How to Treat Alzheimer's Disease

Abstract

The present disclosure relates to antisense oligonucleotides (ASOs) capable of binding to a target site on the pre-mRNA of SORL1. The present disclosure further relates to compositions comprising said ASOs. The present disclosure further relates to ASOs for use in medicine. The present disclosure further relates to ASOs for use in preventing, treating, and / or ameliorating Alzheimer's Disease (AD) or a disease or disorder associated with Alzheimer's Disease. The present disclosure further relates to methods of mediating exon skipping in SORL1 transcripts, determining the efficiency of ASO-mediated SORL1 exon skipping, determining whether a patient identified with a SORL1 mutation would benefit from treatment with ASO-mediated exon skipping, and methods of making ASOs.

Description

[Technical field]

[0001] Disclosed herein are compounds, compositions and methods for modulating splicing of SORL1 mRNA in a cell, tissue or animal. Also provided is the use of the disclosed compounds and compositions in the manufacture of a medicament for the treatment of diseases and disorders, including Alzheimer's disease (AD). Specifically, the disclosure relates to antisense oligonucleotides (ASOs) that induce exon skipping in the transcript of SORL1. [Background technology]

[0002] SORLA is important for the trafficking of amyloid precursor protein (APP) out of endosomes, where amyloidogenic processing of APP into pathogenic fragments (i.e., amyloid β-peptide (Aβ)) occurs if unhindered by SORLA. This SORLA-supported trafficking of APP ensures a reduction in the cleavage of APP by β-secretase, thereby reducing the generation of β-C-terminal fragments (CTFs) that can then be further processed to generate amyloid beta (Aβ) peptides. In AD, Aβ accumulates in amyloid plaques in the brain and is the most important pathological hallmark of the disease. However, the pathogenesis of the disease is rather linked to the levels of β-CTFs and other cargo proteins, which in AD cannot be recycled out of endosomes, leading to endosomal swelling and dysfunctional endosomal activity (i.e., the endosomal traffic jam hypothesis for Alzheimer's disease). The ability of SORLA to participate in endosomal recycling is related to a motif in its cytoplasmic tail (i.e., the FANSHY motif) that is important for its interaction with the retromer complex and serves to transport cargo out of endosomes.

[0003] The SORL1 gene, which encodes the endosomal sorting receptor SORLA, has been linked to the development of Alzheimer's disease for the past 15 years. Recently, large-scale whole-exome sequencing studies have identified SORL1 as the gene harboring the highest genetic variation across the human genome in AD patient populations. The combined group of "loss-of-function" (LOF) variants located in SORL1 is associated with a 36-fold increased odds ratio risk (OR=36) for early-onset AD and a 7-fold increased risk for late-onset AD. The entire group of missense variants has been found to be associated with a 2.7-fold and 1.9-fold increased risk for early-onset and late-onset AD, respectively.

[0004] Interestingly, SORL1 variants from Alzheimer's disease patients are spread throughout the SORL1 gene, and therefore, more than 25% of all variants are located in the genomic region encoding 11 complement repeat (CR) domains. The CR domain is the major ligand-binding site in all known receptors that contain a cluster of CR domains. The cluster of CR domains in SORLA is also involved in binding to ligands, including APP. As a result, mutations in the CR domain can have a significant impact on the function of SORLA, both in terms of ligand binding and in terms of protein misfolding and ER retention.

[0005] While there is knowledge about genetic markers that predict the risk or causation of developing AD, no viable treatment for AD is currently available, and AD remains a significant burden to patients and healthcare systems. Summary of the Invention

[0006] Recently, the inventors of the present disclosure have accumulated evidence indicating that additional SORLA mutations, namely mutations in the CR domain of SORLA, are associated with AD. Two subgroups of missense variants located in a region of SORL1 that encodes a cluster of 11 CR domains, have been identified that are associated with an increased risk of AD (unpublished data).

[0007] The CR domain represents the major ligand-binding site in all known receptors that contain a cluster of CR domains. Also, the cluster of CR domains in SORLA is involved in binding to ligands including APP. However, typical binding of any ligand does not depend on an isolated CR domain, but rather many studies have shown how binding is achieved by the combined interaction of many CR domains with several epitopes on the ligand.

[0008] By carefully examining SORLA domains and their functionality in physiology and pathology, the inventors of the present disclosure have realized that since the presence or absence of individual CR domains only subtly affects the affinity for SORLA ligands, targeting one or more CR domains of SORLA is a feasible approach to remove CR domains that are not functional due to mutations. As disclosed herein, mutated CR domains are removed from SORLA by employing an exon skipping method, in which specifically designed antisense oligonucleotides (ASOs) are used to remove the exons of the CR domains carrying the mutations. As a result, functional SORLA proteins can be generated that lack, for example, one CR domain, or, in the case of mutations in multiple CR domains, lack more than one CR domain. This approach has several advantages.

[0009] First, ASO delivered to the brain of AD patients can restore functional SORLA protein in situ. AD patients carrying CR mutations may produce SORLA protein, but due to the mutation, this protein does not function. For example, this mutated SORLA protein can be misfolded and pathologically retained in the endoplasmic reticulum (ER). By removing the mutated CR exon by exon skipping, a functional SORLA protein can be generated that retains its functionality in terms of ligand binding and ensures that SORLA, which contains many important domains, proceeds through the endosomal pathway in a physiological manner. In summary, instead of a mutated protein that completely abolishes the functionality of the entire protein, a mutant form lacking one or more CR domains can be generated that can function physiologically or close to physiologically.

[0010] Secondly, mutated SORLA protein can have a major negative effect on unmutated SORLA (produced from non-affected alleles) due to SORLA dimer formation. Mutant SORLA can lead to misfolding and retention of unmutated SORLA in dimers. Thus, the level of functional SORLA is even further reduced, so that amyloidogenic processing of APP into pathogenic fragments is no longer prevented. However, by using the teachings of the present disclosure, i.e., ASO-mediated exon skipping of the mutated SORLA CR domain, a SORLA protein lacking one or more CR domains is generated in cells, but this mutant form does not induce misfolding of itself or other unaffected SORLA mutant forms in dimers.

[0011] In a main aspect, the disclosure relates to an antisense oligonucleotide (ASO) that binds to and / or is complementary to a target site on the pre-mRNA of SORL1, wherein the nucleotide sequence of the target site is comprised in a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, or a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0012] In a further aspect, the disclosure relates to a composition comprising said oligonucleotide.

[0013] In a further aspect, the present disclosure is directed to said antisense oligonucleotide (ASO) and / or said composition for use in medicine.

[0014] In a further aspect, the present disclosure is directed to said antisense oligonucleotide (ASO) and / or said composition for use in the prevention, treatment and / or amelioration of Alzheimer's disease (AD) or a disease or disorder related to Alzheimer's disease.

[0015] In a further aspect, the present disclosure is directed to the use of said antisense oligonucleotide or said composition in the manufacture of a medicament for treating Alzheimer's Disease (AD) or a disease or disorder related to Alzheimer's Disease.

[0016] In a further aspect, the disclosure is directed to a method of using the antisense oligonucleotide and / or the composition to mediate exon skipping in a SORL1 transcript in a cell, tissue or organ, wherein the exon is selected from the group consisting of exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32 and exon 33 of SORL1.

[0017] In a further aspect, the disclosure is directed to a method of determining efficiency of ASO-mediated exon skipping of SORL1 in a subject, the method comprising the steps of: a) analyzing the level of excreted SORLA in a first sample comprising cerebrospinal fluid from the patient obtained prior to treatment with an ASO; b) analyzing the level of excreted SORLA in a second sample containing cerebrospinal fluid from the same patient as in a) obtained after treatment with ASO; c) comparing the levels of excreted SORLA in the samples of a) and b); If the level of excreted SORLA in b) is higher than that in a), exon skipping is determined.

