-first strand-optional 2nt overhang sequence-3'. As shown in the examples, the first and second strands of the invention can preferably be incorporated into a pre-miRNA or pri-miRNA scaffold derived from a pri-miRNA or a pre-miRNA scaffold derived from miR-144 (SEQ ID NO: 235). In a preferred embodiment, the third and fourth strands of the invention are incorporated into a pri-miRNA or pre-miRNA scaffold derived from miR-451 (SEQ ID NO: 190). Thus, the first and / or second RNA can be described as a hairpin or double-stranded RNA substantially complementary to itself. In a more preferred embodiment, the first RNA comprises SEQ ID NO: 235 (miR-144) or a variant thereof.

[0050] In some embodiments of the present invention, where the first RNA is incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-144, the first RNA is mutated to reduce the processing and / or expression of the first RNA. In some specific embodiments, SEQ ID NO: 235 or a variant thereof is mutated to reduce the processing and / or expression of the first RNA. In some embodiments, the mutation is a single point mutation. In other words, the first RNA comprises a single point mutation to reduce the processing and / or expression of the first RNA. In some preferred embodiments, the single point mutation is at AT position 19 (SEQ ID NO: 236). The skilled artisan can easily determine whether this is the case by using standard assays and appropriate controls as described in the Examples and known in the art.

[0051] Thus, in some embodiments, the first RNA is processed by Dicer and the second RNA is processed by Ago2, such as AgoshRNA or miR-451 mimicking RNA, which may be said to contain a portion of the second RNA sequence in its loop sequence. Such shRNA structures may also consist of a third strand followed by a fourth strand of the invention.

[0052] The double-stranded RNA according to the present invention can also be incorporated into a pre-miRNA or pri-miRNA scaffold. MicroRNAs, i.e. miRNAs, are guide strands derived from double-stranded RNA molecules that are endogenously expressed, for example, in mammalian cells. Two or more miRNAs can also be included in a miRNA cluster, where the miRNAs in the cluster are transcribed in the same direction and are not separated by transcription units or miRNAs of opposite orientation. miRNAs are processed from pre-miRNA precursor molecules by the RNAi machinery, similar to the processing of shRNAs or extended siRNAs described above, and incorporated into an activated RNA-induced silencing complex (RISC) (Tijsterman M, Plasterk RH. Dicers at RISC; the mechanism of RNAi. Cell. 2004 Apr2; 1 17(1): 1-3). Pre-miRNAs are hairpin RNA molecules that can be part of a larger RNA molecule (pri-miRNA) contained, for example, in an intron, which is first processed by Drosha to form a pre-miRNA hairpin molecule. In one embodiment, the hairpin in the second RNA comprises at least 40 nucleotides. The pre-miRNA molecule is an shRNA-like molecule that can be subsequently processed by Dicer or Ago2 to yield an siRNA-like double-stranded RNA duplex (see Figures 2A and B).

[0053] The miRNA, which is part of the double-stranded RNA duplex, i.e., the guide strand, is then incorporated into RISC. In one embodiment, the second RNA comprises a guide sequence of at least 19 nucleotides, e.g., 20, 21, 22, 23, 24, 25 or 26 nucleotides, substantially complementary to a portion of the APOE gene. In a further embodiment, the second RNA comprises a guide sequence of at least 22 nucleotides, substantially complementary to a portion of the APOE gene, and preferably, the guide sequence is specific to a portion of the APOE4 gene. It has been fortuitously determined that the length of the guide sequence as described herein is the optimal length specific to a portion of the APOE4 gene to have the desired effect. A shorter sequence may result in off-target effects. Naturally occurring RNA molecules, i.e., pri-miRNA, pre-miRNA or miRNA duplex, may be used as a scaffold to create artificial miRNAs that specifically target selected genes. Based on the predicted RNA structure of a naturally occurring RNA molecule, for example, based on the predicted RNA structure of an RNA molecule predicted using m-fold software using standard settings (Zuker. Nucleic Acids Res. 31(13), 3406-3415, 2003), the naturally occurring miRNA sequence present in the RNA structure (i.e., duplex, pre-miRNA or pri-miRNA) and the sequences present in the structure substantially complementary thereto are removed and replaced with the first and second strands according to the invention, i.e., the first and second strands of the second RNA, which may also be referred to as the first and second strands of the first RNA, or the third and fourth strands. Thus, when the first and second strands are used for merely exemplary purposes, the first and second strands are preferably selected such that the predicted secondary RNA structure formed, i.e., the pre-miRNA, pri-miRNA and / or miRNA duplex, resembles the corresponding predicted original secondary structure of the naturally occurring RNA sequence.Pre-miRNA, pri-miRNA and miRNA duplexes (consisting of two separate RNA strands hybridized through complementary base pairing), as found in nature, are often not perfectly base-paired, i.e., not all nucleotides corresponding to the first and second strands as defined above are base-paired, and the first and second strands are often not the same length. Methods for using miRNA precursor molecules as scaffolds for any selected target RNA sequence and a substantially complementary first strand are described, for example, in Liu YP Nucleic Acids Res.2008 May;36(9):281 1-24, which is incorporated herein by reference.

[0054] Pri-miRNAs can be processed by the RNAi machinery of cells. Pri-miRNAs contain flanking sequences at the 5' and 3' ends of the pre-miRNA hairpin and / or shRNA-like molecule. Such pri-miRNA hairpins can be processed by Drosha to produce pre-miRNAs. The length of the flanking sequences can vary, but can be about 80 nt long (Zeng and Cullen, J Biol Chem. 2005 Jul 29, 280(30):27595-603; Cullen, Mol Cell. 2004 Dec 22, 16(6):861-5). The minimum length of the single-stranded flanks can be easily determined as if they are too short, the RNA molecule may lose its function, for example because Drosha processing fails and sequence-specific inhibition is reduced or absent. In one embodiment, a pri-miRNA scaffold carrying the first and second strands according to the invention has 5' and 3' sequence flanks to the predicted pre-miRNA structure of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides. Preferably, the flanking sequences (5' and 3') from the pri-miRNA contained in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence. Preferably, the flanking sequences (5' and 3') and / or loop sequences from the pre-miRNA and / or pri-miRNA contained in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence. Since the (putative) guide strand RNA contained in the endogenous miRNA sequence may be replaced by a sequence comprising (or consisting of) the first strand and the passenger strand sequence may be replaced by a sequence comprising (or consisting of) the second strand, it is understood that the flanking and / or loop sequences of the pri-miRNA sequence or pre-miRNA sequence of the endogenous sequence may contain minor sequence modifications such that the predicted structure (e.g., M-fold predicted structure) of the scaffold miRNA sequence is the same as the predicted structure of the endogenous miRNA sequence.

[0055] The first and second strands and the third and fourth strands are encoded by expression cassettes, for example, to form two double-stranded RNAs, i.e., the first and second RNAs of the present invention. It is understood that if the double-stranded RNAs are, for example, two siRNAs, each consisting of two strands, two or more expression cassettes are required. If each double-stranded RNA is contained in a single RNA molecule, for example encoding an shRNA, a pre-miRNA, or a pri-miRNA, one expression cassette per RNA molecule may be sufficient. The pol II expression cassette may include a promoter sequence that includes a sequence encoding the RNA to be expressed followed by a polyadenylation sequence. If the expressed double-stranded RNA includes a pri-miRNA scaffold, the encoded RNA sequence may code for intron and exon sequences as well as 3'-UTR and 5'-UTR. The pol III expression cassette generally includes a promoter sequence followed by a sequence encoding the RNA, for example an shRNA sequence, a pre-miRNA, or a strand of a double-stranded RNA included in an siRNA or five extended siRNAs. The pol I expression cassette may comprise a pol I promoter followed by an RNA coding sequence and a 3'-sequence. Expression cassettes for double-stranded RNA are well known in the art and any type of expression cassette may suffice, for example, a pol III promoter, a pol II promoter or a pol I promoter may be used (iater Brake et al., Mol Ther. 2008 Mar;16(3):557-64, Maczuga et al., BMC Biotechnol. 2012 Jul24;12:42). In one embodiment, the expression cassette is a DNA molecule. Such DNA molecules may be useful for the application of further techniques and applications providing vectors for the nucleic acids described herein.

[0056] As is clear from the above, the first and second strands, and therefore the third and fourth strands, contained in the double-stranded RNA can contain additional nucleotides and / or nucleotide sequences. The double-stranded RNA can be contained in a single RNA sequence or can be contained in two separate RNA strands. Whatever design is used, the antisense RNA molecule containing the first strand of the present invention, and therefore the third strand in its entirety or a substantial part, from the first and second RNA sequences is designed to be incorporated into the RISC complex, to be processed by the RNAi machinery, and to have its action, i.e., to induce RNAi against the RNA target sequence contained in the RNA encoded by the APOE gene. The sequence comprises or consists of the first strand, which can have a sequence that specifically targets the RNA encoded by the human APOE gene, and therefore also comprises or consists of the third strand in its entirety or a substantial part. Thus, as long as the double-stranded RNA can induce RNAi, such double-stranded RNA is contemplated in the present invention. In one embodiment, the double-stranded RNA according to the present invention is included in a pre-miRNA scaffold, a pri-miRNA scaffold, an shRNA, or an siRNA.Preferably, the first and second strands or the third and fourth strands or all four strands encoded by the expression cassette should be included in a single transcript.It is understood that the subsequent processing, i.e. cleavage, of the expressed transcript results in the processing of a single transcript into multiple separate RNA molecules.

[0057] The term complementary is defined herein as a nucleotide of a nucleic acid sequence that can bind to another nucleic acid sequence through hydrogen bonds, i.e., nucleotides capable of base pairing. Ribonucleotides, the building blocks of RNA, are composed of monomers (nucleotides) that contain a sugar, a phosphate, and a base that is either a purine (guanine, adenine) or a pyrimidine (uracil, cytosine). Complementary RNA strands form double-stranded RNA. Double-stranded RNA can be formed from two separate complementary RNA strands, or two complementary RNA strands can be included in one RNA strand. In complementary RNA strands, the nucleotides cytosine and guanine (C and G) can form base pairs, guanine and uracil (G and U), and uracil and adenine (U and A) can also form base pairs. The term substantial complementarity means that it is not required that the first and second RNA sequences are completely complementary, or that the first RNA sequence and one or more target RNA sequences of the RNA encoded by the human APOE gene are completely complementary.

[0058] Target sequence The second RNA expressed according to the present invention comprises a guide strand, also called the antisense strand, which is complementary ("anti") to the sense target RNA sequence, in its entirety or in a substantial part, and the sense target RNA sequence is comprised in the RNA encoded by the human APOE gene. Thus, the second RNA also comprises a "sense strand" which has substantial sequence identity or may be identical to the target RNA sequence. Thus, the second RNA can be described as a hairpin or double-stranded RNA substantially complementary to itself. The double-stranded RNA according to the present invention is to induce RNA interference, thereby reducing the expression of APOE transcripts, including knockdown of APOE-derived transcripts. Transcripts that can be targeted can include spliced, including splice variants, and unspliced ​​RNA transcripts, such as those encoded by SEQ ID NO: 1. Thus, the RNA encoded by the human APOE gene is understood to include unspliced ​​mRNA, including the 5' untranslated region (UTR), intron and exon sequences, followed by the 3'UTR and polyA tail, and splice variants thereof. The double-stranded RNA according to the present invention may also induce transcriptional silencing. It is understood that, according to the present invention, instead of providing an expression cassette, a third and a fourth strand may be provided which together code for the second RNA.

[0059] In one embodiment, the double-stranded RNA according to the present invention comprises a first RNA sequence and a second RNA sequence, i.e. a third and a fourth RNA strand, the first and second RNA sequences being substantially complementary, the first RNA sequence having a sequence length of at least 19 nucleotides and being substantially complementary to a target RNA sequence of the RNA encoded by the human APOE gene, the first RNA sequence being capable of inducing RNA interference to sequence-specifically reduce the expression of an RNA transcript comprising the target RNA sequence. In a further embodiment, said induction of RNA interference to reduce the expression of an RNA transcript comprising the target RNA sequence means reducing APOE gene expression. As mentioned above, the APOE gene comprises a single nucleotide polymorphism (SNP) that induces a difference in the amino acid residues located at positions 112 and 158 of the APOE isoforms (APOEε2, Cys112 / Cys158; APOEε3, Cys112 / Arg158; APOEε4, Arg112 / Arg158). These single amino acid polymorphisms are believed to have profound effects on the structure and function of APOE, thereby affecting Aβ metabolism, aggregation, deposition and tau phosphorylation. (Liu et al. 2013, Nat Rev Neurol.; 9:106-18). The subtle differences between APOE isoforms mean that targeting using the RNA sequences defined herein can result in downregulation of all APOE, preferably targeting using the RNA sequences defined results in downregulation of all APOE. However, in alternative embodiments, individual isoforms can be targeted. The design of individual RNA sequences is within the expertise of the skilled artisan. In some embodiments, the target RNA sequence targets a portion of the APOE gene, preferably the target RNA sequence targets a portion of the APOE4 gene. Thus, the present invention aims to reduce APOEε4 variants involved in late-onset AD (LOAD), since carriers of APOEε4 are at increased risk of developing AD in an allele-number-dependent manner. Furthermore, the present invention aims to reduce atherosclerosis-related events and adverse outcomes in APOE ε4 variant (APOE4)-related traumatic brain injury (TBI).Said "reduction" of APOE4 includes the use of target RNA sequences described herein that target a portion of the APOE4 gene. The length and target site of the target RNA sequence have been identified by the inventors as having the desired result of improving diseases associated with APOE4 expression, as discussed above and shown in the Examples.

[0060] Therefore, reducing the expression of APOE transcripts is understood herein as preferably reducing the steady-state level of functional APOE mRNA in target cells, so that less mRNA is available in cells for translation into APOE protein, thereby reducing the steady-state level of protein in target cells.Thus, reducing the expression of APOE transcripts does not necessarily involve reducing the de novo transcription of APOE gene, but rather involves increasing the degradation of APOE mRNA and / or its precursors, such as unspliced ​​RNA transcripts.

[0061] For each isotype, the degree of reduction in APOE gene expression can be easily determined by using standard luciferase reporter assays and appropriate controls as described in the Examples and known in the art (Zhuang et al. 2006 Methods Mol Biol. 2006;342:181-7). For example, luciferase reporters containing target RNA sequences can be used to demonstrate that the double-stranded RNA according to the present invention is capable of sequence-specific knockdown. Furthermore, the level of APOE expression can be determined by detecting APOE RNA (nuclear and / or cytoplasmic) or APOE protein, for example, as shown in the Examples section.

[0062] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules that result in the inhibition or "silencing" of expression of corresponding protein-coding genes (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression). RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0063] "Substantially complementary" in this context is understood to mean that all nucleotides of the guide sequence and the target sequence are base-paired, i.e., completely complementary, or that all nucleotides of the guide sequence and the target sequence do not have to be base-paired. Such substantial complementarity is contemplated according to the present invention, so long as the first RNA of the present invention is capable of inducing RNA interference, thereby sequence-specifically targeting a sequence, including the target RNA sequence.

[0064] Substantial complementarity between the strands complementary to the target RNA sequence, also referred to as the part of the APOE gene, preferably consists of at most two mismatched nucleotides, more preferably has one mismatched nucleotide, and most preferably has no mismatches. One mismatched nucleotide is understood to mean that one nucleotide does not base pair with the target RNA sequence over the entire length of the strand complementary to the target RNA sequence when base paired with the target RNA sequence. No mismatches means that all nucleotides of the strand complementary to the target RNA sequence base pair with the target RNA sequence, and two mismatches means that two nucleotides of the strand complementary to the target RNA sequence do not base pair with the target RNA sequence. The strand complementary to the target RNA may also contain additional nucleotides that do not have complementarity to the target RNA sequence, and may be longer than, for example, 21 nucleotides. In such a scenario, substantial complementarity is determined over the entire length of the target RNA sequence. This means that the target RNA sequence in this embodiment has zero, one or two mismatches along its entire length when base-paired with a strand complementary to the target RNA.