[0018] In a further aspect, the disclosure is directed to a method of determining whether a patient identified as having a SORL1 mutation would benefit from treatment with ASO-mediated exon skipping, the method comprising the steps of: a) identifying a mutation in any one of exons 23 to 33 of SORL1; b) introducing a patient-identified SORL1 mutation into the cell line; c) selecting one or more ASOs that target the exon harboring the identified mutation; d) contacting a first aliquot of cells with medium containing one or more selected ASOs and contacting a second aliquot of cells with medium not containing the ASOs; e) analyzing the concentration of excreted SORLA in the first aliquot and the second aliquot; f) comparing the level of excreted SORLA in the first aliquot and the second aliquot; Thereby, if the level of excreted SORLA in the first aliquot is higher than in the second aliquot, it is determined that the patient will benefit from treatment with one or more ASOs.

[0019] In a further aspect, the disclosure is directed to a method of generating an ASO suitable for treating an AD patient, wherein the patient carries a mutation in an exon encoding a complement-type repeat (CR) domain of SORLA, the method comprising the steps of: a) identifying, for example by a computer, the ASO according to any one of claims 1 to 21; b) determining whether the target site of the ASO contains a mutation or whether the target size of the ASO does not contain a mutation; Thereby, it is determined that ASOs that bind to target sites that do not contain mutations are suitable for treating AD patients. [Brief description of the drawings]

[0020] [Figure 1] Schematic diagram of the SORLA domain assembly. The human SORLA polypeptide contains 2214 amino acids that fold into multiple protein domains, including the VPS10p domain, the YWTD-b-propeller domain linked to the EGF domain, 11 CR domains, 6 3Fn domains, a transmembrane domain, and a cytoplasmic tail domain. The CR domain is encoded by exons 23-33. (Ex=exon) [Figure 2A]The structure and sequence of the CR domain of SORLA are shown. A and B: The CR domain sequence contains about 40 amino acids, including six strictly conserved cysteines (cysteine ​​= "C") that form three intradomain disulfides (black bars connecting the cysteines). Four residues with acidic side chains are also conserved, which function in the octahedral coordination of calcium ions. CR1 is shown here as an exemplary CR domain. [Figure 2B] The structure and sequence of the CR domain of SORLA are shown. A and B: The CR domain sequence contains about 40 amino acids, including six strictly conserved cysteines (cysteine ​​= "C") that form three intradomain disulfides (black bars connecting the cysteines). Four residues with acidic side chains are also conserved, which function in the octahedral coordination of calcium ions. CR1 is shown here as an exemplary CR domain. [Figure 2C] The structure and sequence of the SORLA CR domain are shown. A sequence comparison of the 11 SORLA CR domain sequences is shown, with the domain boundaries following their individual exon (i.e., exons 23-33) structures. The numbers above the sequence comparison indicate the relationship between the amino acids in B and C. [Diagram 3] FIG. 1 is a schematic diagram showing how antisense oligonucleotides (ASOs) can be used in exon skipping. Each of the eleven CR domains is encoded by its own exon (exons 23-33). Because each of these exons contains a multiple of three nucleotides, skipping of an exon does not affect the reading frame of downstream exons. Thus, ASOs for individual exons (shown as wavy lines) that target the 3' splice site, 5' splice site, and / or splice enhancer site (ESE) of one or more exons can be used to treat Alzheimer's disease by removing mutated exons from the SORL1 transcript, as exemplified here by exon 23. [Figure 4A]Schematic diagram of ASO treatment and cellular SORLA activity. SORLA is expressed from the wild-type allele, has 11 CR domains (11x), and functions in endosomal cargo recycling. [Figure 4B] Schematic diagram of ASO treatment and cellular SORLA activity. SORLA was expressed from pathogenic SORL1 variants (shown as black CR domains), such as variants characteristic of AD patients, e.g., ONC or CC variants, which may cause receptor misfolding and ER retention and affect translation product from wild-type alleles. [Figure 4C] Schematic diagram of ASO treatment and cellular SORLA activity. Disease allele SORLA treated with exon-skipping ASO will contain 10 functional CR domains (10x) and function indistinguishable from the full-length SORLA protein. [Figure 5A] Figure 1 shows an ASO targeting exon 23. Figure 2 shows the targeting strategy to induce exon skipping of exon 23. The table shows the four ASOs (ASO23.1 to ASO23.4), their respective ASO sequences, and their respective RNA target sequences. [Figure 5B] Figure 1 shows an ASO targeting exon 27. Figure 2 shows the targeting strategy to induce exon skipping of exon 27. The table shows the four ASOs (ASO27.1 to ASO27.4), their respective ASO sequences, and their respective RNA target sequences. [Figure 5C] Figure 1 shows an ASO targeting exon 33. Figure 2 shows the targeting strategy to induce exon skipping of exon 33. The table shows the four ASOs (ASO33.1 to ASO33.4), their respective ASO sequences, and their respective RNA target sequences. [Figure 5D]Four ASOs identified to target exon 23 are shown with respect to their respective binding sites on the precursor mRNA. Bars in various shades of grey indicate splice factor binding sites. The width of each bar represents the sequence putatively bound by a splicing factor. The predicted strength of promoting or suppressing exon splicing is expressed as a score for each splicing factor. Positive scores represent exon splice enhancers (ESEs) while negative scores represent exon splice repressors (ESRs). Numbers below the sequences indicate the nucleotide position within the sequence including the exon + 25 bp of flanking intronic sequence (lowercase) on either side. [Figure 5E] Four ASOs identified to target exon 27 are shown with respect to their respective binding sites on the precursor mRNA. Bars in various shades of grey indicate splice factor binding sites. The width of each bar represents the sequence putatively bound by a splicing factor. The predicted strength of promoting or suppressing exon splicing is expressed as a score for each splicing factor. Positive scores represent exon splice enhancers (ESEs) whereas negative scores represent exon splice repressors (ESRs). Numbers below the sequences indicate the nucleotide position within the sequence including the exon + 25 bp of flanking intronic sequence (lowercase) on either side. [Figure 5F] Four ASOs identified to target exon 33 are shown with respect to their respective binding sites on the precursor mRNA. Bars in various shades of grey indicate splice factor binding sites. The width of each bar represents the sequence putatively bound by a splicing factor. The predicted strength of promoting or suppressing exon splicing is expressed as a score for each splicing factor. Positive scores represent exon splice enhancers (ESEs) while negative scores represent exon splice repressors (ESRs). Numbers below the sequences indicate the nucleotide position within the sequence including the exon + 25 bp of flanking intronic sequence (lowercase) on either side. [Figure 6]Figure 1 shows exon 23 skipping efficiency measured by RT-PCR in human cell lines transfected with ASOs targeting exon 23. We investigated four ASOs (ASO23.1, ASO23.2, ASO23.3, and 23.4) directed against the exon splice enhancer (ESE) element of exon 23 of SORL1 by transfecting HEK293 cells, recovering endogenous SORL1 mRNA, and performing RT-PCR using primer pairs spanning the region around exon 23. PCR products were separated by agarose gel electrophoresis, and data from two independent experiments are shown (top, bottom). In both experiments, we observed clear evidence that ASO23.2 and ASO23.3 induced exon 23 skipping, as demonstrated by the presence of a shorter PCR product (see arrow) that co-migrated with the product generated using as template a plasmid encoding an exon 23 deletion fragment (pΔEx23, a recombinantly produced exon 23 mutant used as a control). pFL, a plasmid encoding full-length SORLA, served as a control template to identify PCR products corresponding to fragments with exon 23 included. [Figure 7]Expression of SORL1 delta-exon 23 is shown. A) Western blot analysis of lysates and media from N2a cells transfected with constructs encoding SORLA full-length or SORLA-delta-exon 23. The level of full-length SORLA in the lysates (left) is indistinguishable from that of SORLA without exon 23 (note: the band in the second replicate of deltaEx23 is partially obscured by air bubbles), yet is as intense as the band in the adjacent lane. The intermediate (right) level of full-length SORLA is indistinguishable from that of SORLA without exon 23. Thus, SORLA without exon 23 is excreted similarly to full-length SORLA, and thus retains its functionality. B) Western blot analysis of lysates and media from N2a cells transfected with SORLA full-length, SORLA-D1105H (pathogenic mutation), SORLA lacking exon 23, or SORLA-R1080C (pathogenic mutation). The levels of sSORLA in the medium of deleted CR1 (i.e., Δexon 23) are similar to those of full-length SORLA, whereas both pathogenic mutations greatly reduce the amount of sSORLA produced (sSORLA = efflux SORLA). [Figure 8A]Functional expression of SORLA delta-exon 23 is shown. Western blot analysis of media from N2a cells transfected with APP alone ("control", lanes 1-2), doubly transfected with APP / full-length SORLA ("full-length", lanes 3-4), or doubly transfected with APP / SORLA deltaEx23 ("deltaExon 23", lanes 5-6) is shown. The amount of shed APP (i.e., sAPPa) is detected using antibody WO2, and the amount of shed SORLA (i.e., sSORLA) is detected using a polyclonal serum against the SORLA luminal fragment. Cells without exogenous SORLA overexpression (control) show a strong signal for sAPP in the media. In contrast, media from cells transfected with either full-length SORLA or a SORLA construct with a deletion of exon 23 show significantly reduced levels of sAPP. In conclusion, SORLA deleted for CR1 (encoded by exon 23) is as effective at inhibiting APP processing as the full-length SORLA receptor. [Figure 8B] We show functional expression of SORLA delta-exon 23. Independent experiments again showed that levels of shed SORLA (sSORLA) were similar between SORLA-WT and SORLA-ΔEx23, indicating that deletion of CR1 (encoded by exon 23) has no observable effect on receptor biology. We also again observed that the two SORLA mutant (full-length and SORLA-delta-exon 23) proteins had indistinguishable effects on reducing sAPPα production by decreasing APP proteolysis. [Figure 8C]We show functional expression of SORLA delta-exon 23. Independent experiments again showed that levels of shed SORLA (sSORLA) were similar between SORLA-WT and SORLA-ΔEx23, indicating that deletion of CR1 (encoded by exon 23) has no observable effect on receptor biology. We also again observed that the two SORLA mutant (full-length and SORLA-delta-exon 23) proteins had indistinguishable effects on reducing sAPPα production by decreasing APP proteolysis. [Figure 8D] Functional expression of SORLA delta-exon 23 is shown. Blots from three independent replicates were quantified and data presented as the mean of replicates relative to levels in cells without exogenous SORLA. This quantification further demonstrates that SORLA delta-Ex23 is as effective as WT in reducing shed APPa (sAPPa) (ns = non-significant). [Figure 9] Expression of SORL1 delta-exon 33 is shown. N2a cells were transfected with either SORL1-WT or SORL1-ΔEx33 constructs, and lysates and conditioned media from the cells were analyzed by Western blot using antibodies against SORLA or actin (in lysate samples), or excreted SORLA (in media samples). Endogenous SORLA expression is low / absent in N2a cells, and therefore undetectable in untransfected cells (blank). SORLA lacking CR domain 11 (encoded by exon 33, which has been removed from construct SORL1-ΔEx33) was detected at similar levels in lysates and media compared to wild-type SORLA. Similar detection in media indicates that deletion of CR11 has no observable effect on SORLA receptor biology, i.e., even SORLA lacking CR11 is processed and excreted similarly to SORLA-WT. [Figure 10]The exon skipping technique was extended to exons of additional SORL1 CR domains. HEK293 cells were transfected with constructs engineered to generate SORLA proteins deleted for individual CR domains, i.e., CR1 (ΔEx23), CR2 (ΔEx24), CR3 (ΔEx25), CR4 (ΔEx26), CR5 (ΔEx27), CR6 (ΔEX28), CR7+8 (ΔEx29+30), CR8 (ΔEx30), CR9 (ΔE31), CR10 (ΔE32) or CR11 (ΔE33). Lysates were prepared from cells harvested 72 hours after transfection, and proteins were separated by 26-lane SDS-PAGE NuPAGE system and analyzed by Western blot analysis using polyclonal SORLA serum from rabbit (sol-SORLA). SORLA blots are known to show a double band when expressing SORLA in HEK cells, with the upper band (which runs slower in the gel due to its larger molecular size) representing mature SORLA and the lower band (which runs faster in the gel due to its smaller molecular size; see arrows). Each of the constructs resulted in the expression of specific CR domain deleted SORLA receptors. Interestingly, some deletions showed surprising results suggesting that potentially not all CR domains can be deleted without disrupting receptor function, i.e., CR4 (ΔE26) and CR9 (ΔEx31), where the SORLA double banding pattern is disrupted. FL=full length SORLA, Δ=delta indicates exon / domain removed, Ex=exon, CR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0022] The term "some embodiments" can include one or more than one embodiments.

[0023] The terms "Alzheimer's disease" and "AD" are used interchangeably throughout this description.

[0024] The use of the words "a" or "an," as used throughout the text or in conjunction with the term "comprising" in the claims and / or this specification, may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more." Thus, for example, a reference to an "ASO" includes a plurality of such ASOs, e.g., one or more ASOs, at least one ASO, or two or more ASOs.

[0025] As used herein, the term "SORLA" is synonymous with the terms SORLA, sortilin-related receptor, sortilin-related receptor 1, SORL1, low density lipoprotein receptor associated with 11 ligand-binding repeats, LDLR associated with 11 ligand-binding repeats, LR11, SorLA-1, sorting protein-associated receptor containing LDLR class A repeats, and gp250. Human sorLA is annotated in UniProt with the accession number Q92673.

[0026] The terms homology, identity and similarity as defined herein with respect to polynucleotides (or polypeptides) are used interchangeably and refer to the percentage of nucleic acids (or amino acids) in a candidate sequence that are homologous, identical or similar, respectively, to the corresponding native nucleic acid (or amino acid) residues after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity / similarity, and considering conservative substitutions as part of sequence identity according to the NCIUB rules (hftp: / / www.chem.qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur. J. Biochem. (1985)). In particular, the percentage of similarity refers to the percentage of conserved residues with similar physicochemical properties. Neither 5' or 3' extensions nor insertions (in the case of nucleic acids), nor N' or C' extensions or insertions (in the case of polypeptides), result in a loss of identity or similarity. Methods and computer programs for sequence comparison are well known in the art. Generally, a given similarity between two sequences means that the identity between those sequences is at least equal to that similarity; for example, if two sequences are 80% similar to each other, they may share 90% identity, but not less than 80% identity with each other.

[0027] As defined herein, the term "at least 80% homology, similarity or identity" means throughout this disclosure at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homology, similarity or identity.