[0065] As shown in the Examples section, double-stranded RNAs were tested that comprise a strand complementary to a target RNA length of 22 nucleotides. These strands complementary to the target RNA have no mismatches and are completely complementary to the target RNA sequence. However, having some mismatches between the strand complementary to the target RNA and the target RNA sequence can be tolerated according to the present invention, as long as the double-stranded RNA according to the present invention can reduce the expression of the transcript that comprises the target RNA sequence, such as a luciferase reporter or the transcript that comprises the target RNA sequence. In this embodiment, the substantial complementarity between the strand complementary to the target RNA and the target RNA sequence consists of no, one or two mismatches over the entire length of either the strand complementary to the target RNA or the target RNA sequence encoded by the RNA of human APOE, whichever is the shortest.

[0066] As mentioned above, mismatch according to the present invention means that the nucleotides of the first or third strand do not base pair with the target RNA sequence encoded by the RNA of human APOE. The non-base paired nucleotides are A and A, G and G, C and C, U and U, A and C, C and U, or A and G. The mismatch can also be due to a deletion of a nucleotide or an insertion of a nucleotide. If the mismatch is a deletion of the first or third strand sequence, this means that the nucleotides of the target RNA sequence are not base paired with the first or third strand sequence when compared with the full length of the first or third strand sequence. The nucleotides that can base pair are AU, GC and GU. GU base pairs are also called GU wobble or wobble base pairs. In one embodiment, the number of GU base pairs between the first or third strand sequence and the target RNA sequence is 0, 1 or 2. In one embodiment, there is no mismatch between the first or third strand sequence and the target RNA sequence, and GU base pairs or GU pairs are allowed. Preferably, there may be no GU base pairs between the first or third strand sequence and the target RNA sequence, or the first or third strand sequence and the target RNA sequence only have AU or GC base pairs. Preferably, there are no GU base pairs and no mismatches between the first or third strand sequence and the target RNA sequence. The first or third strand sequence of the double-stranded RNA according to the invention is preferably fully complementary to the target RNA sequence, said complementarity consisting of GU, GC and AU base pairs. The first or third strand sequence of the double-stranded RNA according to the invention is more preferably fully complementary to the target RNA sequence, said complementarity consisting of GC and AU base pairs. More preferably, it is the third strand, i.e. the first strand of the second RNA of the invention.

[0067] Thus, in one embodiment, the first strand of the second RNA and the target RNA sequence have at least 15, 16, 17, 18 or 19 nucleotides that are base-paired. Preferably, the first strand of the second RNA and the target RNA sequence are substantially complementary, said complementarity comprising at least 19 base pairs. In another embodiment, the first strand of the second RNA has at least 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides that are base-paired with consecutive nucleotides of the target RNA sequence. In another embodiment, the first strand of the second RNA has at least 19 consecutive nucleotides that are base-paired with consecutive nucleotides of the target RNA sequence. In another embodiment, the first strand of the second RNA comprises at least 19 consecutive nucleotides that are base-paired with 19 consecutive nucleotides of the target RNA sequence. In yet another embodiment, the first strand of the second RNA has at least 17 nucleotides that are base-paired with the target RNA sequence and at least 15 consecutive nucleotides that are base-paired with consecutive nucleotides of the target RNA sequence. The sequence length of the first strand is preferably at most 21, 22, 23, 24, 25, 26 or 27 nucleotides. In another embodiment, the first strand of the second RNA has at least 20 consecutive nucleotides that base pair with 20 consecutive nucleotides of the target RNA sequence. In another embodiment, the first strand of the second RNA comprises at least 21 consecutive nucleotides that base pair with 21 consecutive nucleotides of the target RNA sequence.

[0068] As mentioned above, it may not be necessary to have perfect complementarity (i.e., perfect base pairing (no mismatches) and no GU base pairing) between the first strand of the second RNA and the target RNA sequence, since the first strand of the second RNA can still allow sufficient inhibition of gene expression. Also, not having perfect complementarity may be contemplated, for example, to avoid or reduce off-target RNA sequence-specific gene inhibition while maintaining sequence-specific inhibition of transcripts containing the target RNA sequence. However, having perfect complementarity may be preferred, since it may result in more potent inhibition. Without being bound by theory, having perfect complementarity between the first strand of the second RNA and the target RNA sequence may allow an activated RISC complex containing said first strand of the second RNA (or a substantial portion thereof) to cleave its target RNA sequence, while having mismatches may prevent cleavage and may mainly allow inhibition of translation, so that the latter may result in less potent inhibition.

[0069] With respect to the second strand on the second RNA, the second strand is substantially complementary to the first strand. The second strand combined with the first strand forms a double-stranded RNA. As mentioned above, this is to form a suitable substrate for the RNA interference mechanism so that the guide sequence from the first strand is included in the RISC complex to sequence to specifically inhibit the expression of its target, i.e. the RNA encoded by the human APOE gene. The sequence of the second strand has sequence similarity with the target RNA sequence. However, the substantial complementarity of the second strand with the first strand can be selected to be less substantial complementarity compared to the substantial complementarity between the first strand and the target RNA sequence. Thus, the second strand can contain 0, 1, 2, 3, 4 or more mismatches, 0, 1, 2, 3 or more GU wobble base pairs, and can contain 0, 1, 2, 3, 4 nucleotide insertions and / or 0, 1, 2, 3, 4 nucleotide deletions. Preferably, the first strand and the second strand are substantially complementary, said complementarity comprising 0, 1, 2 or 3 GU base pairs, and / or said complementarity comprising at least 17 base pairs. These mismatches, GU wobble base pairs, insertions and deletions are with respect to the first strand, i.e. the double-stranded region formed between the first strand and the second strand. Such substantial complementarity is permissible according to the present invention, as long as the first and second strands can substantially base pair and induce sequence-specific inhibition of the RNA encoded by the human APOE gene. It is also understood that the substantial complementarity between the first strand and the second strand may depend on the double-stranded RNA design selected. It may depend, for example, on the miRNA scaffold selected for the double-stranded RNA to be incorporated.

[0070] As is clear from the above, substantial complementarity between the first and second strands of the second RNA may include mismatches, deletions and / or insertions relative to the first and second RNA sequences being fully complementary (i.e. fully base-paired). In one embodiment, the first and second strands of the second RNA have at least 11 consecutive base pairs. Thus, at least 11 consecutive nucleotides of the first strand and at least 11 consecutive nucleotides of the second strand are fully complementary. In another embodiment, the first and second strands of the second RNA have at least 15 nucleotides that form base pairs. Said base pairing between at least 15 nucleotides of the first strand and at least 15 nucleotides of the second strand may consist of GU, GC and AU base pairs, or may consist of GC and AU base pairs. In another embodiment, the first and second RNA sequences have at least 15 nucleotides that form base pairs and have at least 11 consecutive base pairs. In another embodiment, the first RNA sequence and the second RNA sequence are substantially complementary, said complementarity comprising at least 17 base pairs, said 17 base pairs may preferably be 17 contiguous base pairs, said base pairs consisting of GU, GC and AU base pairs, or consisting of GC and AU base pairs.

[0071] As mentioned above, the present invention now provides an expression cassette encoding a first strand and a second strand of a second RNA, wherein the first strand and the second strand are substantially complementary, and the first strand has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence contained in the RNA encoded by the human APOE gene. As shown in the examples, suitable target RNA sequences according to the present invention are provided (see, for example, Table 1). Thus, in one embodiment, an expression cassette is provided encoding a first strand and a second strand, wherein the first strand and the second strand are substantially complementary, and the first strand has a sequence length of at least 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides and is substantially complementary to a target RNA sequence selected from the group listed in Table 1 contained in the RNA encoded by the human APOE gene.

[0072]

Table 1

[0073]

Table 2

[0074]

Table 3

[0075]

Table 4

[0076]

Table 5

[0077] As shown in the examples, the third and fourth strands of the present invention may be preferably incorporated into a pre-miRNA or pri-miRNA scaffold derived from miR-101, or a pri-miRNA or pre-miRNA scaffold derived from miR-451. In a preferred embodiment, the third and fourth strands of the present invention are incorporated into a pri-miRNA or pre-miRNA scaffold derived from miR-451. These scaffolds have been found to be particularly useful, since both of these scaffolds can induce RNA interference and can be combined into a single transcript. These scaffolds also allow to induce RNA interference, which can mainly result in guide strand-induced RNA interference. The pri-miR451 scaffold does not yield a passenger strand, as its processing is distinct from the canonical miRNA processing pathway (Cheloufi et al., 2010 Jun 3;465(7298):584-9 and Yang et al., Proc Natl Acad Sci US A. 2010 Aug 24;107(34):15163-8). The pri-miR-101 scaffold is produced by the canonical miRNA processing pathway, whereas many of the miR-101 scaffolds were found to produce mainly the guide strand (see, e.g., C2, C4, C32, and C33) and very small amounts of the passenger strand. Thus, both scaffolds represent excellent candidates for developing gene therapy products, since potential undesirable off-targeting by the passenger strand can be largely, if not completely, avoided. The passenger strand (corresponding to the second sequence) may result in targeting of transcripts other than APOE RNA, and the use of such a scaffold may allow such undesired targeting to be avoided. Therefore, it is preferred to select a scaffold that produces less than 15%, less than 10%, less than 5%, less than 4%, or less than 3% passenger strand.In some embodiments, the second RNA is derived from a pri-miRNA scaffold selected from the group consisting of pri-miR-21, pri-miR-22, pri-miR-26a, pri-miR-30a, pri-miR-33, pri-miR-122, pri-miR-375, pri-miR-199, pri-miR-99, pri-miR-194, pri-miR-155 and pri-miR-451. In a preferred embodiment, the second RNA is derived from a scaffold comprising SEQ ID NO: 190 (miR-451) or a variant thereof. Thus, in one embodiment, the first RNA comprises SEQ ID NO: 235 (miR-144) or a variant thereof, and the second RNA comprises SEQ ID NO: 190 (miR-451) or a variant thereof. The experimental data appended hereto support the surprising discovery that improved silencing of APOE is achieved when the miRNA cluster approach is applied. The particular combination of 144 / 451 scaffolds in the nucleic acids of the present invention is particularly useful in the context of gene therapy and RNA silencing. miR-144 and miR-451 are examples of trans-regulated clustered miRNAs, with miR-144 regulating miR-451 processing by Ago2. Specifically, miR-144 enhances the biogenesis of miR-451 in trans by suppressing Dicer, which in turn suppresses global canonical miRNA processing (Kretov et al., 2020, Molecular Cell 78, 317-328). It has been demonstrated that miR451 is the most abundant miRNA in erythrocytes and is clustered with miR144, which plays a complex role during erythropoiesis, but the inventors have further developed this cluster, where it is shown for the first time that these miRNAs can also be used as scaffolds for targeting RNAs that affect their expression when clustered in a similar manner. Thus, when this combination of scaffolds is used, the first RNA of the invention plays an important role in enhancing the biogenesis of the second RNA of the invention and thus the delivery of the guide sequence contained in said first and second RNA.Thus, in one embodiment, reduction and / or silencing of APOE expression is increased when the second RNA is expressed with the first RNA, as compared to the reduction in APOE expression achieved with expression of the second RNA alone.

[0078] As shown in the examples, a first strand of a second RNA of 22 nucleotides in length (e.g., for miR-451) or a third strand of the invention can be selected and incorporated into a miRNA scaffold. Such miRNA scaffold sequences are then processed by the RNAi machinery present in the cell. When referring to a miRNA scaffold, it is understood to include a pri-miRNA structure or a pre-miRNA structure. As shown in the examples, such miRNA scaffolds, when processed in a cell, result in a guide sequence that includes a first strand of a second RNA in the range of 21-30 nucleotides in length for the miR-451 scaffold, or a substantial portion thereof. Such a guide strand can reduce APOE transcript expression by targeting a selected target sequence. As is clear from the above and as shown in the examples, the first strand of a second RNA encoded by an expression cassette of the invention, when processed by the RNAi machinery of a cell, is partially or entirely included in the guide strand. Thus, the guide strand generated from the RNA encoded by the expression cassette, which comprises the first strand of the second RNA and the second strand of the second RNA, comprises at least 18 nucleotides of the first RNA sequence. Preferably, such guide strand comprises at least 19, 20, 21 or 22 nucleotides. The guide strand can also comprise the first strand of the second RNA sequence as a whole. When selecting a miRNA scaffold, the first strand of the second RNA sequence can be selected so that it replaces the original guide strand. As shown in the exemplary section, this does not necessarily mean that the guide strand generated from such an artificial scaffold is identical in length to the first strand of the selected second RNA, or that the first strand of the second RNA is found in its entirety in the guide strand generated.The miR-451 scaffold, as shown in the examples and in FIG. 2a, preferably comprises, from 5' to 3', first, 5'-CUUGGGAAUGGCAAGG-3' (SEQ ID NO: 233), followed by a 22 nucleotide sequence comprising or consisting of a first RNA sequence, followed by a 17 nucleotide sequence, which can be considered as a second RNA sequence that is complementary to nucleotides 2 to 18 of said 22 nucleotide sequence over its entire length, followed by the sequence 5'-MWCUUGCUAUACCCAGA-3' (wherein M is A or C and W is A or U) (SEQ ID NO: 234). Preferably, the first 5'-C nucleotide of the latter sequence does not base pair with the first nucleotide of the first strand of the second RNA. Such a scaffold may contain further flanking sequences as found in the original pri-miR-451 scaffold. Alternatively, the flanking sequences 5'-CUUGGGAAUGGCAAGG'-3' and 5'-MWCUUGCUAUACCCAGA-3' may be replaced by flanking sequences of other pri-mRNA structures. As is clear from above, the sequence of the scaffold may not only differ with respect to the (presumed) guide strand sequence and the sequence complementary thereto, as present in the wild-type scaffold (Figure 2a), but may also contain additional mutations in the 5', loop and 3' sequences, and additional mutations may be required to provide an RNA structure predicted to mimic the secondary structure of the wild-type scaffold. Such a scaffold may be included in a larger RNA transcript, for example a pol II expressed transcript containing 5'UTR and 3'UTR and polyA. The flanking structures may also be absent. Thus, an expression cassette according to the invention expressing an shRNA-like structure having a 22 nucleotide sequence comprises or consists of a first strand of a second RNA followed by a 17 nucleotide sequence which can be considered to be the second strand of the second RNA and is complementary over its entire length to nucleotides 2 to 18 of said 22 nucleotide sequence. The latter shRNA-like structure derived from the miR-451 scaffold can be referred to as a pre-miRNA scaffold derived from miR-451.

[0079] Alternatively, the flanking sequences may be replaced by flanking sequences of other pre-miRNA structures. The flanking structures may also be absent. Thus, the expression cassette according to the invention expresses an shRNA-like structure, i.e., the pre-miRNA structure of miR-101. Such an shRNA-like structure consists of, starting from the 5' end, a second RNA sequence of 22 nucleotides in length, followed by a loop sequence, the last 3' two nucleotides of the loop sequence being base-paired with the last 3' nucleotide of the second strand of the second RNA, followed by a first strand of the second RNA of 21 nucleotides in length, the first 20 consecutive nucleotides being complementary to the second strand of the second RNA. The second strand of the second RNA contains a bulge (non-base-paired nucleotide) at position 5, counting from the 3' end of said 22 nucleotides.

[0080] In one embodiment, an expression cassette according to the present invention is provided, wherein the first strand of the second RNA is substantially complementary to a target RNA sequence contained in an antisense RNA transcript encoded by the human APOE gene. Preferably, the first strand of the second RNA is substantially complementary to SEQ ID NO: 4, 16, 24, 41, 44, 46, 54, 59, 93. More preferably, the first strand of the second RNA has a length of 19, 20, 21 or 22 nucleotides. More preferably, the first strand of the second RNA is fully complementary to the target sequence over its entire length. Most preferably, the first strand of the second RNA has a length of 19, 20, 21 or 22 nucleotides, and the first strand of the second RNA is fully complementary to the target sequence over its entire length. The first strand of the second RNA may be SEQ ID NO: 94-185, Table 2.