[0028] ASO-mediated exon skipping Antisense oligonucleotides can be used to induce exon skipping in pre-mRNA (also called precursor mRNA) transcripts. This type of antisense-mediated splicing regulation uses antisense oligonucleotides (ASOs) to manipulate splicing, for example, by sterically blocking the binding of splicing factors to pre-mRNA (also called precursor mRNA) transcripts.

[0029] In a main aspect, the disclosure relates to an antisense oligonucleotide (ASO) that binds to and / or is complementary to a target site on the pre-mRNA of SORL1, the nucleotide sequence of the target site being comprised in a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, or a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0030] In an alternative aspect, the disclosure relates to an antisense oligonucleotide (ASO) capable of binding to a target site on the pre-mRNA of SORL1, the nucleotide sequence of the target site being comprised in a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, or a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0031] One of skill in the art will understand that sufficient binding of an ASO to a target site requires binding via complementary bases.

[0032] Those skilled in the art will recognize that a SORL1 pre-mRNA is understood to be a transcript from one or more SORL1 exons with or without additional nucleotides transcribed from upstream and / or downstream sequences flanking each exon(s). For example, a SORL1 pre-mRNA may include: - exon 23 transcript (SEQ ID NO: 36) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 24 transcript (SEQ ID NO: 37) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 25 transcript (SEQ ID NO: 38) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 26 transcript (SEQ ID NO: 39) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 27 transcript (SEQ ID NO: 40) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 28 transcript (SEQ ID NO: 41) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 29 transcript (SEQ ID NO: 42) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 30 transcript (SEQ ID NO: 43) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 31 transcript (SEQ ID NO: 44) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 32 transcript (SEQ ID NO: 45) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, - exon 33 transcript (SEQ ID NO: 46) containing 100 nucleotides transcribed from the upstream / downstream adjacent introns, In this regard, exon 23 encodes CR domain 1 of SORLA, exon 24 encodes CR domain 2 of SORLA, exon 25 encodes CR domain 3 of SORLA, exon 26 encodes CR domain 4 of SORLA, exon 27 encodes CR domain 5 of SORLA, exon 28 encodes CR domain 6 of SORLA, exon 29 encodes CR domain 7 of SORLA, exon 30 encodes CR domain 8 of SORLA, exon 31 encodes CR domain 9 of SORLA, exon 32 encodes CR domain 10 of SORLA, and exon 33 encodes CR domain 11 of SORLA.

[0033] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:36, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:36, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0034] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:37, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:37, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0035] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:38, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:38, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0036] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:39, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:39, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0037] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:40, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:40, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0038] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:41, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:41, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0039] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:42, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:42, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0040] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:43, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:43, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0041] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:44, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:44, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0042] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:45, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:45, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0043] In some embodiments of the present disclosure, the nucleotide sequence of the target site comprises the nucleotide sequence of SEQ ID NO:46, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0044] In some embodiments of the present disclosure, an oligonucleotide that binds to and / or is complementary to a target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence selected from the group consisting of exon 23 as set forth in SEQ ID NO:1, exon 24 as set forth in SEQ ID NO:2, exon 25 as set forth in SEQ ID NO:3, exon 26 as set forth in SEQ ID NO:4, exon 27 as set forth in SEQ ID NO:5, exon 28 as set forth in SEQ ID NO:6, exon 29 as set forth in SEQ ID NO:7, exon 30 as set forth in SEQ ID NO:8, exon 31 as set forth in SEQ ID NO:9, exon 32 as set forth in SEQ ID NO:10, and exon 33 as set forth in SEQ ID NO:11, or a nucleotide sequence having at least 80% sequence identity or homology to any one of SEQ ID NOs:1-11, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0045] In some embodiments of the present disclosure, the exon comprises or consists of a nucleotide sequence selected from the group consisting of exon 23 set forth in SEQ ID NO:1, exon 24 set forth in SEQ ID NO:2, exon 25 set forth in SEQ ID NO:3, exon 26 set forth in SEQ ID NO:4, exon 27 set forth in SEQ ID NO:5, exon 28 set forth in SEQ ID NO:6, exon 29 set forth in SEQ ID NO:7, exon 30 set forth in SEQ ID NO:8, exon 31 set forth in SEQ ID NO:9, exon 32 set forth in SEQ ID NO:10, and exon 33 set forth in SEQ ID NO:11, or a nucleotide sequence having at least 80% sequence identity or homology to any one of SEQ ID NOs:1-11, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0046] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 23 as set forth in SEQ ID NO:1.

[0047] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 24 as set forth in SEQ ID NO:2.

[0048] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 25 as set forth in SEQ ID NO:3.

[0049] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 26 as set forth in SEQ ID NO:4.

[0050] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 27 as set forth in SEQ ID NO:5.

[0051] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 28 as set forth in SEQ ID NO:6.

[0052] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 29 as set forth in SEQ ID NO:7.

[0053] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 30 as set forth in SEQ ID NO:8.

[0054] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 31 as set forth in SEQ ID NO:9.

[0055] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 32 as set forth in SEQ ID NO:10.

[0056] In some embodiments of the present disclosure, the target site causes exon skipping of an exon encoding the complement-type repeat (CR) domain of SORLA, wherein the exon comprises or consists of a nucleotide sequence consisting of exon 33 as set forth in SEQ ID NO:11.

[0057] In some embodiments of the disclosure, the oligonucleotide is between 10-30 nucleotides in length, such as between 10-13 nucleotides, such as between 10-16 nucleotides, such as between 10-19 nucleotides, such as between 10-22 nucleotides, such as between 10-23 nucleotides, such as between 10-26 nucleotides, such as between 10-29 nucleotides, such as between 10-30 nucleotides.

[0058] In some embodiments of the present disclosure, the oligonucleotides are at least 10 nucleotides in length, e.g., at least 12 nucleotides in length, and / or at least 14 nucleotides in length, and / or at least 16 nucleotides in length, and / or at least 18 nucleotides in length, and / or at least 20 nucleotides in length, and / or at least 22 nucleotides in length, and / or at least 24 nucleotides in length, and / or at least 26 nucleotides in length, and / or at least 28 nucleotides in length, and / or at least 30 nucleotides in length.

[0059] In some embodiments of the present disclosure, the oligonucleotide is 21 nucleotides in length.

[0060] In some embodiments of the present disclosure, the oligonucleotide has a GC content of 40-60%, for example, 45-55%.

[0061] In some embodiments of the present disclosure, the oligonucleotide comprises a backbone that comprises phosphorothioate (PS).

[0062] In some embodiments of the present disclosure, the oligonucleotide further comprises a modification at at least one nucleotide position, or at each nucleotide position.

[0063] In some embodiments of the present disclosure, the modifications are modifications of the nucleic acid backbone, the nucleobase, the ribose sugar and / or the 2'-ribose substitution.

[0064] In some embodiments of the present disclosure, the oligonucleotides comprise 2'-O-methoxyethyl sugar modifications.

[0065] In some embodiments of the present disclosure, the oligonucleotide comprises a 2'-O-methyl ribose modification.

[0066] In some embodiments of the disclosure, the target sites are at a 3' splice site boundary, a 5' splice site boundary, and / or an exon splice enhancer (ESE) site.

[0067] In some embodiments of the present disclosure, the target site is a nucleotide sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, or SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. 1, consisting of or comprising a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity or homology thereto.

[0068] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:12, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:12, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0069] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:13, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:13, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0070] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:14, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:14, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0071] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:15, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:15, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0072] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:20, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:20, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0073] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:21, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:21, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0074] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:22, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:22, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0075] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:23, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:23, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0076] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:28, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:28, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0077] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:29, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:29, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0078] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:30, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:30, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0079] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:31, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:31, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0080] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:36, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:36, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0081] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:37, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:37, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0082] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:38, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:38, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0083] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:39, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:39, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0084] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:40, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0085] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:41, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0086] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:42, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:42, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0087] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:43, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0088] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:44, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:44, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0089] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:45, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:45, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0090] In some embodiments of the present disclosure, the target site consists of or comprises the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:46, e.g., at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0091] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, or a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0092] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO: 16, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0093] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO: 17, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0094] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:18, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:18, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0095] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:19, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:19, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0096] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:24, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:24, for example at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0097] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:25, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:25, for example at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0098] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:26, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:26, for example at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0099] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:27, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:27, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0100] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO: 32, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0101] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO: 33, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0102] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:34, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:34, for example at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0103] In some embodiments of the present disclosure, the oligonucleotide consists of or comprises the nucleotide sequence set forth in SEQ ID NO:35, or a nucleotide sequence having at least 80% sequence identity or homology to SEQ ID NO:35, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity or homology thereto.

[0104] In some embodiments of the present disclosure, the oligonucleotide specifically hybridizes to the target site under highly stringent solution hybridization conditions.

[0105] In some embodiments of the present disclosure, upon binding to the target site, the oligonucleotide prevents splicing factors from binding.

[0106] In some embodiments of the present disclosure, the oligonucleotide is conjugated to a moiety or a nanoparticle formulation.

[0107] In some embodiments of the present disclosure, the moiety is a cell targeting moiety and / or a cell penetrating moiety.

[0108] In some embodiments of the present disclosure, the oligonucleotide is conjugated to a triantennary N-acetylgalactosamine (GalNAc) moiety and / or a peptide.

[0109] In some embodiments of the present disclosure, the oligonucleotides target the 5' splice site, the 3' splice site, and / or the exon splice enhancer site (ESE).

[0110] 2. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide further comprises at least one additional nucleotide, at least two additional nucleotides, at least three additional nucleotides at one or both ends of the oligonucleotide.

[0111] In a further aspect, the disclosure is directed to a composition comprising said oligonucleotide.

[0112] In some embodiments of the present disclosure, the composition is a pharmaceutical composition.

[0113] In some embodiments of the present disclosure, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0114] In some embodiments of the present disclosure, a composition comprises one or more of the above oligonucleotides.

[0115] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16 and SEQ ID NO:17.

[0116] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16 and SEQ ID NO:18.

[0117] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16 and SEQ ID NO:19.

[0118] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:17 and SEQ ID NO:18.

[0119] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:17 and SEQ ID NO:19.

[0120] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:18 and SEQ ID NO:19.

[0121] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0122] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:19.

[0123] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.

[0124] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0125] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24 and SEQ ID NO:25.

[0126] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24 and SEQ ID NO:26.

[0127] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24 and SEQ ID NO:27.

[0128] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:25 and SEQ ID NO:26.

[0129] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:25 and SEQ ID NO:27.

[0130] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:26 and SEQ ID NO:27.

[0131] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

[0132] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:27.

[0133] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0134] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0135] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32 and SEQ ID NO:33.

[0136] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32 and SEQ ID NO:34.

[0137] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32 and SEQ ID NO:35.

[0138] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:33 and SEQ ID NO:34.

[0139] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:33 and SEQ ID NO:35.

[0140] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:34 and SEQ ID NO:35.

[0141] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.

[0142] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:35.

[0143] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35.

[0144] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:32 and SEQ ID NO:33 and SEQ ID NO:34 and SEQ ID NO:35.

[0145] In a further aspect, the present disclosure is directed to said antisense oligonucleotide (ASO) and / or said composition for use in medicine.

[0146] In a further aspect, the present disclosure is directed to said antisense oligonucleotide (ASO) and / or said composition for use in the prevention, treatment and / or amelioration of Alzheimer's disease (AD) or a disease or disorder related to Alzheimer's disease.

[0147] Those skilled in the art will understand that the invention disclosed herein can be used to prevent and / or treat AD and / or alleviate symptoms of AD in AD patients carrying a pathogenic mutation in the CR domain of SORLA. AD is a destructive disease that affects the brain at a structural level. Therefore, it may be beneficial to provide ASO-mediated exon skipping of the pathogenic mutated CR domain at an early stage, for example, before the onset of symptoms, or at the early stage of AD, or before substantial structural brain remodeling. For example, the approach described herein may be applicable to the treatment of family members of AD patients before the onset of disease.

[0148] In some embodiments of the disclosure, one or more ASOs of the invention or a composition comprising one or more ASOs of the invention are administered to an individual prior to the onset of AD symptoms.

[0149] In some embodiments of the disclosure, one or more ASOs of the invention or a composition comprising one or more ASOs of the invention are administered to an individual prior to the onset of AD symptoms.

[0150] In some embodiments of the disclosure, one or more ASOs of the invention, or a composition comprising one or more ASOs of the invention, are administered to an individual when a family member is diagnosed with AD.

[0151] Alternatively, the invention disclosed herein may be applicable to treating patients with early stage symptoms.

[0152] In some embodiments of the disclosure, one or more ASOs of the invention or a composition comprising one or more ASOs of the invention are administered to an individual with early AD symptoms.

[0153] Alternatively, the invention disclosed herein may be applicable to the treatment of patients at various disease stages of AD.

[0154] In some embodiments of the disclosure, one or more ASOs of the invention or a composition comprising one or more ASOs of the invention are administered to individuals with various degrees of AD symptoms.

[0155] Furthermore, the invention disclosed herein, due to its property of restoring physiological SORLA function, may be applicable to the treatment of patients in the later stages of AD.

[0156] In some embodiments of the disclosure, one or more ASOs of the invention or a composition comprising one or more ASOs of the invention are administered to a patient in the later stages of AD.

[0157] In some embodiments of the present disclosure, an effective amount of an antisense oligonucleotide is administered to the eye, spinal cord, cerebrospinal fluid, brain, and / or liver.

[0158] In some embodiments of the present disclosure, an effective amount of an antisense oligonucleotide is administered to an individual when a close relative of the individual has been diagnosed with Alzheimer's disease.

[0159] In some embodiments of the present disclosure, an effective amount of an antisense oligonucleotide is administered to an individual if a close relative of the individual is found to currently have or have long had Alzheimer's disease.

[0160] For example, it may be known that an individual's relative has AD when the relative has been diagnosed by a clinician, or it may be known that the individual's relative currently has or has had Alzheimer's disease by obtaining information from other sources, such as family records or memories.

[0161] An individual's next of kin may be understood as members of that individual's family.

[0162] A close relative of an individual may be understood as someone who shares genetic material with said individual.

[0163] For example, an individual's next of kin is his great-grandmother. For example, an individual's next of kin is his great-grandfather. For example, an individual's next of kin is his grandmother. For example, an individual's next of kin is his grandfather. For example, an individual's next of kin is his mother. For example, an individual's next of kin is his father.

[0164] For example, close relatives of an individual are siblings. For example, a close relative of an individual is a sibling. For example, an individual's next of kin is a daughter. For example, an individual's next of kin is a son.

[0165] In some embodiments of the present disclosure, an effective amount of an antisense oligonucleotide is administered to an individual once one or more members of the individual's family have been documented to be afflicted with Alzheimer's disease.

[0166] In some embodiments of the present disclosure, an effective amount of an antisense oligonucleotide is administered to an individual once one or more members of the individual's family have been documented to be afflicted with Alzheimer's disease.