[0081] [Table 6]

[0082] [Table 7]

[0083] [Table 8]

[0084] [Table 9]

[0085] [Table 10]

[0086] As mentioned above, such a first strand of the second RNA should be combined with the second strand of the second RNA, which may be referred to as the third and fourth strands of the present invention. As described herein, the skilled artisan can design and select a suitable second strand of the second RNA to provide a first and second strand for the second RNA that can induce RNA interference when expressed in a cell. A suitable second strand of the second RNA is complementary to nucleotides 2-15, 2-16, 2-17, or 2-18 over its entire length to the first strand of the second RNA having a length of 19, 20, 21, or 22 nucleotides.

[0087] Said first strand of the second RNA is preferably comprised in a miRNA scaffold as shown in the examples, more preferably in a miR-451 scaffold. A suitable scaffold comprising the first and second strands for the second RNA according to the invention may be a sequence such as SEQ ID NO: 190.

[0088] Such a first strand of the second RNA as described above can be included in an expression cassette. Such a first strand of the second RNA can be included in an RNA structure encoded by an expression cassette.

[0089] Such first and second strands of the second RNA sequence as described above may be included in an expression cassette. Such first and second strands of the second RNA may be included in an RNA structure encoded by an expression cassette. Thus, in some embodiments, the sequence encoding the first RNA and the sequence encoding the second RNA are included in an intron sequence. By incorporating sequences encoding the first and second RNA sequences, the inventors have identified the beneficial effect of deriving smaller constructs for the expression of the RNA of the present invention, particularly useful for downstream processing steps such as the application of viral vector technology. In a further embodiment, the sequence encoding the first RNA and the sequence encoding the second RNA are present in a promoter, preferably the intron sequence has SEQ ID NO: 231.

[0090] Therefore, it has been found that using such a first strand and a second strand of a second RNA to target these target RNA sequences is particularly useful for reducing the expression of the RNA transcripts encoded by human APOE gene.By targeting human APOE in this way, the inventors can reduce human APOE gene expression very efficiently, and thus reduce the formation of amyloid plaques.Finally, this can reverse, prevent, slow down or completely stop the progression of neuropathology, such as neurodegeneration and / or tauopathy.

[0091] APOE Expression In a further aspect, an expression cassette is provided encoding at least one of APOE2 and APOE3. APOEε2 and / or APOEε3 alleles exert protective or neutral effects against AD development (Yamazaki et al. 2016, CNS Drugs.; 30: 773-89). Thus, in some embodiments, the expression cassette comprises a nucleic acid encoding at least one of APOE2 and APOE3 having an amino acid sequence selected from SEQ ID NOs: 249-254. Expression of at least one of APOE2 and APOE3 is neuroprotective against tauopathy. As used herein, the term "tauopathy" refers to a neurodegenerative disorder characterized by the deposition of abnormal tau protein in the brain. In one embodiment, the expression cassette encoding at least one of APOE2 and APOE3 further comprises a promoter and a polyA signal. In a further embodiment, the expression cassette comprises a nucleic acid encoding one or more APOE2 and APOE3 proteins having an amino acid sequence selected from one or more of SEQ ID NOs: 249-254 or variants thereof for use in gene therapy.

[0092] In some embodiments, the expression cassette comprises one or more nucleic acid sequences comprising at least a partial wild type sequence. By at least a partial wild type sequence is meant a sequence with codon optimization only in a specific region, such as can be seen in SEQ ID NOs: 212-217. In a preferred embodiment, the expression cassette comprises one or more nucleic acid sequences comprising a full length wild type coding sequence (e.g. SEQ ID NOs: 195-201). The invention of the present application surprisingly demonstrated that (at least a partial) wild type version of the nucleic acid sequence exhibited the highest protein expression among the different variants. In a more preferred embodiment, the expression cassette comprises a nucleic acid selected from one of SEQ ID NOs: 195, 197, 200. In an even more preferred embodiment, the expression cassette comprises a nucleic acid comprising SEQ ID NOs: 197 and 200 due to the known beneficial properties of the encoded protein. In an alternative embodiment, the expression cassette comprises a nucleic acid selected from one of SEQ ID NOs: 197 and 200. The inventors have surprisingly shown that the wild-type version of the nucleic acid sequence encoding the APOE2 or APOE3 protein is the one that results in the highest protein expression and secretion among the different variants. Codon optimization of the entire length of the nucleic acid sequence results in lower levels of expression of the APOE2 or APOE3 protein.

[0093] In a further embodiment, the expression cassette encoding at least one of APOE2 and APOE3 comprises one or more nucleic acid sequences selected from one or more of SEQ ID NOs: 195-217 or variants thereof. For example, in a still further embodiment, the expression cassette encoding at least one of APOE2 and APO3 comprises two or three or four or more nucleic acid sequences selected from one or more of SEQ ID NOs: 195-217 or variants thereof. In a further aspect, an expression cassette as disclosed herein is provided comprising a nucleic acid comprising a sequence encoding a first RNA as disclosed herein and a sequence encoding a second RNA, the second RNA comprising a guide sequence of at least 19 nucleotides substantially complementary to a portion of the APOE gene, the first RNA and the second RNA each comprising a hairpin, and further comprising a second nucleic acid encoding at least one of APOE2 and APOE3. In one embodiment, the first and second nucleic acids are operably linked to a promoter and a polyA signal. In further embodiments, the second nucleic acid encodes a protein comprising one of SEQ ID NOs: 249-254, and / or the second nucleic acid comprises one of SEQ ID NOs: 195-217. Thus, in some embodiments, the second nucleic acid encodes a protein having an amino acid sequence as set forth in SEQ ID NOs: 249-254. In some embodiments, the second nucleic acid is a gene product encoded by a coding portion (e.g., cDNA) of a naturally occurring gene. In some embodiments, the gene product is a protein or fragment thereof encoded by the APOE2 and / or APOE3 isoforms of the APOE gene. In further embodiments, the second nucleic acid does not comprise a sequence substantially complementary to a guide sequence as defined herein. In further embodiments, the second nucleic acid comprises a nucleotide sequence that is codon-optimized for expression in a human cell. In further embodiments, the second nucleic acid is codon-optimized to be sufficiently different from endogenous APOE2 and / or APOE3 sequences in the cell such that it is not recognized by shRNAs targeting wild-type APOE, APOE2 and / or APOE3. Those skilled in the art will understand the requirements necessary to design such a nucleic acid. In a further embodiment, the second nucleic acid comprises a sequence selected from Table 3 (SEQ ID NOs: 195-217).

[0094]

Table 11

[0095]

Table 12

[0096]

Table 13

[0097]

Table 14

[0098]

Table 15

[0099]

Table 16

[0100]

Table 17

[0101]

Table 18

[0102]

Table 19

[0103]

Table 20

[0104] [Table 21]

[0105] [Table 22]

[0106] As known in the art, expression of a gene product requires the presence of expression control and / or control sequences, such as one or more promoters and any other nucleic acid sequences, such as introns, necessary for the expression of the selected nucleic acid sequence, all operably linked to the selected sequence and may include enhancer sequences. Thus, in one embodiment, an expression construct comprising an expression cassette encoding at least one of APOE2 and APOE3 comprises a promoter. In an alternative embodiment, the expression cassette disclosed herein comprises a nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, the second RNA comprising a guide sequence of at least 19 nucleotides substantially complementary to a portion of the APOE gene, the first RNA and the second RNA each comprising a hairpin, further comprising a second nucleic acid encoding at least one of APOE2 and APOE3, and comprising one or more promoters, preferably one promoter. Such a design has been identified by the inventors as being advantageous in view of the ability of subsequent downstream processing associated with transfection and expression.

[0107] Promoter The nucleotide sequence comprising the expression cassette defined herein above for expression in mammalian cells further preferably comprises at least one mammalian cell-compatible expression control sequence, such as a promoter, operably linked to the sequence encoding the gene product of interest, thus forming an expression cassette for expression of the gene product of interest in mammalian target cells treated by gene therapy with the gene product of interest. Many such promoters are known in the art (see Sambrook and Russel, supra, 2001). Constitutive promoters that are broadly expressed in many cell types, such as the CMV promoter, can be used. However, promoters that are inducible, tissue-specific, cell type-specific or cell cycle-specific would be more preferred. Preferably, pol II promoters such as CAG promoter (SEQ ID NO: 191) (ia Miyazaki et al. Gene. 79(2):269-77; Niwa, Gene. 108(2):193-9), PGK promoter, CMV promoter (e.g., as shown in FIG. 2 of WO2016102664, which is incorporated herein by reference) or adapted / synthetic promoters (P1, SEQ ID NO: 192 and P2, SEQ ID NO: 193) are used. Since Alzheimer's disease mainly affects the brain, it may be useful to use a neurospecific promoter in particular. Thus, in some embodiments, the promoter is a promoter capable of driving transcription in brain cells. Examples of suitable neurospecific promoters are neuron-specific enolase (NSE), human synapsin 1, caMK kinase and tubulin (Hioki et al. Gene Ther. 2007 Jun;14(11):872-82). Other suitable promoters that may be contemplated are inducible promoters, ie promoters that initiate transcription only when the host cell is exposed to some particular stimulus.

[0108] As described above, the expression cassette for expressing at least APOE further preferably encodes a polyA signal contained in a DNA expression cassette operably linked to the 3' end of the RNA molecule encoded by the transgene, as described above. Preferably, said polyA signal is the simian virus 40 polyadenylation (SV40 polyA, SEQ ID NO: 194), a synthetic polyadenylation signal, the bovine growth hormone polyadenylation signal (BGH polyA), or the human growth hormone polyadenylation signal (HGH polyA).

[0109] vector The isolated nucleic acid described herein may be present by itself, as part of an expression cassette, and / or as part of a vector. In general, the vector may be a viral vector, such as a plasmid, a cosmid, a phagemid, a bacterial artificial chromosome (BAC), or a gene therapy vector. The expression cassette or vector according to the present invention may be transferred into a cell, for example, using a transfection method. Any suitable means may be sufficient to transfer the expression cassette according to the present invention. Preferably, a viral vector is used that stably transfers the expression cassette into the cell, so that stable expression of the double-stranded RNA that induces sequence-specific inhibition of the APOE gene described above can be achieved. A suitable vector may be a lentiviral vector, a retrotransposon-based vector system, or an AAV vector. For example, when the lentiviral vector has an RNA genome, it is understood that the RNA genome codes for the expression cassette, such that after transduction of the cell, the DNA sequence and the expression cassette are formed. Preferably, a viral vector, such as an AAV, is used. Thus, in some embodiments, the expression cassette disclosed herein is flanked by inverted terminal sequences. Preferably, the AAV vector used is a serotype 5 AAV vector. Serotype 5 AAV (also called AAV5) can be particularly useful for transducing human neurons and human astrocytes, as shown in the examples. Thus, in some embodiments, there is an AAV that comprises the expression cassette disclosed herein. Thus, AAV5 can efficiently transduce various human cell types of the CNS, including FBN, dopaminergic neurons, motor neurons and astrocytes, and is therefore a suitable vector candidate for delivering therapeutic genes to the CNS to treat neurodevelopmental diseases (including, but not limited to, the treatment of Alzheimer's disease by targeting APOE as described herein).The production of AAV vectors containing any expression cassette of interest is fully described in WO 2007 / 046703, WO 2007 / 148971, WO 2009 / 014445, WO 2009 / 104964, WO 2011 / 122950, ​​WO 2013 / 036118, which are incorporated herein in their entireties.

[0110] AAV sequences that may be used in the present invention for the production of AAV vectors, for example produced in insect or mammalian cell lines, may be derived from the genome of any AAV serotype. In general, AAV serotypes share significant genomic sequence homology at the amino acid and nucleic acid levels, provide the same set of gene functions, produce essentially physically and functionally equivalent virions, and replicate and assemble by substantially identical mechanisms. For an overview of the genomic sequences and genomic similarities of various AAV serotypes, see, for example, GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chlorini et al. (1997, J. Vir. 71:6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74:8635-47). AAV serotypes 1, 2, 3, 4 and 5 are preferred sources of AAV nucleotide sequences for use in the context of the present invention. Preferably, the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and / or AAV5. Similarly, the Rep52, Rep40, Rep78, and / or Rep68 coding sequences are preferably derived from AAV1, AAV2, and AAV5. However, the sequences encoding the VP1, VP2, and VP3 capsid proteins for use in the context of the present invention may be taken from any of the 42 known serotypes, more preferably from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or from newly developed AAV-like particles obtained, for example, by capsid shuffling techniques and AAV capsid libraries. The AAV capsid may consist of VP1, VP2, and VP3, but may also consist of VP1 and VP3. In some embodiments, the AAV vector according to the present invention comprises AAV5 or AAV9 capsid proteins. In some embodiments, the AAV vector according to the present invention comprises AAV5 capsid proteins.In some embodiments, an AAV vector according to the invention comprises an AAV9 capsid protein.

[0111] In another embodiment, a host cell is provided comprising said nucleic acid or said expression cassette according to the invention. For example, said expression cassette or nucleic acid may be comprised in a plasmid contained in a bacterium. Said expression cassette or nucleic acid may also be comprised in a producer cell, for example producing a viral vector. Said expression cassette may also be provided in a baculovirus vector.

[0112] Various modifications of the above-defined nucleotide sequences, including, for example, the wild-type AAV sequence, for appropriate expression in a host cell are achieved by application of well-known genetic engineering techniques, as described, for example, in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Various further modifications of the coding region are known to those skilled in the art and can increase the yield of the encoded protein. These modifications are within the scope of the present invention.

[0113] In one embodiment, any mammalian cell can be infected with an AAV vector of the invention, including but not limited to muscle cells, liver cells, neuronal cells, glial cells, and epithelial cells. In a preferred embodiment, the cell to be infected is a human cell.

[0114] It is understood that the capsid amino acid sequences and the nucleotide sequences encoding them can be engineered, e.g., the sequences may be in hybrid form or may be codon optimized, e.g., according to codon usage in AcmNPv or Spodoptera frugiperda. The capsid proteins can be engineered, e.g., by DNA shuffling, error-prone PCR, bioinformatics rational design or site-saturation mutagenesis. The resulting capsids are based on existing serotypes but contain various amino acid or nucleotide changes that improve the characteristics of such capsids. The resulting capsids can be combinations of various parts of existing serotypes, "shuffled capsids," or can contain entirely novel changes, i.e., additions, deletions, or substitutions of one or more amino acids or nucleotides, organized into groups or spanning the entire length of the gene or protein. See, e.g., Schaffer and Maheshri; Proceedings of the 26th Annual International Conference of the IEEE EMBS San Francisco, CA, USA; September 1-5, 2004, pages 3520-3523; Asuri et al., 2012 Molecular Therapy 20(2):329-3389; Lisowski et al., 2014, Nature 506(7488):382-386, which are incorporated by reference herein.

[0115] In some embodiments, the ITRs and capsid proteins (or portions thereof) in an AAV vector of the invention can be derived from different AAV serotypes. By way of example and not limitation, the ITRs can be derived from AAV2, while the capsid proteins can be derived from a different serotype, such as AAV5 or AAV9.