[0167] Those skilled in the art will understand the genetic predisposition of the risk of developing AD.Those skilled in the art will understand that administering the antisense oligonucleotide of the present invention to individuals at risk of developing AD, for example, to individuals at risk when they are close relatives of people diagnosed with AD, provides the possibility of preventing AD before the onset of disease, or alleviating the symptoms of AD.

[0168] In a further aspect, the disclosure is directed to the use of said antisense oligonucleotide or said composition in the manufacture of a medicament for treating Alzheimer's Disease (AD) or a disease or disorder related to Alzheimer's Disease.

[0169] In a further aspect, the present disclosure is directed to the use of said antisense oligonucleotides for the preparation of a medicament for treating Alzheimer's Disease (AD) or a disease or disorder related to Alzheimer's Disease.

[0170] In a further aspect, the disclosure is directed to a method of using the antisense oligonucleotides (ASOs) and / or compositions to mediate exon skipping in a SORL1 transcript in a cell, tissue or organ, wherein the exon is selected from the group consisting of exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32 and exon 33 of SORL1.

[0171] In some embodiments of the disclosure, one ASO is used in the method.

[0172] In some embodiments of the present disclosure, two or more ASOs are used in the method, such as two ASOs, such as three ASOs, such as four ASOs, such as five ASOs, such as six ASOs.

[0173] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:16 and SEQ ID NO:17.

[0174] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:16 and SEQ ID NO:18.

[0175] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:16 and SEQ ID NO:19.

[0176] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:17 and SEQ ID NO:18.

[0177] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:17 and SEQ ID NO:19.

[0178] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:18 and SEQ ID NO:19.

[0179] In some embodiments of the disclosure, the composition comprises the ASOs set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0180] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:19.

[0181] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0182] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0183] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:24 and SEQ ID NO:25.

[0184] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:24 and SEQ ID NO:26.

[0185] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:24 and SEQ ID NO:27.

[0186] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:25 and SEQ ID NO:26.

[0187] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:25 and SEQ ID NO:27.

[0188] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:26 and SEQ ID NO:27.

[0189] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

[0190] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:27.

[0191] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0192] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0193] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO: 32 and SEQ ID NO: 33.

[0194] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:32 and SEQ ID NO:34.

[0195] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO: 32 and SEQ ID NO: 35.

[0196] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:33 and SEQ ID NO:34.

[0197] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO: 33 and SEQ ID NO: 35.

[0198] In some embodiments of the present disclosure, the ASO used in the method is the ASO shown in SEQ ID NO:34 and SEQ ID NO:35.

[0199] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

[0200] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:35.

[0201] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0202] In some embodiments of the present disclosure, the ASO used in the method is the ASO set forth in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0203] In some embodiments of the disclosure, exon skipping of one exon is mediated by the method.

[0204] In some embodiments of the disclosure, exon skipping of more than one exon, such as two exons, such as three exons, such as four exons, is involved in the method.

[0205] In an alternative further aspect, the disclosure is directed to a method of determining efficiency of ASO-mediated exon skipping of SORL1 in a subject, the method comprising the steps of: a) analyzing a level of excreted SORLA in a first sample comprising cerebrospinal fluid from the patient obtained prior to treatment with the ASO; b) analyzing the level of excreted SORLA in a second sample containing cerebrospinal fluid from the same patient as in a) obtained after treatment with the ASO; c) comparing the levels of excreted SORLA in the samples of a) and b); If the level of excreted SORLA in b) is higher than that in a), exon skipping is determined.

[0206] In an alternative aspect, the disclosure is directed to a method of determining the efficiency of ASO-mediated exon skipping of SORL1 in a subject, the method comprising the steps of: a) analyzing a level of excreted SORLA in a first sample comprising cerebrospinal fluid from the patient obtained prior to treatment with the ASO; b) analyzing the level of excreted SORLA in a second sample containing cerebrospinal fluid from the same patient as in a) obtained after treatment with the ASO; c) comparing the levels of excreted SORLA in the samples of a) and b); d) determining that exon skipping has occurred if the level of excreted SORLA in b) is higher than that in a).

[0207] Those skilled in the art will understand that the level of excreted SORLA may be used as an indicator of functional SORLA that can be physiologically processed in cells and excreted according to physiological processes, as described in the examples disclosed herein.For example, non-functional SORLA that is misfolded due to mutations in one or more CR domains will not be physiologically processed and will not be excreted or will be excreted to a low extent.For example, functionality is restored by exon skipping methods using ASOs disclosed herein, thus removing the mutated exon and restoring the processing and excretion of SORLA.

[0208] In some embodiments of the present disclosure, the method optionally includes obtaining samples b) at several time points after treatment with the ASO, thereby monitoring the efficiency of ASO-mediated exon skipping over time.

[0209] In a further aspect, the disclosure is directed to a method of determining whether a patient identified as having a SORL1 mutation would benefit from treatment with ASO-mediated exon skipping, the method comprising the steps of: a) identifying a mutation in any one of exons 23 to 33 of SORL1; b) introducing the SORL1 mutation identified in the patient into the cell line; c) selecting one or more ASOs that target exons harboring the identified mutations; d) contacting a first aliquot of cells with medium containing one or more selected ASOs and contacting a second aliquot of cells with medium not containing the ASOs; e) analyzing the level of excreted SORLA in the first aliquot and the second aliquot; f) comparing the level of excreted SORLA in the first aliquot and the second aliquot; Thereby, if the level of excreted SORLA in the first aliquot is higher than in the second aliquot, it is determined that the patient will benefit from treatment with one or more ASOs.

[0210] In some embodiments of the present disclosure, the method is an in vitro method.

[0211] In a further aspect, the disclosure is directed to a method of generating an ASO suitable for treating an Alzheimer's disease (AD) patient, wherein the patient carries a mutation in an exon encoding a complement-type repeat (CR) domain of SORLA, the method comprising the steps of: a) identifying, for example by a computer, the ASO according to any one of claims 1 to 21; b) determining whether the target site of the ASO contains a mutation or whether the target size of the ASO does not contain a mutation; This determines that ASOs that bind to target sites that do not contain mutations are suitable for treating AD patients.

[0212] One of skill in the art will understand that mutations in the ASO target site may impair effective binding of the ASO.

[0213] In some embodiments of the disclosure, the mutation is a calcium cage mutation or an odd cysteine ​​mutation.

[0214] In some embodiments of the disclosure, the mutation is an arginine to cysteine ​​substitution at position 1080 of human SORL1 (R1080C), or the mutation is an aspartic acid to histidine substitution at position 1105 of human SORL1 (C1105H). EXAMPLES

[0215] Example 1: Development of SORL1-specific exon-skipping ASO the purpose To provide proof of concept, we identified individual ASOs that induce or are candidates for inducing exon skipping of SORL1 exon 23, SORL1 exon 27, and SORL1 exon 33.

[0216] background: The human SORLA polypeptide contains 2214 amino acids that fold into multiple protein domains, including the VPS10p domain, the YWTD-b-propeller domain linked to the EGF domain, 11 CR domains, 6 3Fn domains, a transmembrane domain, and a cytoplasmic tail domain (Figure 1). The CR domains are encoded by exons 23-33. The CR domain sequences contain approximately 40 amino acids, including six strictly conserved cysteines that form three intradomain disulfides (Figures 2A and B). Also conserved are four residues with acidic side chains, which function in the octahedral coordination of calcium ions. Figure 2C shows a sequence comparison of the 11 CR domain sequences of SORLA, with the domain boundaries following their individual exon structures (i.e., exons 23-33).