[0116] composition In another aspect, the invention relates to a pharmaceutical composition comprising an AAV vector according to the invention, i.e. an AAV vector comprising a nucleic acid according to the invention or an expression cassette according to the invention. In one embodiment, the invention provides a composition comprising an AAV vector according to the invention and suitable excipients such as buffers and stabilizers, antioxidants, etc. In a particular embodiment, these compositions are used to transduce cells in vitro or ex vivo, in which case the excipients must be compatible with cell culture. In another preferred embodiment, the composition is used for the treatment of a (human) subject. To that end, the invention provides a pharmaceutical composition comprising an AAV vector according to the invention and at least one pharma- ceutically acceptable carrier. In the case of an AAV gene delivery vehicle, the pharmaceutical composition typically comprises a physiological buffer, e.g. PBS, with further stabilizers, e.g. sucrose. Such compositions are compatible, suitable and intended for use in subsequent intravenous, intrathecal, intraparenchymal, intravitreal, subretinal administration, or for use in organ-targeted vascular delivery, e.g. oral or intracoronary delivery, or isolated limb perfusion. Thus, in some embodiments the invention provides a nucleic acid according to the invention, or an expression cassette according to the invention, or an AAV vector according to the invention, or a pharmaceutical composition according to the invention for the uses disclosed herein, wherein the nucleic acid, expression cassette, AAV vector or pharmaceutical composition is administered to the central nervous system, preferably by intracerebral, intraparenchymal, intrathecal, intracisternal, intraventricular injection or a combination thereof, more preferably by convection-enhanced delivery.

[0117] use Another aspect of the invention relates to the use of an AAV vector according to the invention, or a composition comprising an AAV vector. In one embodiment, there is provided an expression cassette according to the invention, an AAV vector according to the invention, or a pharmaceutical composition according to the invention for use as a medicament. In a further embodiment, there is provided a nucleic acid according to the invention, an expression cassette according to the invention, an AAV vector according to the invention, or a pharmaceutical composition according to the invention for use in the treatment of and / or prevention of a tauopathy in a subject. In a further embodiment, there is provided a nucleic acid according to the invention, an expression cassette according to the invention, an AAV vector according to the invention, or a pharmaceutical composition according to the invention for use in the treatment of and / or prevention of Alzheimer's disease ... a subject that is a carrier of the APOE4 allele, preferably where the subject is homozygous for the APOE4 allele.

[0118] Host cells and methods and kits for producing AAV vectors In a further aspect, the present invention relates to a method for producing a nucleic acid according to the invention, an expression cassette according to the invention, an AAV vector according to the invention, or a pharmaceutical composition according to the invention.

[0119] Methods for producing the nucleic acids of the invention include any method for producing nucleic acids, including but not limited to de novo synthesis, all of which would be apparent to one of skill in the art.

[0120] A method for producing an AAV vector, preferably comprising the steps of: a) culturing a host cell as defined above under conditions such that the AAV vector is produced; and b) optionally one or more steps of recovery, purification and formulation of the AAV vector.

[0121] The host cell is preferably a host cell suitable for the production of AAV vectors. Thus, the host cell is preferably a host cell suitable for large-scale in vitro culture. Host cells suitable for the production of AAV vectors are well known in the art and are typically mammalian or insect cell lines. Mammalian cell lines for producing AAV vectors are selected from any mammalian species, including, but not limited to, cells from mammals including human, monkey, mouse, rat, rabbit and hamster, such as A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK293 cells (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblasts, hepatocytes and myoblasts. The choice of mammalian species providing the cells is not a limitation of the present invention, nor are the types of mammalian cells, i.e., fibroblasts, hepatocytes, tumor cells. Mammalian cell lines for producing AAV vectors particularly include the wide range of HEK293 cell lines, of which the HEK293T cell line is preferred.

[0122] The insect cell line for producing the AAV vector can be any cell line suitable for the production of heterologous proteins. Preferably, the insect cell allows the replication of the baculovirus vector and can be maintained in culture, more preferably in suspension culture. In a preferred embodiment, the insect cell allows the replication of the recombinant parvovirus vector, including the rAAV vector. For example, the cell line used can be derived from Spodoptera frugiperda, Drosophila melanogaster, or a mosquito, such as Aedes albopictus, derived cell line. Preferred insect cells or cell lines are cells from insect species susceptible to baculovirus infection, including, for example, S2 (CRL-1963, ATCC), Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (U.S. Pat. No. 6,103,526; Protein Sciences Corp., CT, USA).

[0123] Thus, in one embodiment, the expression cassette or construct is an insect cell-compatible vector or a mammalian cell-compatible vector. A "mammalian cell-compatible vector" is understood to be a nucleic acid molecule capable of productive transformation or transfection of a mammalian cell or cell line. Mammalian cell-compatible vectors are well known in the art. An "insect cell-compatible vector" is understood to be a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary insect cell-compatible vectors include plasmids, linear nucleic acid molecules, and recombinant viruses such as baculoviruses. Any vector can be used as long as it is compatible with insect cells. A mammalian or insect cell-compatible vector can be integrated into the genome of the cell, but the presence of the vector in the cell need not be permanent, and also includes transient episomal vectors. The vector can be introduced by any known means, such as chemical treatment, electroporation, or infection of the cell.

[0124] The AAV in the supernatant can be recovered and / or purified using suitable techniques known to those skilled in the art. For example, monolith columns (e.g., in ion exchange, affinity or IMAC mode), chromatography (e.g., capture chromatography, immobilized chromatography, and expanded bed chromatography), centrifugation, filtration and precipitation can be used for purification and concentration. These methods can be used alone or in combination. In one embodiment, a capture chromatography method, including a column-based or membrane-based system, is utilized in combination with filtration and precipitation. For example, a suitable precipitation method utilizing polyethylene glycol (PEG) 8000 and NH3SO4 can be easily selected by the skilled artisan. The precipitate can then be treated with benzonase and purified using suitable techniques. Furthermore, the recovery can preferably include a step of affinity purification of the recombinant parvovirus (rAAV) vector (including virions) using an anti-AAV antibody, preferably an immobilized antibody. The anti-AAV antibody is preferably a monoclonal antibody. Particularly suitable antibodies are single chain camelid antibodies or fragments thereof, which can be obtained, for example, from camels or llamas (see, for example, Muyldermans, 2001, Biotechnol. 74:277-302). Antibodies for affinity purification of rAAV are preferably antibodies that specifically bind to an epitope on an AAV capsid protein, whereby preferably the epitope is present on the capsid protein of more than one AAV serotype. For example, an antibody can be produced or selected based on specific binding to the AAV2 capsid, but can also specifically bind to AAV1, AAV3 and AAV5 capsids.

[0125] In general, suitable methods for producing AAV vectors according to the invention in mammalian or insect host cells, and means therefor (e.g., expression constructs for expression of AAV rep proteins), can be found in Clark et al. (1995, Hum. Gene Ther. 6, 1329-134), Gao et al. (1998, Hum. Gene Ther. 9, 2353-2362), Inoue and Russell (1998, J. Virol. 72, 7024-7031), Grimm et al. (1998, Hum. Gene Ther. 9, 2745-2760), Xiao et al. (1998, J. Virol. 72, 2224-2232) and Judd et al. (Mol Ther Nucleic Acids. 2012;1:e54) for mammalian cells and in Urabe et al. al. (2002, Hum. Gene Ther. 13:1935-1943), WO 2007 / 046703, WO 2007 / 148971, WO 2009 / 014445, WO 2009 / 104964, WO 2011 / 122950, ​​WO 2013 / 036118, WO 2015 / 137802, WO 2019 / 016349, and co-pending applications EP 21177449.2, PCT / EP2021 / 058794 and PCT / EP2021 / 058798, all of which are incorporated herein in their entirety.

[0126] In a further aspect, there is provided a kit comprising a nucleic acid according to the invention, an expression cassette according to the invention, an AAV vector according to the invention or a pharmaceutical composition according to the invention, the kit further comprising an immunosuppressant.

[0127] In certain embodiments of the invention, the immunosuppressant compound may reduce and / or prevent the immune response induced by administration of a nucleic acid, AAV vector, or pharmaceutical composition of the invention.

[0128] In another aspect, the present invention relates to a cell or a host cell comprising a nucleic acid of the invention or an AAV of the invention.

[0129] In some embodiments, the cell of the invention is a prokaryotic cell. In some specific embodiments, the cell of the invention is a bacterial cell. In some embodiments, the cell of the invention is a eukaryotic cell. In some embodiments, the cell of the invention is a mammalian cell. In some embodiments, the cell of the invention is an insect cell.

[0130] The nucleic acid of the present invention or the AAV vector of the present invention can be delivered to the cell of the present invention by any suitable method, including but not limited to transfection, transformation, transduction, nucleofection, electroporation, microinjection. For example, the expression cassette or nucleic acid of the present invention can be contained in a plasmid contained in a bacterium. The expression cassette or nucleic acid of the present invention can also be contained in a producer cell, for example, producing a viral vector. The expression cassette can also be provided in a baculovirus vector.

[0131] Further details regarding host cells comprising the AAV vectors according to the invention are provided elsewhere in this application. Any mammalian cell can be infected with the AAV vectors of the invention, including but not limited to muscle cells, hepatic cells, neuronal cells, glial cells or epithelial cells. In some preferred embodiments of the invention, the cells to be infected are human cells.

[0132] In another aspect, the present invention relates to a method for treating or preventing a disorder, the method comprising administering a nucleic acid of the invention or an AAV of the invention to a subject, thereby treating or preventing the disorder.

[0133] In another aspect, the present invention relates to a nucleic acid of the invention or an AAV of the invention for use in the manufacture of a medicament for the treatment of a disorder as detailed herein.

[0134] The invention has been described above with reference to a number of exemplary embodiments shown in the drawings. Modifications and alternative implementations of some parts or elements are possible and fall within the scope of protection defined in the appended claims. EXAMPLES

[0135] Materials and Methods Design of miRNA guide strands targeting APOE miAPOE, miRNA guide strands were designed to target the coding or non-coding RNA sequences of one of the human APOE transcripts (Figure 1, SEQ ID NO: 1). Conserved regions between humans and non-human primates were identified and 22 nt target sequences were selected (SEQ ID NO: 2-93, Table 1) to generate different miRNA guide strands (SEQ ID NO: 94-185, Table 2). Scrambled miRNA guide strands were designed to generate negative controls (SEQ ID NO: 186-189). In addition, a scrambled miRNA guide previously designed for a different research program was used for in vivo testing (SEQ ID NO: 294).

[0136] DNA constructs miAPOE and scrambled control guide sequences were embedded in a human pri-miR-451 scaffold (Figure 2a, SEQ ID NO: 190) flanked by 178 or 206 nt 5' and 139 or 205 nt 3' flanking sequences. The pri-miAPOE cassette was expressed from a CMV immediate early enhancer fused to a chicken β-actin promoter (CAG promoter, SEQ ID NO: 191) or an adapted / synthetic promoter (P1, SEQ ID NO: 192 and P2, SEQ ID NO: 193) and terminated by a simian virus 40 polyadenylation signal (SV40 polyA, SEQ ID NO: 194).

[0137] The APOE transgene was expressed from a CAG promoter (SEQ ID NO: 191) or an adapted / synthetic promoter (P1, SEQ ID NO: 192 and P2, SEQ ID NO: 193) and terminated by SV40 polyA (SEQ ID NO: 194). The APOE sequence was codon optimized for Homo Sapiens using the online codon optimization tool from ThermoFisher, with NheI, NotI and SpeI sites left intact during codon optimization.

[0138] miAPOE (SEQ ID NO: 108, 116, 146) or scrambled negative control (SEQ ID NO: 186) and APOE transgene (SEQ ID NO: 195, 197, 200, 196, 198, 201, 206, 208, 210, 207, 209, 211, 214, 216, 216) were combined to facilitate simultaneous knockdown of APOE4 and overexpression of protective APOE variants (combinatorial approach). Plasmid DNA constructs containing the intronic elements of the P2 promoter with the pri-miAPOE cassette and the APOE transgene cassette were synthesized with the addition of 5' (BlpI) and 3' (EcoRV) sequences and subcloned by Genewiz (Azenta Life Sciences). Expression of the combinatorial approach constructs was driven by the P4 promoter (P3, SEQ ID NO:232 + intronic element, SEQ ID NO:231) and terminated by the SV40 polyA signal (SEQ ID NO:194). The identity of each construct was confirmed by sequencing.

[0139] An APOE4 luciferase reporter was generated containing a complementary APOE target region (1166 bp, SEQ ID NO: 218) fused to the Renilla luciferase (RL) gene (Figure 3). The target region sequence was synthesized with the addition of 5' (XhoI) and 3' (PmeI) sequences and cloned into the 3'UTR of the Renilla luciferase (RL) gene in the psiCHECK-2 vector (Promega, Madison, WI) by GeneWiz (Azenta Life Sciences).

[0140] AAV5 vectors Recombinant AAV5 particles were produced by infecting serum-free SF+ insect cells (Protein Sciences Corporation, Meriden, Connecticut, USA) with two baculoviruses, one encoding the Rep / Cap combination and the other carrying the transgene construct. The titer of purified AAV was determined using QPCR following standard protein purification procedures on a fast protein liquid chromatography system (AKTA Avant 150, GE30 Healthcare) using AVB Sepharose (GE Healthcare).

[0141] Recombinant AAV5 and AAV9 were also produced by PEI transfection of HEK293T cells with two plasmids encoding Rep-Cap and the transgene (Sirion Biotech). After two-step purification by primary capture with POROS™ CaptureSelect™ AAV-X resin (ThermoFisherScientific) and an iodixanol gradient, the titers of purified AAV were determined using QPCR.

[0142] Transfection experiments Human hepatocellular carcinoma (Huh7), human embryonic kidney 293T (HEK293T) or U118 astrocytoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum without antibiotics. The day before transfection or transduction experiments, cells were seeded into 24-well plates at a density of 1E+05 cells / well.

[0143] Transfections were performed using Lipofectamine® 2000 or Lipofectamine® 3000 according to the manufacturer's protocol.

[0144] Luciferase assay HEK293T cells were co-transfected with miAPOE or the above combination constructs together with a luciferase reporter containing both the RL gene and the firefly luciferase (FL) gene fused to the APOE target sequence. Equal amounts of DNA were introduced by adding pBluescript. Transfected cells were harvested 48 hours after transfection in 100 μl of 1x passive lysis buffer (Promega, Thermo Fisher Scientific) by gentle rocking at room temperature for 15 minutes. Cell lysates were centrifuged at 4,000 rpm for 5 minutes, and 10 μl of the supernatant was used to measure FL and RL activities with the Dual-Luciferase Reporter Assay System (Promega, Thermo Fisher Scientific). Relative luciferase activity was calculated as the ratio of RL activity to FL activity.

[0145] Transduction experiments For transduction, HEK293T cells were seeded in 24-well plates at a density of 1E+05 cells per well one day before transduction. The next day, cells were incubated with AAV vectors at multiplicities of infection (MOI) of 1E+04, 1E+05E, and 1E+06 gc / cell. Cells and culture supernatants were harvested two days after transduction.

[0146] For transduction of U-118 MG cells (ATCC® HTB-15™), cells were cultured at 2.63E+4 cells / cm 1 day prior to transduction. 2 The cells were seeded in 12- or 24-well plates at a density of 1.55E+06 gc / cell. The next day, the cells were incubated with AAV vectors at a multiplicity of infection (MOI) of 1.55E+06 gc / cell. The cell medium was changed 2 days after transduction, and the cells were harvested 3 days after transduction for DNA and RNA isolation.

[0147] Pulverization of mouse tissue using a cryogenic grinding system Frozen tissue samples were ground using an automated cryogenic sample grinding system. Snap-frozen tissues were disrupted by applying one or more punches of various impacts with a CryoPREP system type CP02 (Covaris). Before and after each impact, the tissue TUBE (Covaris) containing the tissue was immersed in liquid nitrogen and the procedure was repeated until the sample was ground. The powder was stored at -80°C in cryovials (Covaris or Corning) until further use (e.g., DNA and RNA extraction for APOE protein measurement, lysate production).

[0148] Isolation and quantification of vector DNA and mRNA DNA and RNA extraction for in vitro experiments was performed using either the All prep 96 DNA / RNA Isolation Kit (Qiagen, 80311), the All Prep DNA / RNA Mini Kit (Qiagen, 80204) or the MagMAX™ mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were harvested in lysis buffer according to the manufacturer's protocol.

[0149] DNA and RNA extraction for in vivo experiments was performed using the All prep 96 DNA / RNA isolation kit (Qiagen, 80311) or the All Prep DNA / RNA Mini kit (Qiagen, 80204). Tissue lysates were generated by adding ground tissue to Lysis Matrix D tubes (MP Biomedicals) containing the supplied RLT lysis buffer and β-mercaptoethanol (Sigma) and homogenizing in a TissueLyser II (QIAGEN) for 60 seconds at a frequency of 30 Hz according to the manufacturer's protocol.

[0150] DNA and RNA concentrations and purity ratios were quantified in duplicate using a spectrophotometer (either NanoPhotometer® N120 (IMPLEN) or SynergyHT in Take3MicrovolumePlate (BioTek) or NanoDropOne or 2000 (ThermoScientific)). Samples were stored at -80°C until further use.

[0151] After extraction, DNA concentrations were normalized to ensure equal input into quantitative polymerase chain reaction (qPCR). Vector genome copies were quantified by using TaqMan qPCR assays (SEQ ID NOs: 219-222, and SEQ ID NO: 277; Table 4) using linearized plasmid standard stocks (5E+0-5E+7) and ACTB TaqMan [Human ACTB Primer / Probe Mix Hs01060665_g1 (Thermo Fisher Scientific) and (SEQ ID NOs: 225-227; Table 4) or SybrGreen qPCR (SEQ ID NOs: 223-224; Table 4) assays as loading controls.

[0152] After extraction, RNA concentrations were normalized prior to DNase treatment to ensure equal input for cDNA synthesis and subsequent qPCR. DNase treatment of isolated RNA was performed by using TURBO DNAse™ provided in the RNA isolation kit (Thermo Fisher Scientific) or DNase with the Maxima First Strand cDNA Synthesis Kit (K1672) for cDNA synthesis (Thermo Fisher Scientific) according to the manufacturer's instructions. WT and codon-optimized variants V2 and V3 APOE mRNA expression was quantified using single or duplex qPCR using TaqMan qPCR assays (SEQ ID NOs: 228-230 for WT APOE and SEQ ID NOs: 287-291 for codon-optimized variants; Table 4) and β-actin ACTB TaqMan [Human ACTB Primer / Probe Mix Hs01060665_g1 (Thermo Fisher Scientific), Mouse ACTB Primer / Probe Mix mm01205647_g1 (Thermo Fisher Scientific)] or SybrGreen qPCR assays (SEQ ID NOs: 223-224; Table 4) as reference genes for normalization. Either absolute or relative gene expression levels were calculated using either ddCT (Livak's method) or linearized plasmid standard strains (1E+8-1E+3 or 1E+2).

[0153] qPCR was performed using a QuantStudio 5 Real-Time PCR System or a 7500 Fast Real-Time PCR System (ThermoFisher). The lower limit of quantification (LLOQ) was determined by calculating the copy amount at the mean Ct value of the lowest point of the standard line, corrected according to the input of (c)DNA (ng) used.

[0154] [Table 23]

[0155] miRNA RT-qPCR and quantification DNase treatment of RNA isolated from in vivo experiments and cDNA synthesis were performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and the E. coli poly(A) polymerase kit (New England Biolabs) according to the manufacturer's instructions. For reverse transcription, the RT primers shown in Table 5 were developed.

[0156] miRNA expression was quantified using SybrGreen qPCR assays specific for each miRNA (SEQ ID NOs: 278-286 and 292-293 in Table 5). For in vivo hAPOE4-Tr mouse studies, relative miRNA expression was calculated using standard lines obtained from each RNA oligo (22NT for miAPOE-016, -037 and -145, and 23NT for miSCR1-C9O). Based on the in vitro small RNA sequencing data, the assay was optimized for in vivo WT mouse studies, and standard lines were generated from RNA oligos with the sizes of the most abundant isoforms (24NT for miAPOE-016, 25NT for miAPOE-037, 24NT for miAPOE-145 and 23NT for miAPOE_SCR).

[0157] The lower limit of quantification (LLOQ) is the lowest amount of analyte in a sample that can be reliably quantified with an acceptable level of precision. It is calculated using the lower limit of the standard line (reliably quantifiable) and the corresponding copies per ng of RNA.

[0158] The lower limit of detection (LLOD) is the lowest amount of analyte in a sample that can be reliably detected, but not necessarily quantified. It is calculated using the lowest Ct value detectable by the machine and the corresponding copies per ng of RNA.

[0159] [Table 24]

[0160] RNA isolation for small RNA sequencing and polyA-enriched mRNA sequencing For in vivo, ground tissues were lysed in Lysing Matrix D tubes (MP Biomedicals) containing RLT lysis buffer and β-mercaptoethanol (Sigma) using a TissueLyser II (QIAGEN) at a frequency of 30 Hz for 60 seconds. RNA was extracted using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN) according to the manufacturer's protocol. For in vitro, transduced cells were harvested and lysed in TRIzol™ Reagent (Invitrogen). RNA was extracted using the Direct-zol™ RNA MiniPrep Kit (Zymo Research R2052) according to the manufacturer's protocol. RNA quantification was performed by measuring nucleic acid concentration and purity ratios in duplicate using a NanoDrop™ One (Thermo Scientific). The quality of the RNA samples was analyzed using the Agilent High Sensitivity RNA ScreenTape (Agilent) before shipping. Samples were stored at -80°C and transported on dry ice to GenomeScan for small RNA sequencing (Illumina NovaSeq6000 sequencing, Paired-End, 150bp. Approximately 3Gb per sample, 10 million paired-end reads) and polyA enriched mRNA sequencing (Illumina NovaSeq6000 sequencing, Paired-End, 150bp. Approximately 9Gb per sample, 30 million paired-end reads).

[0161] RNAseq data analysis Raw RNA-seq data were provided by GenomeScan. For data analysis, CLC Genomics Workbench version 21.0.5 was used. For small RNA sequencing analysis of both in vivo and in vitro derived samples (representing the caudal cortex of administered hAPOE4-Tr mice or transduced U-118MG cells, respectively), the obtained data were aligned and annotated to the miAPOE or miSCR reference sequences (SEQ ID NOs: 295-297; Table 7). The length and matching of mature miRNA isoforms were identified and their abundance was calculated as the percentage of all reads mapping to the reference miAPOE or miSCR sequences. During the analysis, a threshold of 2% of the mature form was adopted for convenience. Ranking of endogenously expressed miRNA levels together with miAPOE or miSCR transcript levels was performed by correcting the reads in counts per million (CPM) using the CLC Genomics Workbench toolbox for small RNA sequencing analysis. miR-29a-3p and miR-16-5p were used as internal controls, which are miRNAs expressed in the brain.

[0162] For mRNA sequencing analysis of in vitro derived samples (transduced U-118MG cells), the obtained data were aligned and annotated to the host species (Homo Sapiens for the transduced U-118MG experiment). Using the CLC Genomics Workbench Toolbox for Differential Expression of RNA-Seq, the data sets obtained from samples transduced with the AAV5 combinatorial approach or single constructs were compared to the data set obtained from samples transduced with AAV5-miSCR (control).

[0163] [Table 25]

[0164] APOE protein measurement APOE concentrations in cell culture supernatants were quantified using a human apolipoprotein E (APOE) ELISA kit (ab108813, Abcam). This kit recognizes all three human APOE isoforms (APOE2, APOE3 and APOE4). Several dilutions of cell culture supernatant samples were tested to ensure accurate measurements within the range of the provided standards. For HEK293T cells transfected with an APOE expression construct, cell culture supernatants were diluted 100-fold in the provided ELISA assay buffer. After sample preparation, APOE was measured using the provided kit protocol and concentrations were quantified using a four-parameter logistic curve fit in PRISM.

[0165] Human Apolipoprotein E (APOE) MSD R-PLEX assay (K1512IR-2, Meso Scale Discovery) was used to quantify APOE concentration in tissue lysates. Tissues were lysed in MSD Tris Lysis Buffer (R60TX-2, Meso Scale Discovery) and corrected for total protein concentration using Pierce™ 660nm Protein Assay Reagent (22660, ThermoFisher). Lysates at 2mg / ml total protein were diluted 50-fold with MSD Diluent 100 (R50AA-2, Meso Scale Discovery). After sample preparation, APOE was measured using the kit's supplied protocol and concentrations were quantified using MSD Discovery Workbench (software version 4.0).

[0166] Phosphorylated tau (pTau181) and total tau protein measurement Human pTau181 MSD S-PLEX assay (K151AGMS, Meso Scale Discovery) was used to quantify pTau181 protein concentration, and multispot phospho(Thr231) / total tau assay (K15121D, Meso Scale Discovery) was used to measure total tau in mouse hippocampal tissue lysates. Tissues were lysed in MSD Tris Lysis Buffer (R60TX-2, Meso Scale Discovery) and normalized for total protein concentration using Pierce™ 660nm Protein Assay Reagent (22660, ThermoFisher). Hippocampal lysates were diluted 10000-fold in dPBS for pTau181 assay and 200-fold in dPBS for total tau assay. Samples were then further diluted 2-fold in blocking buffer supplied with the respective kit. After sample preparation, pTau181 and total tau protein concentrations were measured using MSD Methodological Minds software using the supplied protocols of the two kits and further processed using MSD Discovery Workbench (software version 4.0).

[0167] SDS-PAGE and Western Blot Samples were prepared in 1x Laemmli sample buffer (1610747; Bio-Rad) and heated for 5 min at 95°C. Proteins were separated on Mini-PROTEAN TGX Stain-Free Protein Gel 4-20% (4568093; Bio-Rad) and transferred to PVDF membranes using Trans-blot Turbo Mini PVDF Transfer packs (1704156; Bio-Rad) and the Trans-Blot Turbo™ Transfer System (1704150; Bio-Rad).

[0168] PVDF membranes were blocked with SuperBlockT20 (PBS) Blocking Buffer (37516; Bio-Rad) and stained with primary antibodies in blocking buffer. After washing with 0.5% Tween-20 in phosphate-buffered saline (PBS), membranes were incubated with secondary antibodies in blocking buffer. After extensive washing with PBS-0.5% Tween 20, proteins were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34580; ThermoScientific) and the ChemiDoc Touch Gel Imaging System (1708370; Bio-Rad).

[0169] The following primary and secondary antibodies were used against each protein: APOE (Sigma Aldrich; HPA065539 & HPA068768), Polyclonal Goat Anti-Rabbit Immunoglobulins-HRP (DAKO, P0448) (DAKO), Polyclonal Rabbit Anti-Mouse Immunoglobulins-HRP (DAKO, P0260).

[0170] Known concentrations of recombinant human APOE protein (Sigma Aldrich; SRP4760, SRP4696, A3234) or apolipoprotein E from human plasma (Sigma Aldrich; SRP6303) were used as references.

[0171] Animal testing To study the efficacy of miAPOE, a transgenic model carrying the human APOE4 gene, B6.129P2-APOE tm3(APOE*4)Mae N8 was used. At 8 weeks of age, male B6.129P2-Apoe tm3(APOE*4)MaeN8 mice were treated with a single bilateral intrastriatal (IS) administration of empty AAV5, AAV5-miSCR (SEQ ID NO: 186), or AAV5-miAPOE_016 (SEQ ID NO: 108), (medium and high doses), or AAV5-miQURE-miAPOE_037 (SEQ ID NO: 116), (medium and high doses), or AAV5-miAPOE_145 (medium and high doses), or AAV9-miAPOE_145 (medium dose) (SEQ ID NO: 146). Each group contained 8 animals. Body weight (individual) was determined before treatment and every week thereafter. Before treatment, 3, 6 and 8 weeks after treatment (sacrifice), the mice were subjected to blood sampling for plasma preparation. Plasma total cholesterol, triglycerides, low density lipoprotein (LDL), high density lipoprotein (HDL) (all per individual mouse) were determined for all time points in collected plasma samples. Mice were sacrificed after 8 weeks of treatment. Multiple organs and CSF were collected for analysis of AAV transduction by qPCR, APOE mRNA expression, hAPOE4 expression levels, and miAPOE transgene expression.

[0172] To test the expression of APOE3ch in C57Bl6 mice, eight mice per group were intracerebroventricularly injected with vehicle, a single bilateral intrastriatal dose of AAV5 or AAV9 vector, or a single bilateral dose of AAV5 or AAV9 vector. All AAV vectors carry an APOE3ch expression cassette with a double HA tag. Blood samples were taken before administration, 2 weeks after administration, and 4 weeks after (sacrifice). Four weeks after treatment, animals were sacrificed. After perfusion, terminal blood and CSF samples were taken for each animal. For five animals per group, several brain regions, spinal cord, and liver were collected and snap frozen. For three animals per group, brains were fixed for immunohistochemistry and FISH purposes.

[0173] The efficacy of AAV5 vectors encoding different APOE variants to modify the phenotype of tauopathy model P301S mice (PS19, JAX strain: 008169) was evaluated by a single bilateral intrastriatal injection of AAV vectors. Twelve male mice per group are treated at 8 weeks of age. Body weight is measured before treatment and once a week thereafter. Blood samples are taken before treatment and at 2, 4, 12 and 20 weeks after treatment. A panel of markers is analyzed from the plasma for the following parameters: LDL, HDL, cholesterol and triglycerides on terminal plasma samples. Mice are subjected to behavioral tests to evaluate motor and cognitive function. At sacrifice 28 weeks after treatment, terminal blood and CSF samples are taken. Several different brain regions, separate spinal cord segments and livers of the animals are collected and snap frozen for 8 mice of each group. The brains of the remaining 4 animals are perfused and fixed for IHC and FISH purposes.

[0174] In vivo expression levels of transgenes with the combination approach constructs compared to single constructs were assessed by single bilateral intrastriatal administration of AAV vectors in WT mice. Six mice per group were treated at 8 weeks of age. Animals were sacrificed one month after treatment. Multiple brain regions and livers were collected and snap frozen for 5-6 animals per group.

[0175] The in vivo expression levels and effects on tauopathy of AAV encoding transgenes derived from the combination approach constructs compared to single constructs were examined in a tauopathy model P301S mouse (PS19, JAX strain: 008169) that also carries hAPOE4 (B6.129P2- APOEtm3(APOE*4)MaeN8 The efficacy of AAV in mice is evaluated using a single bilateral intrastriatal administration of AAV vectors in mice (Table 1). Six mice per group are treated at 8 weeks of age. Animals are sacrificed several months after treatment. Multiple brain regions, liver, and biofluids are collected and snap frozen for all animals per group.

[0176] In silico evaluation of potential off-target transcripts in miAPOE guide In silico off-target prediction for the miAPOE miRNA guide sequences (SEQ ID NOs: 108, 116, and 146) was performed using BLASTN against the human reference transcriptome (ENSEMBL, https: / / www.ensembl.org / Homo_sapiens / Tools / Blast, see Table 8 for specifications; Bedell et al., 2003) to identify transcripts that are partially complementary to the 22-nucleotide miAPOE guide sequence.

[0177] [Table 26]

[0178] The “siRNA Seed Potential of Off-Target Reduction”-tool (siSPOTR, Boudreau et al., 2013) was used to predict binding of miAPOE miRNA guide seed sequences (nucleotides 2–8) to off-target transcripts.

[0179] result Example 1: In vitro testing of miAPOE constructs on the APOE4Luc reporter system To test the knockdown efficiency of the designed miAPOE constructs, HEK293T cells were co-transfected with a miAPOE (SEQ ID NOs: 94-185) expression construct and an APOE4 luciferase reporter (SEQ ID NOs: 2-93) with complementary APOE targeting regions.