[0217] FIG. 3 is a schematic diagram showing how antisense oligonucleotides (ASOs) can be used in exon skipping. Each of the eleven CR domains is encoded by its own exon (exons 23-33). Because each of these exons contains a multiple of three nucleotides, skipping of an exon does not affect the reading frame of downstream exons. Thus, ASOs against individual exons targeting the 3' splice site, 5' splice site, and / or splice enhancer site (ESE) of one or more exons can be used to treat Alzheimer's disease by removing mutated exons from the SORL1 transcript, as exemplified herein by exon 23.

[0218] Interestingly, SORL1 variants from Alzheimer's disease patients are spread throughout the SORL1 gene, such that >25% of all variants are located in the genomic region encoding the 11 CR domains ( Holstege, 2020 ).

[0219] We recently analyzed the distribution of ONC (odd cysteine) and CC (calcium cage) variants in the CR domain sequence (data not shown). SORL1 variants identified in AD patients from recently published exome sequencing data were mapped from AD patients and non-demented controls. Each of the 11 CR domains harbors mutations from AD patients.

[0220] Figure 4 shows the evidence for ASO treatment and the subsequent effect on SORLA activity. Figure 4A illustrates wild-type (WT) SORL1 gene expression resulting in normal protein function and endosomal processing. SORLA expressed from the wild-type allele has 11 CR domains (11x) and functions in endosomal cargo recycling. Figure 4B illustrates SORLA expressed from alleles with ONC- or CC-type AD mutations (shown as black CR domains) that lead to receptor misfolding and ER retention that may also affect translation products from the wild-type allele. As a result, mutated SORLA cannot undergo normal endosomal processing and prevent amyloidogenic processing of APP, leading to AD. Figure 4C illustrates SORLA from disease alleles treated with exon-skipping ASOs that contains 10 functional CR domains (10x) and has functions indistinguishable from full-length SORLA protein, i.e., normal protein function and endosomal processing. The result would be a reduction in AD symptoms and, ideally, a cure for AD.

[0221] Identification of ASO candidates targeting exon 23, exon 27, or exon 33 Materials and Methods: Based on predicted ESE (exon splice enhancer) elements in exons 23, 27, and 33, we identified ASO candidates targeting exon 23 (ASO23.1, 23.2, 23.3, 23.4 described below), exon 27 (ASO27.1, 27.2, 27.3, 27.4), and exon 33 (ASO33.1, 33.2, 33.3, 33.4 described below). In the first set of experiments, we purchased standard backbone phosphorothioate (PS) ASOs with 2'-O-methyl (2'OMe) ribose modifications (ASO23.1, 23.2, 23.3, 23.4) as these molecules are suitable for initial cell experiments and we are examining these molecules in cell culture experiments. ASO candidates targeting exons 27 and 33 were also purchased and will be examined in follow-up experiments.

[0222] To improve the skipping efficiency, we will design a set of new ASOs based on the results from the primary evaluation to systematically define the optimal sequences, explore the effects of different backbone and ribose chemistries, and select ASOs targeting ESEs with the greatest effect on exon skipping in additional experiments.

[0223] Regarding ribose chemistry, we plan to investigate the effect of 2'-O-methoxyethyl modifications. In particular, we will examine ASOs with modified backbone chemistries, including phosphorodiamidate morpholine oligomers (PMOs), peptide nucleic acids (PNAs), and locked nucleic acids (LNAs) (reviewed in Dhuri 2020).

[0224] result: We performed computational analysis to identify (predicted) strong exonic splice enhancers (ESEs) using the online tool SpliceAid (Piva 2009). Due to the high sequence similarity between the SORLA CR domains (14 of 40 positions contain amino acids conserved in 11 CR domains), we decided to prioritize sequences targeting the 3'ss boundary. An additional advantage of targeting transcripts at the 3'ss is that ASOs should be able to target exons containing disease variants affecting the calcium cage, so that in an optimal situation they are not part of the ASO sequence, but rather the distance to the sequence coding for residues that are part of the calcium cage, which would be mismatched for the disease allele. Further experiments will explore other options, for example targeting the 3'ss boundary, the 5'ss boundary, and / or one or more exonic splice enhancer (ESE) sites for any one of exons 23-33.

[0225] For each of the three exons 23, 27, and 33, we selected a total of four sequences targeting the ESE with the highest positive scores predicting the presence of splicing enhancing sequences (negative scores predict splicing repressing sequences): ASO23.1, ASO23.2, ASO23.3, ASO23.4 (for Ex23 see Fig. 5A,D), ASO27.1, ASO27.2, ASO27.3, ASO27.4 (for Ex27 see Fig. 5B,E), and ASO33.1, ASO33.2, ASO33.3, and ASO33.4 (for Ex33 see Fig. 5C,F) as outlined in Fig. 5 and Tables 1-3. In Figure 5D–F, bars in different shades of grey indicate binding sites for splice factors. If ASOs target these sites, they are likely to induce exon skipping because they block access of splice factors to the target sequence, resulting in inhibition of splicing and exon skipping.

[0226] [Table 1]

[0227] The following sequence (5'-3', sense strand) shows exon 23 (uppercase letters) and the 5' and 3' flanking intronic sequences (lowercase letters): tcccctgccgcactctgatgggtagAGAACACCTGTCTTCGCAACCAGTATCGCTGCAGCAACGGGAACTGTATCAACAGCATTTGGTGGTGTGACTTTGACAACGACTGTGGAGACATGAGCGATGAGAGAAACTGCCgtgagtcttctggattggacgttaa

[0228] The respective (partial) precursor mRNA transcripts containing exon 23 are (underlined: target sites for ASO23.1-ASO23.4): ucccugccgcacucu gauggguagAGAACACCUGUC UUCGCAACCAG UAUCGCUGCAGCAACGGGAACUGUAUCAACAGC AUUUGGU GGUGUGACUUUGACAACGACU GUGGAGACAUGAGCGAUGAGAGAAACUGCCgugagucuucuggauuggacguuaa

[0229] [Table 2]

[0230] The following sequence (5'-3', sense strand) shows exon 27 (uppercase letters) and the 5' and 3' flanking intronic sequences (lowercase letters): tctgtgttgttgaattctatttcagAGAAGAAGTGCAATGGATTCCGCTGCCCAAACGGCACTTGCATCCCATCCAGCAAACATTGTGATGGTCTGCGTGATTGCTCTGATGGCTCCGATGAACAGCACTGCGgtgagttcattccttgcccccagga

[0231] The respective (partial) precursor mRNA transcripts containing exon 27 are (underlined: target sites for ASO27.1-ASO27.4): ucu guguguugaauucuauuucagAGAGAAGUGC AAU GGAUUCCGCUGCCCAAACGGC A CUUGCAUCCCAUCCAGCAAAC AUUGUGAUGGUCUGCGUGAUUGCUCUGAUGGCUCCGAUGAACAGCACUGCGgugaguucauuccuugcccccagga

[0232] [Table 3]

[0233] The following sequence (5'-3', sense strand) shows exon 33 (uppercase letters) and the 5' and 3' flanking intronic sequences (lowercase letters): cttaagaagcctctctgtgtttcagCCACACACAGCACCTTGACTTGCATGAGCAGGGAGTTCCAGTGCGAGGACGGGGAGGCCTGCATTGTGCTCTCGGAGCGCTGCGACGGCTTCCTGGACTGCTCGGACGAGAGCGATGAAAAGGCCTGCAGTGgtgagtgccggtccacgggctgggc

[0234] The respective (partial) precursor mRNA transcripts containing exon 33 are (underlined: target sites for ASO33.1-ASO33.4): Cuua agaagccucucuguguuucagCCACACACAGCACCUUGACUUGCAUGAGCA GGGAGUU CCAGUGCGAGGACGGGGAGGC CUGCAUUGUGCUCUCGGAGCGCUGCGACGGCUUCCUGGACUGCUCGGACGAGAGCGAUGAAAAGGCCUGCAGUGgugagugccgguccacgggcugggc

[0235] To assess the effect of intentional exon skipping, we designed a set of primers spanning the targeted exons to amplify the SORL1 transcript in cells transfected with increasing doses of various ASOs (20, 50, 100, and 200 nmol). Exon skipping results in smaller amplicons of 114 bp, 108 bp, or less than 132 bp for exons 23, 27, and 33, respectively.