[0180] First, because exogenous expression of miR-144 (SEQ ID NO: 235) can result in side effects (e.g., miR-144 target binding) (Huang et al. 2021; H. Li et al. 2016; Lin et al. 2020), we designed a novel strategy to introduce modifications into the scaffold to abrogate miR-144 expression. We engineered a mutant form of the miR-144 (SEQ ID NO: 236) hairpin in which the adenosine at position 5 of the Drosha cleavage site was replaced by thymidine (T), creating a bulge near the cleavage site, which disrupted miR-144 expression (S. Li et al. 2020, 2021).

[0181] A mismatch, bulge, or GU wobble introduced at positions 4-8 from the Drosha cleavage site impairs the enzymatic activity of Drosha. Double and triple mismatches, bulges, or wobbles within the 4-8 stretch further reduce Drosha activity. Thus, any of the following SNPs and their combinations within the 4-8 nucleotide stretch of miR-144 can alter (pre-)miR-144 expression. These mutations include U>G at position 4, and / or A>U or G at position 5, and / or U>A at position 6, and / or C>G or U at position 7, and / or A>U or G at position 8.

[0182] Next, a set of scaffolds was created expressing synthetic miRNA6 (=miHTT) derived from miR-451, expressing miRNA6 as a single hairpin (SEQ ID NO: 238) or associated with either miR-144 wild type (miR-144WT helper) (SEQ ID NO: 239) or -144 mutant (miR-144A>T helper) (SEQ ID NO: 240). miRNA6 expression was assessed across the different scaffolds by small RNA sequencing (Figure 3A). The introduced bulge repressed the expression of miR-144 5p and 3p without affecting its helper function for miR-451, as shown by the expression values ​​of miRNA6 remaining unaffected when associated with either miR-144WT helper or miR-144A>T (Figure 3A).

[0183] Subsequently, the expression values ​​of a series of different synthetic miRNA sequences (SEQ ID NO: 241-248) expressed from the miR-451 scaffold (with or without miR-144A>T helper) (SEQ ID NO: 190) were also analyzed, confirming an unexpected and significant improvement of miR-451 expression synthetic miRNA in the presence of miR-144A>T (SEQ ID NO: 236) (Figure 3B). Thus, the use of the mutant helper miR-144 allows for the exploitation of miR-144-miR-451 activity without the expression of undesired microRNAs, allowing for higher synthetic miRNA expression levels. Thus, the miR-144A>T mutant-miR-451 cluster was selected for use.

[0184] Initial screening was performed on HEK293T cells co-transfected with 250 ng of miAPOE (SEQ ID NOs: 94-185) or miSCR constructs (SEQ ID NOs: 186-189) and 50 ng of reporter construct. Two days after transfection, cells were harvested and luciferase expression was measured. Luciferase expression in the presence of miSCR was set to 100%.

[0185] Most of the transfected miAPOE constructs showed a relative reduction in luciferase activity compared to the miSCR construct (SEQ ID NO: 186). miAPOE with SEQ ID NOs: 96, 105, 106, 108, 113, 115, 116, 131, 132, 133, 134, 135, 136, 137, 139, 146, 149 resulted in a knockdown of reporter activity of ≧70% ( FIG. 4 ).

[0186] To more precisely define the efficacy of miAPOE constructs, potent miAPOE constructs with knockdown efficiency of 60% or more were tested in titration experiments using 2ng, 10ng or 50ng of miAPOE (SEQ ID NO: 96, 99, 100, 105, 106, 108, 109, 113, 114, 115, 116, 117, 131, 132, 133, 134, 135, 136, 137, 139, 143, 146, 149, 151, 185) or miSCR constructs (SEQ ID NO: 186) and 10ng of reporter construct (n=2). Luciferase expression in the presence of miSCR was set to 100%. All constructs showed dose-dependent reporter knockdown (Figure 5).

[0187] Example 2: In vitro testing of miAPOE - knockdown of endogenous APOE (mRNA and protein) The ability of miAPOE candidates to silence endogenously expressed APOE mRNA and reduce APOE protein levels was tested in Huh7 cells.

[0188] Huh7 cells were co-transfected with 250 ng of miAPOE constructs (SEQ ID NOs: 94-185) or miSCR constructs (SEQ ID NOs: 186-189). APOE mRNA expression was determined using RT-QPCR and calculated using the ddCT method using β-actin gene expression as a reference gene. APOE mRNA was reduced in most of the transfected miAPOE constructs compared to the average of the four SCR constructs, pBluescript and the non-transfected set, which was set to 100%. miAPOE SEQ ID NOs: 96, 98, 99, 108, 109, 113, 115, 116, 136, 138, 144, 146, 149, 151, 160, 163, 171 and 185 reduced endogenous APOE mRNA expression by >50% or more (Figure 6).

[0189] Nine miAPOE constructs that showed a reduction in reporter activity of ≥60% in previous experiments were selected to test targeting at lower doses. Huh7 cells were co-transfected as above with 50 or 250 ng of miAPOE (SEQ ID NOs: 96, 108, 116, 133, 136, 138, 146, 151, 185) or miSCR constructs (SEQ ID NOs: 186-187) and 10 ng of pFI reporter construct. APOE mRNA expression of the average miSCR sample was set to 100% expression. All constructs showed a reduction in APOE mRNA of ≥60% after transfection with 250 ng of plasmid, and a reduction of ≥50% after transfection with 50 ng of plasmid (Figure 7). Additionally, APOE protein levels in culture supernatants were quantified in 250 ng transfected samples using Western blot. APOE protein levels were quantified in the supernatants of transfected cells compared to untransfected cells and showed a 70-90% reduction for all miAPOEs tested (Figure 8).

[0190] Example 3: In vitro study of endogenous APOE mRNA expression in Huh7 cells upon transduction with AAV-miAPOE Recombinant AAV (5 or 9) vectors were generated carrying expression cassettes for miAPOE_016 (SEQ ID NO: 108), miAPOE_037 (SEQ ID NO: 116), miAPOE_145 (SEQ ID NO: 146) and miSCR_144 (SEQ ID NO: 186). The ability of the resulting AAV to transduce and deliver the packaged expression cassettes was tested by transducing Huh7 cells at a multiplicity of infection (MOI) of 5E+06, 3.6+06, 1E+06, 5E+05 or 5E+04 gc / cell. Vector DNA copies and residual APOE mRNA were assessed 48 hours after transduction by qPCR and RT-QPCR using TaqMan qPCR duplexes. Vector DNA levels were quantified using a standard line ranging from 1E+07 to 5E+01 copies. A dose-dependent increase in detected vector genomic DNA copies was observed. The transduction efficiency of Huh7 cells was comparable for all AAV5 batches with the same MOI. AAV9 vectors show lower vector genome copies in Huh7 cells upon transduction. Similar to AAV5 vectors, there is a dose-dependent increase in vector copies detected upon transduction (Figure 9A).

[0191] Endogenous APOE mRNA expression was calculated by using the ddCT method, using β-actin as the reference gene. APOE mRNA expression in the presence of AAV5-miSCR was set to 100% expression. The results are shown in Figure 9B.

[0192] All AAV batches resulted in a dose-dependent reduction in APOE mRNA expression, confirming the functionality of the AAV-delivered expression cassette and the potency of miAPOE. AAV5-miAPOE_037 (AAV5) and AAV5-miAPOE_016 also showed a clear dose-dependent reduction: miAPOE_037 showed the greatest potency in reducing APOE mRNA expression at MOI 5E+06 (about 55% reduction) and MOI 1E+06 (about 35% reduction), with little reduction observed at MOI 5E+05 (about 10% reduction), and no reduction at all at 5E+04gc / cell. Similarly, miAPOE_016 showed the highest reduction at MOI 5E+06 (about 35% reduction), MOI 1E+06 (about 25% reduction), and MOI 5E+05 (about 15% reduction).

[0193] For AAV5-miAPOE_145, a reduction in APOE mRNA expression of approximately 15% for MOI5E+06 and approximately 25% for MOI1E+06 was observed, with a similar range of reduction seen for AAV9-miAPOE_145.

[0194] Example 4: Testing of AAV-miAPOE targeting APOE in hAPOE4TR mice To investigate the ability of selected miAPOE candidates to silence human APOE4 expression in vivo, a hAPOE4 targeted replacement (TR) mouse model was used. Male mice were treated with a single bilateral intrastriatal (IS) administration of empty AAV5, AAV5-miSCR (previously mislabeled as SEQ ID NO: 186, SEQ ID NO: 294), or AAV5-miAPOE_016 (SEQ ID NO: 108), (medium and high doses), or AAV5-miQURE-miAPOE_037 (SEQ ID NO: 116), (medium and high doses), or AAV5-miAPOE_145 (medium and high doses), or AAV9-miAPOE_145 (medium dose) (SEQ ID NO: 146). The medium dose was set to a total of 6E10 gc per mouse, and the high dose was set to a total of 3E11 gc per mouse. Each group contained 8 animals. Eight weeks after treatment, animals were sacrificed and brain levels of vector DNA, APOE mRNA, miAPOE or miSCR, and hAPOE4 protein were determined.

[0195] AAV transduction was analyzed by quantification of vector DNA levels by qPCR. Mice treated with AAV5-miSCR, AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, and AAV9-miAPOE_145 all showed a significant increase in vector DNA levels in the striatum and cortex, reaching a maximum of 2.05E+08 gc / μg DNA and 2.54E+07 gc / μg DNA, respectively (Figure 16A-B). The lack of a dose-dependent increase in vDNA indicates that both doses reach transduction saturation.

[0196] Silencing of human APOE mRNA by selected miAPOE candidates (miAPOE_016, miAPOE_037, and miAPOE_145) was evaluated by RT-qPCR. In the striatum, AAV5-miSCR showed similar mRNA levels compared to the empty AAV5 group, at 3.82E+07 copies / μg RNA. A significant reduction in hAPOE4 mRNA levels of up to >80-90% was observed in mice injected with AAV-miAPOE candidates compared to AAV5-miSCR (Figure 17A-B). In the frontal cortex, variation was observed between hAPOE4 mRNA levels in mice injected with empty AAV5 or AAV5-miSCR. A reduction of >50-60% in hAPOE4 mRNA was observed in mice injected with AAV-miAPOE candidates compared to AAV5-miSCR (Figure 17C-D).

[0197] Furthermore, miRNA (miAPOE or miSCR) levels were quantified by SYBR green RT-qPCR assay using specific primers for 22-nucleotide or 23-nucleotide mature miAPOE and miSCR. The miRNA levels in the striatum of AAV-miAPOE-injected mice reached a maximum of 2.45E+11 copies / μg total RNA (FIG. 18). 1.45E+08 miRNA copies / μg total RNA were found for mice injected with AAV5-miSCR. Striatal tissues of mice injected with empty AAV5 were also employed in all RT-qPCR experiments using specific primers for either miAPOE or miSCR. In all experiments, the amount of miRNA copies / μg total RNA in the striatum of mice injected with empty AAV5 was below the LLOQ (not shown).

[0198] To investigate whether silencing of hAPOE4 mRNA correlates with a reduction in hAPOE4 protein levels, hAPOE-MSD was performed in frontal cortex tissue. hAPOE4 protein levels in mice injected with AAV5-miSCR were equal to control levels (empty AAV5) at 41 ng / mg, again confirming that AAV5-miSCR does not affect hAPOE4 protein levels (Figure 19). A significant protein reduction of up to 50-60% was observed in mice injected with AAV-miAPOE candidate compared to AAV5-miSCR.

[0199] To examine the processing and abundance of expressed miAPOE relative to the expression levels of endogenous miRNAs, RNA was isolated from the tail cortex of two mice in the high dose group (AAV5-miAPOE_016, AAV5-miAPOE_037, and AAV5-miAPOE_145) and analyzed by small RNA sequencing. Data analysis revealed expected miAPOE transcripts with read counts ranging from 4.57 to 0.32% when compared to total miRNA reads (Figure 20B). The most abundant isomIRs produced by miAPOE_016, miAPOE_037, and miAPOE_145 are 23, 25, and 24 nucleotides long, respectively (Figure 20A). Total miSCR reads and total miAPOE reads of all samples were compared to the expression levels of endogenous miRNAs (Figure 20C). Considering that miAPOE is not one of the most abundant endogenously expressed miRNAs, the risk of oversaturation of the RNAi pathway by miAPOE is very low. Two internal controls, miR-29a-3p and miR-16-5p, showed consistent abundance rankings of 10.5±1 (SD) and 37.37±2.44 (SD) among all samples, respectively (marked in Figure 20C). Taken together, these results demonstrate high levels of vDNA and miRNA in vivo, as well as a significant reduction in hAPOE4 mRNA and protein levels of the AAV-miAPOE candidate. Thus, these results reveal that upon miAPOE expression delivered via AAV, high levels of miRNA are expressed, resulting in a significant reduction in hAPOE4 mRNA and protein levels.

[0200] Example 5: In vitro testing of overexpression of APOE variants APOE expression constructs were transfected into HEK293T cells and APOE protein expression was measured in the supernatants 2 days after transfection. The following APOE constructs were tested: APOE2ch WT (R136S mutation, SEQ ID NO: 197), APOE2ch v1 and APOE2ch v2 (codon optimized for Homo Sapiens using ThermoFisher's online codon optimization tool) variants, SEQ ID NO: 204, 208), APOE2 WT (SEQ ID NO: 195) and APOE2 v1 and v2 (2 codon optimized variants, SEQ ID NO: 202, 206), APOE3 WT (SEQ ID NO: 196) and APOE3 v1 and v2 (codon optimized variants, SEQ ID NO: 203, 207) and APOE3ch WT (R136S mutation, SEQ ID NO: 198), APOE3ch v1 and APOE3ch v2 (codon optimized variants, SEQ ID NO: 205, 209).

[0201] APOE protein expression was detected in all cells transfected with the different APOE constructs (Figure 10A). Recombinant APOE3 protein served as a positive control. The WT forms of all constructs, APOE2ch, APOE3ch, APOE2 and APOE3, showed the highest protein expression among the different variants. Furthermore, the expression of APOE constructs was also confirmed in astrocytoma cells (Figure 10B).

[0202] Example 6: In vitro studies - Expression of APOE variants in HEK293T cells upon transduction with AAV5-APOE variants To examine the ability of AAV5-APOE variants to transduce and deliver packaged expression cassettes, HEK293T cells were transduced with a multiplicity of infection (MOI) of 1E+04, 1E+05, 1E+06gc / cell (n=1). Vector DNA levels in cells were quantified by qPCR. APOE variant protein expression was analyzed in the culture supernatant of cells transduced with 1E+05, 1E+06gc / cell via Western blot as previously described. The results are shown in Figure 11A. All five AAV5-APOE batches tested resulted in dose-dependent vector DNA levels in cell lysates. Untransduced cells showed background levels of approximately E3gc / ug DNA. APOE variant protein expression was confirmed by Western blot in the culture supernatant of cells transduced with 1E+06gc / cell (Figure 11B).

[0203] Example 7: WT mouse study to examine AAV5 / AAV9 APOE3ch-HA expression To test the expression of APOE3ch in C57Bl6 mice, eight mice per group were injected intracerebroventricularly with a single bilateral intrastriatal dose of AAV5 or AAV9 vectors, or a single bilateral dose of AAV5 or AAV9 vectors. All AAV vectors carry an APOE3ch expression cassette with a double HA tag. Four weeks after treatment, animals were sacrificed and vector DNA and mRNA levels were determined in the striatum of the brain. On average, the AAV5 and AAV9 groups that received vectors intrastriatal (IS) expressed 6e7 and 2e7 genome copies per ug of DNA. The intracerebroventricular (ICV) delivered AAV groups showed slightly lower vector genome copies, 4e6 copies for AAV5 and 3e6 copies for AAV9, respectively (12A). The mRNA copy number was determined in the striatum of the brain for all animals. The IS AAV5 and AAV9 groups expressed an average of 6e8 and 8e8 copies of mRNA in the striatum of the brain, while the ICV group showed slightly lower mRNA expression levels; 1.1e8 and 1.5e8 copies for the AAV5 and AAV9 groups (Figure 12B).

[0204] Example 8: Effects of APOE overexpression in different brain regions in the P301S tauopathy model The efficacy of AAV5 vectors encoding different APOE variants to modify the phenotype of tauopathy model P301S mice was evaluated by a single bilateral intrastriatal injection of AAV5 vectors. Two-month-old male mice were treated with vehicle (0.001% Pluronic F-68) and a single bilateral intrastriatal injection of empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2 and AAV5-APOE3b V2 (SEQ ID NOs: 197, 208, 210, 209 and 211) at a dose of 3E11gc / mouse. An untreated wild-type (WT) littermate control group was also included. The effect of AAV5-APOE vectors was evaluated by behavioral testing. Seven months after injection, molecular analyses of different brain regions, CSF, plasma and liver were performed in 8-11 animals per group. Brains were fixed for histological purposes in 1-4 animals per group.

[0205] Vector DNA and APOE mRNA levels in the striatum were quantified by RT-qPCR to analyze AAV transduction and APOE transcript expression. Mice treated with AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2 all showed a significant increase in vector DNA levels up to 4.67E+07 gc / μg DNA and APOE mRNA levels up to 4.48E+09 copies / μg RNA compared to WT, vehicle, and empty AAV5 groups (Figures 21A-B).

[0206] To examine whether the elevated mRNA copy levels in the APOE variant treatment groups were consistent with increased APOE protein expression, hAPOE-MSD was performed in hippocampus and frontal cortex tissues (Figure 21C-D). No hAPOE protein levels were detected in the WT, vehicle, and empty AAV5 groups. Elevated hAPOE protein levels were detected in the AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2 groups in both the hippocampus (up to 511.09 ng / mg) and frontal cortex (up to 2086.41 ng / mg). AAV5-APOE2ch WT administration resulted in the highest yield of APOE protein compared to the other groups, a pattern previously seen in vitro (Figure 15).

[0207] To understand whether expression of APOE variants affects the levels of pathogenic phosphorylated tau, soluble total tau and phosphorylated tau were examined. Separate MSD assays were performed to quantify total tau and p-tau181 on hippocampal tissue. Total tau was detected at comparable levels in all P301S mouse groups in addition to the WT group (Figure 22A). Significant reductions were detected in p-tau181 levels in the AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2 and AAV5-APOE3b V2 groups when compared to the vehicle group (up to 55%, Figure 22B-C). Examination of the quantitative relationship between pTau181 and total tau levels (Figures 22D-E) revealed up to a 70% decrease in pTau181 / total tau ratios for the AAV5-APOE2ch V2, AAV5-APOE2b V2 and AAV5-APOE3b V2 groups compared to the vehicle group.

[0208] Based on these results, we expect that immunohistochemistry will reveal changes in phosphorylated tau levels, neurofibrillary tangle levels, and astrocyte and microglia numbers in the hippocampus of mice injected with AAV5 vectors encoding different APOE variants compared to vehicle and / or empty AAV5 injection groups. As a joint finding of these immunohistochemistry studies, we expect to detect changes in the size of the ventricular cavity in the brains of vehicle and / or empty AAV5 injection groups in mice injected with AAV5 vectors encoding different APOE variants compared to the WT group and compared to the vehicle and / or empty AAV5 injection groups.

[0209] Taken together, these results demonstrate that treatment with AAV5-APOE variants results in high and comparable vDNA and mRNA levels, leading to increased APOE protein levels and reduced pTau181 levels.

[0210] Example 9: In vitro testing of combination construct-reporter reduction To test the ability to silence APOE4 and simultaneously overexpress APOE2 or APOE3 variants, HEK293T cells are co-transfected with the combined expression construct and an APOE luciferase reporter. miAPOE or APOE variant expression plasmids are used as controls.

[0211] In the initial screen, HEK293T cells were co-transfected with 86.54 fmol of combination constructs (SEQ ID NOs: 255-276), APOE variants (SEQ ID NOs: 197, 208, 210, 211) or miAPOE (SEQ ID NOs: 108, 116, 146, 186) and 10 fmol of reporter. Expression of all combination constructs (SEQ ID NOs: 255-276; Table 6) resulted in knockdown of the luciferase reporter (Figure 13). To further evaluate the potency of the different constructs, titration experiments were performed using DNA constructs (0.69, 3.46 and 17.31 fmol (corresponding to 2, 10 or 50 ng, see Example 1). A dose-dependent decrease was evident for all constructs tested (Figure 14).

[0212] [Table 27]

[0213] [Table 28]

[0214] [Table 29]

[0215] [Table 30]

[0216] [Table 31]

[0217] [Table 32]

[0218] [Table 33]

[0219] [Table 34]

[0220] [Table 35]

[0221] [Table 36]

[0222] [Table 37]

[0223] [Table 38]

[0224] [Table 39]

[0225] Example 10: Combination constructs - In vitro testing of APOE expression Furthermore, APOE protein expression was quantified by ELISA in the supernatant of cells transfected with the combination constructs. Protein quantification was evaluated only in the supernatant of cells transfected with the highest amount of DNA construct (e.g., 86.54 fmol). APOE protein expression was confirmed for all transfected cells (Figure 15).

[0226] Example 11: In vivo testing of AAV-delivered combination constructs in P301S / hAPOE4-TR mice The efficacy of the AAV5 vectors encoding the combined expression constructs and the single constructs to modify the phenotype of the tauopathy model P301S mice (P301S / hAPOE4-TR mice) is evaluated by a single bilateral intrastriatal injection of the AAV vectors. The effect of the AAV vectors is evaluated by behavioral tests and molecular analysis of different brain regions, CSF, plasma and liver.

[0227] We expect that vDNA levels and APOE variant mRNA / miRNA expression will be detected in the brain in the group administered the AAV vector.Indeed, high levels of miRNA are expected in the brain of the group injected with the construct containing miAPOE, whereas the group administered with the construct containing the APOE variant transgene is expected to show an increase in APOE variant mRNA.

[0228] Furthermore, it is expected that reduced or modified levels of hAPOE4, tau and phosphorylated tau protein will be observed in the brains of the AAV vector-injected group in contrast to the vehicle group.

[0229] Example 12: In vivo testing of AAV-delivered combination approach constructs in WT mice Transgene expression levels of the combination approach constructs and single constructs (4.3E10gc / mouse) mediated by AAV5 delivery were assessed by single bilateral intrastriatal injection in WT mice. Male mice were treated with IDs 2, 3, 16 and 18 of the AAV5 combination approach constructs (SEQ ID NO: 256, 257, 270 and 272, respectively) and single constructs including AAV5-APOE2ch V2 expression variant (SEQ ID NO: 208), AAV5-miSCR (SEQ ID NO: 186), AAV5-miAPOE_016 (SEQ ID NO: 108) and AAV5-miAPOE_037 (SEQ ID NO: 116). Empty AAV5 was included as a control. Animals were sacrificed one month after treatment. Multiple brain regions and livers were collected and flash frozen for 5-6 animals per group.

[0230] AAV transduction was analyzed by quantifying vector DNA levels by qPCR. In addition to the empty AAV5 treatment group, all groups showed comparable significant increases in vector DNA levels in the striatum, reaching up to 3.74E+07 gc / μg DNA (Figure 23A). miRNA levels were quantified by SYBR green RT-qPCR assay using specific primers for mature miSCR and miAPOE of 23, 24 or 25 nucleotides in length (Figure 23B). The selected primers and standard strains were optimized based on previous small RNA sequencing results (Figure 20A). miRNA levels in the striatum of the group injected with the combination approach vector reached up to 1.48E+09 copies / μg total RNA (Figure 23B). The miRNA levels of the AAV5-miSCR group were similar to those of the combination approach construct group. The miAPOE copy level in the AAV5-miAPOE_016 group reached a maximum of 7.06E+09 copies / μg total RNA, while the miRNA levels for the AAV5-miAPOE_037 group and the empty AAV5 group did not result in levels above the LLOQ. Tissues from the AAV5-APOE2ch V2 group were not subjected to this assay because miRNAs were not expected.

[0231] hAPOE mRNA expression in the striatum of all groups was assessed by RT-qPCR (Figure 23C). Elevated expression of hAPOE mRNA was detected up to 1.22E+10 copies / μg RNA in groups expressing the combination approach construct, and 7.08E+08 copies / μg RNA in the AAV5-APOE2ch V2 group. Although no mRNA detection was expected in the WT, AAV5-miSCR, AAV5-miAPOE_016, and AAV5-miAPOE_037 groups, the assay detected mRNA copies slightly above the LLOQ.

[0232] To examine whether the increase in mRNA copy levels in the groups injected with the combination approach vector and AAV5-APOE2ch V2 was consistent with increased APOE protein expression, hAPOE-MSD was performed in rostral cortex tissue (Figure 23D). hAPOE protein levels were not detected in the empty AAV5, AAV5-miSCR, AAV5-miAPOE_016 and AAV5-miAPOE_037 groups. hAPOE protein levels up to 478.83ng / mg were detected in the groups injected with the combination approach vector, while hAPOE protein levels in the AAV5-APOE2ch V2 group reached 129.48ng / mg protein levels.

[0233] Taken together, these in vivo results show high levels of vDNA across all groups, and similar or superior transcript and protein levels for the combinatorial approach constructs compared to constructs expressing single transcripts.

[0234] Example 13: AAV5 combinatorial approach to investigate miAPOE processing and off-target effects. Constructs and in vitro transduction of AAV5-miAPOE To investigate the processing and abundance of miAPOE expressed by the combinatorial approach constructs and single miAPOE constructs in human-derived glioblastoma cells, U-118MG cells were transduced with AAV5 combinatorial approach constructs ID2 and 16 (SEQ ID NO: 256 and 270), AAV5-miSCR (SEQ ID NO: 186), AAV5-miAPOE_016 (SEQ ID NO: 108), AAV5-miAPOE_037 (SEQ ID NO: 116) and AAV5-miAPOE_145 (SEQ ID NO: 146). miAPOE_016 is encoded in combinatorial approach construct ID2 and miAPOE_145 is encoded in combinatorial approach construct ID16. Three days after transduction, RNA was isolated from these cell cultures and analyzed by small RNA sequencing and polyA-enriched mRNA sequencing.

[0235] Data analysis revealed that for the combinatorial approach construct, miAPOE_016 and miAPOE_145 predicted miAPOE transcripts with read counts ranging from 2.68 to 0.05% when compared to total miRNA reads (Figure 24B). There were no read counts for miAPOE_037.

[0236] The most abundant isomIRs produced by the combinatorial approach constructs, miAPOE_016 and miAPOE_145, are all 24 nucleotides long (Figure 24A). The total miAPOE reads of all constructs, except miAPOE_037, were compared with the expression levels of endogenous miRNAs (Figure 24C). Considering that miAPOE is not one of the most abundant endogenously expressed miRNAs, the risk of oversaturation of the RNAi pathway by miAPOE is very low. Two internal controls, miR-29a-3p and miR-16-5p, showed consistent abundance rankings of 6.33±1.55 (SD) and 19.75±6.13 (SD), respectively, among all samples (marked in Figure 24C).

[0237] Differential expression analysis was performed to clarify potential off-target effects of the combination approach, miAPOE_016 and miAPOE_145 constructs. Using miSCR as a control, analysis was performed to clarify the extent to which the other constructs differentiated at the transcript level. This revealed very few differentially expressed transcripts for all samples with FDR p-value >0.05 (Figure 24D). As expected, the combination approach constructs significantly increased APOE transcripts. The combination approach construct ID16 had no other differentially expressed transcripts, while ID2 had two genes whose transcript levels deviated from the control. Both miAPOE_016 and miAPOE_145 had the same differentially expressed genes.

[0238] Taken together, these in vitro results demonstrated normal processing of miAPOE expressed by the combinatorial approach and by the single constructs, with minimal or no off-target effects observed within the transcriptomes of these miAPOE-expressing samples.

[0239] Example 14: In silico evaluation of potential off-target transcripts of miAPOE miRNA guides In silico analysis of potential off-target gene transcripts was performed on the miAPOE miRNA guide sequence (BLASTN) and guide seed sequence (siSPOTR). BLASTN against short sequences was performed against the human reference transcriptome (via ENSEMBL) to identify transcripts with partial complementarity of at least 7 consecutive nucleotides within the 22-nucleotide miAPOE guide sequence (SEQ ID NOs: 108, 116, and 146). No gene transcripts were found that were fully complementary to the 22-nucleotide miAPOE miRNA guide sequence outside of APOE. Gene transcripts with partial complementarity were ranked by alignment score, calculated from the sum of reward for matched nucleotides and penalty for mismatches and gaps (match / mismatch: 1 / -3). The top 15 hits are shown below (Table 9).

[0240] The siSPOTR tool (Boudreau et al., 2013) was used to predict the binding of miAPOE miRNA guide seed sequences (nucleotides 2-8 of the 5'-3' guide sequence) to potential off-target gene transcripts that are mainly found in the 3'UTR of the target transcripts. The potential off-targeting score (POTS) is calculated using the seed site type frequencies (8mer, 7mer-M8, 7mer-1A and 6mer) (Table 10). The list of possible off-target genes was ranked by individual transcript probability of off-target score (tPOTS). tPOTS was calculated based on the number and type of seed matches found in individual transcripts. The top 15 hits of potential off-target genes are listed in Table 11.

[0241] None of the top 15 hits for potential off-targets for the miAPOE guide seed sequence overlapped with the top 15 hits of in silico off-target predictions of the miAPOE guide sequence by BLASTN for short sequences. No in silico predicted transcripts were found to be significantly differentially expressed in the RNA-seq data from AAV5 combinatorial approach constructs ID2 and 16 (SEQ ID NO: 256 and 270), AAV5-miAPOE_016 (SEQ ID NO: 108) and AAV5-miAPOE_145 (SEQ ID NO: 146) (Figure 24D).

[0242] [Table 40]

[0243] [Table 41]

[0244] [Table 42]

[0245] [Table 43]

[0246] References Knouff C, Hinsdale ME, et al.Apo E structure determines VLDL clearance and atherosclerosis risk in mice.J Clin Invest.1999 Jun;103(11):1579-86.doi:10.1172 / JCI6172.PMID:10359567;PMCID:PMC408371. Sullivan PM,Mezdour H,et al.Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis.J Biol Chem.1997 Jul 18;272(29):17972-80.doi:10.1074 / jbc.272.29.17972.PMID:9218423. Arboleda-Velasquez JF,Lopera F,et al.Resistance to autosomal dominant Alzheimer’s disease in an APOE3 Christchurch homozygote:a case report.Nat Med.2019 Nov;25(11):1680-1683.doi:10.1038 / s41591-019-0611-3.Epub 2019 Nov 4.PMID:31686034. van den Maagdenberg AM,Weng W,et al.Characterization of five new mutants in the carboxyl-terminal domain of human apolipoprotein E:no cosegregation with severe hyperlipidemia.Am J Hum Genet.1993 May;52(5):937-46.PMID:8488843. Liu CC,Murray ME,et al.APOE3-Jacksonville(V236E)variant reduces self-aggregation and risk of dementia.Sci Transl Med.2021 Sep 29;13(613):eabc9375.doi:10.1126 / scitranslmed.abc9375.Epub 2021 Sep 29.PMID:34586832. BLAST.Joseph Bedell,Ian Korf and Mark Yandell[OReilly&Associates,2003],Altschul SF,Gish W,Miller W,Myers EW,Lipman DJ.Basic local alignment search tool.J Mol Biol.1990;215(3):403-410.doi:10.1016 / S0022-2836(05)80360-2 Boudreau RL,Spengler RM,Hylock RH,Kusenda BJ,Davis HA,Eichmann DA,Davidson BL.siSPOTR:a tool for highly designing specific and potent siRNAs for human and mouse.Nucleic Acids Res.2013 Jan 7;41(1):e9.doi:10.1093 / nar / gks797.Epub 2012 Aug 31.PMID:22941647;PMCID:PMC3592398. [Brief description of the drawings]

[0247] [Figure 1-1] 1 is a schematic diagram of a portion of the APOE4 cDNA sequence that is part of the NCBI Reference Sequence: NM_000041. The sequence shown is referred to herein as SEQ ID NO:1, corresponds to nucleotides 1 to 1166 thereof, and represents the DNA sequence of (part of) the spliced ​​APOE transcript (transcript 2). [Figure 1-2] 1 is a schematic diagram of a portion of the APOE4 cDNA sequence that is part of the NCBI Reference Sequence: NM_000041. The sequence shown is referred to herein as SEQ ID NO:1, corresponds to nucleotides 1 to 1166 thereof, and represents the DNA sequence of (part of) the spliced ​​APOE transcript (transcript 2). [Diagram 2] Schematic diagrams of the scaffold RNA structure (a) and (b) showing the first RNA sequence as designed. [Diagram 3]Validation of the miR-144A>T mutant scaffold by small molecule mRNA sequencing. A) Comparison of expression levels of miRNA6 (=miHTT), miR-144 5p, miR-144 3p and miR-16 across miR-451, miR-144 wild-type (WT) and miR-144A>T mutant scaffolds. B) Comparison of expression values ​​of a panel of different miRNA sequences inserted within the miR-451 and miR-144A>T mutant scaffolds. [Figure 4] FIG. 13 shows knockdown of APOE4Luc reporter by miAPOE in vitro upon co-transfection with 250 ng of DNA construct (sequence numbers 94-185) in HEK293T cells. [Diagram 5] Figure 1. Knockdown of APOE4Luc reporter by 25 miAPOE constructs (SEQ ID NO: 96, 99, 100, 105, 106, 108, 109, 113, 114, 115, 116, 117, 131, 132, 133, 134, 135, 136, 137, 139, 143, 146, 149, 151, 185) in vitro by co-transfection with 2, 10 or 50 ng of DNA construct. Renilla relative expression (RL / FL) of miSCR441 (SEQ ID NO: 186) was set to 100%. [Figure 6] FIG. 1 shows endogenous APOE mRNA expression in Huh7 cells and its reduction in vitro upon transfection with 250 ng of miAPOE constructs (SEQ ID NOs: 94-185). [Figure 7] Figure showing endogenous APOE mRNA expression and its reduction in vitro upon transfection with 50 or 250 ng of miAPOE constructs (sequence numbers 96, 108, 116, 133, 136, 138, 146, 151, 185) or miSCR constructs (sequence numbers 186-187) (n=2). [Figure 8] (A) Western blot and (B) relative quantification showing (reduction of) secreted endogenous APOE protein in the supernatant of Huh7 cells transfected with miAPOE constructs (sequence numbers 96, 108, 116, 136, 146, 185) or miSCR constructs (sequence number 186). [Figure 9] Illustrates vector DNA copy and knockdown of APOE mRNA expression in Huh7 cells upon transduction with AAV5 or AAV9 containing miAPOE expression cassettes (SEQ ID NO: 108, 116, 146). Huh7 cells were transduced at a multiplicity of infection (MOI) of 5E+06, 3.6+06, 1E+06, 5E+05 or 5E+04 gc / cell. (A) Vector DNA copy number was determined 48 hours after transduction and (B) APOE mRNA expression. APOE mRNA expression in the presence of AAV5-miSCR (SEQ ID NO: 186) was set to 100%. [Figure 10] FIG. 1 shows a Western blot of APOE protein in culture supernatants of HEK293T and astrocytoma cells 48 hours after transfection with 250 ng of an APOE variant expression construct in vitro. [Figure 11] Illustrates vector DNA copies and APOE protein expression in culture supernatants of HEK293T cells transduced with AAV5 containing APOE variant expression cassettes (SEQ ID NOs: 197, 208, 210, 209, 211). HEK293T cells were transduced at a multiplicity of infection (MOI) of 1E4, 1E5, or 1E6. (A) Vector DNA copies in transduced HEK293T cells. (B) Detection of APOE protein by Western blot in supernatants of AAV-transduced HEK293T cells. [Figure 12] Detection of vector DNA (A) and mRNA copies (B) in the striatum of the brain of C57Bl6 mice injected with AAV-APOE3ch.-HA. [Figure 13] Figure 14. Silencing of APOE4Luc reporter in vitro by combination approach and miAPOE constructs upon co-transfection with reporter (10 fmol) and combination approach (sequence numbers 255-276, labeled 1-22) or miAPOE constructs (sequence numbers 108, 116, 146). [Figure 14]Figure 1. Silencing of APOE4Luc reporter by combination approach constructs in vitro upon co-transfection of reporter (2.18 fmol) with combination approach DNA constructs (SEQ ID NOs: 255-276, labeled 1-22) or miAPOE constructs (SEQ ID NOs: 108, 116, 146) at 0.69.3.46 or 17.31 fmol. Relative Renilla expression (RL / FL) of the combination approach containing miSCR441 was set to 100%. [Figure 15] FIG. 14. APOE transgene expression in supernatants of Hek293T cells transfected with combination approach constructs (SEQ ID NOs: 255-276, labeled 1-22) or APOE transgene constructs (SEQ ID NOs: 197, 208, 210, 211). [Figure 16] FIG. 14. Expression of vector DNA in the striatum (A) and frontal cortex (B) of mice injected with high (gray bars) or medium (white bars) doses of empty AAV5, AAV5-miSCR, AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, or AAV9-miAPOE_145. [Figure 17-1] Knockdown of APOE4 mRNA in the striatum (A-B) and frontal cortex (C-D) of mice injected with high (gray bars) or medium (white bars) doses of AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, or AAV9-miAPOE_145 compared to empty AAV5 and AAV5-miSCR. (B, D) Relative AAV5-miSCR copy levels were set to 100%. [Figure 17-2] Knockdown of APOE4 mRNA in the striatum (A-B) and frontal cortex (C-D) of mice injected with high (gray bars) or medium (white bars) doses of AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, or AAV9-miAPOE_145 compared to empty AAV5 and AAV5-miSCR. (B, D) Relative AAV5-miSCR copy levels were set to 100%. [Figure 18] FIG. 11. MiRNA copy levels in the striatum of mice injected with high (gray bars) or medium (white bars) doses of AAV5-miSCR, AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, or AAV9-miAPOE_145. [Figure 19] FIG. 13 shows the reduction of hAPOE4 protein in the frontal cortex of mice injected with high (gray bars) or medium (white bars) doses of AAV5-miAPOE_016, AAV5-miAPOE_037, AAV5-miAPOE_145, or AAV9-miAPOE_145 compared to empty AAV5 and AAV5-miSCR. [Figure 20-1] (A) Read length abundance compared to total miAPOE reads found in the caudal cortex of hAPOE4-Tr mice injected with AAV5-miAPOE_016, AAV5-miAPOE_037, and AAV5-miAPOE_145. The most abundant read lengths for miAPOE_016, miAPOE_037, and miAPOE_145 were 23, 25, and 24 nucleotides, respectively. (B) Read counts of miAPOE transcripts. (C) Ranking of endogenous miRNAs and miAPOE abundance detected in the caudal cortex of hAPOE4-Tr mice injected with AAV5-miSCR, AAV5-miAPOE_016, AAV5-miAPOE_037, and AAV5-miAPOE_145. The internal controls miR-29a-3p and miR-16-5p are marked with one or two asterisks, respectively. [Figure 20-2](A) Read length abundance compared to total miAPOE reads found in the caudal cortex of hAPOE4-Tr mice injected with AAV5-miAPOE_016, AAV5-miAPOE_037, and AAV5-miAPOE_145. The most abundant read lengths for miAPOE_016, miAPOE_037, and miAPOE_145 were 23, 25, and 24 nucleotides, respectively. (B) Read counts of miAPOE transcripts. (C) Ranking of endogenous miRNAs and miAPOE abundance detected in the caudal cortex of hAPOE4-Tr mice injected with AAV5-miSCR, AAV5-miAPOE_016, AAV5-miAPOE_037, and AAV5-miAPOE_145. The internal controls miR-29a-3p and miR-16-5p are marked with one or two asterisks, respectively. [Figure 21-1] Figure 1. Expression of vector DNA (A) and APOE mRNA (B) in the striatum of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. The dashed line indicates the LLOQ. (C-D) hAPOE protein levels detected in the hippocampus (C) and frontal cortex (D) of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. [Figure 21-2]Figure 1. Expression of vector DNA (A) and APOE mRNA (B) in the striatum of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. The dashed line indicates the LLOQ. (C-D) hAPOE protein levels detected in the hippocampus (C) and frontal cortex (D) of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. [Figure 22-1](A) Total tau protein levels in the hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. (B-C) pTau181 protein levels in the hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. Measured values ​​(B) and normalized values ​​(C) demonstrate a significant decrease in pTau181 levels in all groups injected with AAV5-APOE variants. **P<0.01, ***P<0.001 by one-way ANOVA. Dashed lines indicate 50%. (D-E) pTau181 / total Tau ratio in hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2 and AAV5-APOE3b V2. Measured values ​​(D) and normalized values ​​(E) reveal a significant decrease in pTau181 / total Tau ratio in AAV5-APOE2ch V2, AAV5-APOE2b V and AAV5-APOE3b V2 injected groups. **P<0.01, ****P<0.0001 by one-way ANOVA. Dashed lines indicate 50%. [Figure 22-2](A) Total tau protein levels in the hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. (B-C) pTau181 protein levels in the hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2, and AAV5-APOE3b V2. Measured values ​​(B) and normalized values ​​(C) demonstrate a significant decrease in pTau181 levels in all groups injected with AAV5-APOE variants. **P<0.01, ***P<0.001 by one-way ANOVA. Dashed lines indicate 50%. (D-E) pTau181 / total Tau ratio in hippocampus of WT or P301S mice injected with vehicle, empty AAV5, AAV5-APOE2ch WT, AAV5-APOE2ch V2, AAV5-APOE2b V2, AAV5-APOE3ch V2 and AAV5-APOE3b V2. Measured values ​​(D) and normalized values ​​(E) reveal a significant decrease in pTau181 / total Tau ratio in AAV5-APOE2ch V2, AAV5-APOE2b V and AAV5-APOE3b V2 injected groups. **P<0.01, ****P<0.0001 by one-way ANOVA. Dashed lines indicate 50%. [Figure 23-1] Vector DNA expression (A) and miAPOE copy levels (B) in the striatum of WT mice injected with the combination approach and single constructs encapsidated in AAV5. APOE mRNA copy levels in the striatum (C) and APOE protein levels in the cortex (D) of WT mice injected with the combination approach and single constructs encapsidated in AAV5. The dashed line indicates the LLOQ. [Figure 23-2]Vector DNA expression (A) and miAPOE copy levels (B) in the striatum of WT mice injected with the combination approach and single constructs encapsidated in AAV5. APOE mRNA copy levels in the striatum (C) and APOE protein levels in the cortex (D) of WT mice injected with the combination approach and single constructs encapsidated in AAV5. The dashed line indicates the LLOQ. [Figure 24-1] (A) Read length abundance compared to total miAPOE reads found in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miAPOE_016 and AAV5-miAPOE_145. The most abundant read length for all constructs is 24 nucleotides. (B) Read counts of miAPOE transcripts. (C) Ranking of endogenous miRNAs and miAPOE abundance detected in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miSCR, AAV5-miAPOE_016 and AAV5-miAPOE_145. Internal controls miR-29a-3p and miR-16-5p are marked with one or two asterisks, respectively. (D) Combinatorial approach, differential expression analysis of miAPOE_016 and miAPOE_145 constructs. [Figure 24-2](A) Read length abundance compared to total miAPOE reads found in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miAPOE_016 and AAV5-miAPOE_145. The most abundant read length for all constructs is 24 nucleotides. (B) Read counts of miAPOE transcripts. (C) Ranking of endogenous miRNAs and miAPOE abundance detected in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miSCR, AAV5-miAPOE_016 and AAV5-miAPOE_145. Internal controls miR-29a-3p and miR-16-5p are marked with one or two asterisks, respectively. (D) Combinatorial approach, differential expression analysis of miAPOE_016 and miAPOE_145 constructs. [Figure 24-3] (A) Read length abundance compared to total miAPOE reads found in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miAPOE_016 and AAV5-miAPOE_145. The most abundant read length for all constructs is 24 nucleotides. (B) Read counts of miAPOE transcripts. (C) Ranking of endogenous miRNAs and miAPOE abundance detected in U-118MG cell cultures transduced with AAV5 combinatorial approach constructs ID2 and 16, AAV5-miSCR, AAV5-miAPOE_016 and AAV5-miAPOE_145. Internal controls miR-29a-3p and miR-16-5p are marked with one or two asterisks, respectively. (D) Combinatorial approach, differential expression analysis of miAPOE_016 and miAPOE_145 constructs.

Claims

1. A nucleic acid comprising a sequence encoding a first RNA and a sequence encoding a second RNA, The second RNA comprises a guide sequence of at least 19 nucleotides that is substantially complementary to a portion of the ApoE gene. The first RNA and the second RNA each contain a hairpin, The aforementioned guide sequence is substantially complementary to the sequence selected from sequence numbers 16, 24, and 54. A nucleic acid in which the guide sequence comprises at least 22 nucleotides.

2. The nucleic acid according to claim 1, wherein in the 5' to 3' direction, a spacer follows the sequence encoding the first RNA, and the sequence encoding the second RNA follows the spacer, and the spacer is at least 50 nucleotides.

3. The nucleic acid according to claim 1, wherein the first RNA comprises SEQ ID NO: 235 or a variant thereof, and the second RNA comprises SEQ ID NO:

190.

4. The nucleic acid according to claim 1, wherein the first RNA is mutated such that it reduces the processing and / or expression of the first RNA.

5. An expression cassette comprising the nucleic acid described in claim 1, wherein the expression cassette is a DNA molecule.

6. The expression cassette according to claim 5, further comprising a second nucleic acid encoding at least one of APOE2 and APOE3, wherein the first and second nucleic acids are operably linked to a promoter and optionally to a polyA signal, the second nucleic acid encoding a protein comprising one of sequence numbers 249 to 254, and / or the second nucleic acid comprising one of sequence numbers 195 to 217.

7. The expression cassette according to claim 6, wherein the sequence encoding the first RNA and the sequence encoding the second RNA are included in an intron sequence, and the intron sequence is present in the promoter.

8. The expression cassette according to claim 6, wherein the promoter is a promoter capable of driving transcription in brain cells, and the promoter includes one of sequence numbers 191, 192, 193, or 232.

9. The expression cassette according to claim 5, wherein the expression cassette is adjacent to an inverted terminal sequence (ITR).

10. An adeno-associated virus (AAV) vector comprising the expression cassette described in claim 9, and comprising AAV5 capsid protein or AAV9 capsid protein.

11. An expression cassette for use in gene therapy, comprising nucleic acids encoding one or more APOE2 and APOE3 proteins selected from SEQ ID NOs: 249 to 254.

12. An adeno-associated virus (AAV) vector comprising the expression cassette described in Claim 11, and comprising AAV5 capsid protein or AAV9 capsid protein.

13. A pharmaceutical composition comprising the nucleic acid described in claim 1 and at least one pharmaceutically acceptable excipient.

14. The nucleic acid according to claim 1, for use as a pharmaceutical product.

15. The nucleic acid according to claim 1, for use in the treatment and / or prevention of a target Alzheimer's disease.

16. The nucleic acid according to claim 15, wherein the subject is a carrier of the ApoE4 allele.

17. The nucleic acid according to claim 14, wherein the nucleic acid is administered to the central nervous system by intracerebral injection, intraparenchymal injection, intrathecal injection, or convective-enhanced delivery.

18. A kit comprising the nucleic acid described in claim 1, further comprising an immunosuppressant.

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