[0236] ASO affinity, specificity, efficiency, stability and tolerance can be increased by chemical modifications of the monomer and the internal structure of the backbone. Therefore, in a final set of experiments, we will repeat the experiments using the most effective ASO sequence with a number of chemical modifications intended to increase its effect on exon skipping. Also, if possible, without losing exon specificity, we will optimize the sequence for length (shorter, up to 18 bases, or longer, up to 25 bases), GC content (aiming for 45-55%), RNA secondary structure (aiming for optimal melting temperature), prediction of dimer structures to be preferred over multimers, etc.

[0237] Additionally, Table 4 shows the intron / exon boundaries and 3' splice acceptor or 5' splice donor sites that can be used to target any one of exons 23-24 using the ASOs and procedures described in this example. The underlined nucleotides represent the exon, while the non-underlined nucleotides represent a portion of the adjacent intron. The bold nucleotides represent the boundary between the intron and the exon, i.e., the terminal nucleotide of the intron (ag / gt). [Table 4]

[0238] ASO-mediated exon skipping Materials and Methods: ASOs are transfected into human lymphoblastoid cell lines (LCLs) with wild-type SORL1 genotype using standard transfection reagents, e.g., lipofectamine. Cells are harvested either 24 or 48 hours after transfection, and total RNA is extracted using the RNAeasy isolation kit. Transcripts are amplified using one-step SuperScript RT with total RNA as template. To assess exon 23 skipping, SORL1 transcripts are amplified using primers Ex20fw (fw=forward) and Ex26rev (rev=reverse) and standard PCR conditions. To assess exon 27 skipping, SORL1 transcripts are amplified using primer pair Ex24fw and Ex30rev, and to assess exon 33 skipping, SORL1 transcripts are amplified using primers Ex30fw and Ex36rev. PCR amplicons are fractionated on a 2% agarose gel in Tris-acetate-EDTA buffer. Relative exon skipping efficiency is estimated via densitometric analysis of images using ImageJ image analysis software.

[0239] In another experiment, human HEK293 cells are transfected with ASOs and cells and media are harvested 24 hours after transfection. In another experiment, iPSC-derived neurons are used for transfection and RT-PCR is performed as described above.

[0240] For the above-described ASO candidates targeting exon 23, exon skipping was examined in vitro. For ASO treatment, HEK cells were seeded at a density of 1 × 105 cells per well on 4-well dishes. The next day, cells were transfected with ASOs targeting exon 23 splicing at a final concentration of 250 nM using Oligofectamine transfection reagent (Thermo Fischer, #12255011) diluted in serum-free medium according to the manufacturer's protocol. Cells were harvested 48 hours after transfection, after which RNA extraction was performed using the RNeasy Kit (Qiagen) according to the manufacturer's protocol, and then cDNA was prepared by the High-Capacity RNA to cDNA kit (Applied Biosystems, #4387406). Exon 23 skipping was verified by RT-PCR using the following primers: forward, 5'-ACACTGGAAGCAATGCCTGT-3' (SEQ ID NO: 47); reverse: 5'-CGGCACTGGTGCATTTCAC-3' (SEQ ID NO: 48).

[0241] result: We tested four ASOs (ASO23.1, ASO23.2, ASO23.4, and 23.4) directed against the exon splice enhancer (ESE) element of exon 23 of SORL1 by transfecting HEK293 cells, recovering endogenous SORL1 mRNA, and performing RT-PCR using primer pairs spanning the region around exon 23. PCR products were separated by agarose gel electrophoresis, and data from two independent experiments are shown (Figure 6, top and bottom panels). In both experiments, we observed clear evidence that ASO23.2 and ASO23.3 induce exon 23 skipping, as demonstrated by the presence of a shorter PCR product (see arrow) that co-migrated with the product generated using a plasmid encoding an exon 23 deletion fragment (pΔEx23, a recombinantly generated mutant lacking exon 23 used as a control) as a template. pFL served as a control template to identify a PCR product corresponding to a fragment with included exon 23. Faint bands of shorter PCR products were also seen, potentially indicating that ASO23.1 and ASO23.4 also have specific exon skipping efficiencies.

[0242] Conclusion: Our results indicate that ASO-mediated exon skipping can be achieved, as exemplified by the skipping of exon 23 by at least two of our candidate ASOs. We will further identify ASOs that can induce the skipping of exon 27 or exon 33 of the human SORL1 transcript, as well as other exons encoding CR domains. This approach can be used to target and remove any one or several of exons 23-33.

[0243] Example 2: Retained functionality of modified SORLA proteins (lacking one or more CR domains) the purpose The goal was to determine whether the CR domain, exemplified by CR domain 1 (corresponding to the deleted exon 23), is dispensable for SORLA.

[0244] Materials and Methods: Using specific primers and an expression construct for full-length SORL1, fragments leading to the deletion of exon 23 were generated by PCR. These fragments were joined using Gibson assembly. N2a cells were transfected with plasmids encoding either full-length or exon 23 deleted SORLA proteins. Western blots of lysates and conditioned media from transfected cells were performed as described. Note that in N2A cells SORLA appears as a single band of approximately 250 kDa on western blots, in contrast to the characteristic doublet band in HEK cells.

[0245] Generation of cDNA constructs of SORL1 Deletion of individual exons in the SORL1 cDNA was performed by Gibson assembly technique using SORL1-wt cDNA as template in pcDNA3.1 / zeo vector (Jacobsen et al. 2001). Vector and SORL1 fragments were amplified using Herculase II fusion DNA polymerase (Agilent) with specific primer sets (Table 5). PCR products were first digested with Dpnl enzyme (New England Biolabs) to remove methylated DNA template and then purified using PCR purification kit (Qiagen, #28104). Purified fragments were ligated using Gibson assembly kit (NEB, #5510S) and ligated products were transformed into NEB 5α competent E. coli according to the manufacturer's protocol (NEB, #C29871). Correct deletion of exons in each plasmid was verified by Sanger sequencing (Eurofins). [Table 5] TIFF2024524332000006.tif119162

[0246] Transfection of cells with SORL1 exon deletion constructs For analysis of APP processing and SORLA shedding, N2a cells were cultured in DMEM supplemented with 10% FBS and penicillin / streptomycin and plated at 5 × 10 cells per well the day before transfection. 5 Cells were seeded in 6-well plates. Cells were then transiently co-transfected with a myc-flagged construct encoding APP and either SORL1-wt or exon deleted constructs using Fugene HD transfection reagent according to the manufacturer's protocol (Promega). After 48 h of recovery, culture medium was replaced with conditioned serum-free medium, and lysates and medium were harvested after a further 48 h.

[0247] Western blot Equal amounts of protein from lysates and media of N2a-transfected cells were loaded onto Nupage 4-12% Bis-Tris gels (Invitrogen, #NP0321BOX) and transferred to nitrocellulose membranes using an iBlot2 gel transfer apparatus (Life Technologies). Membranes were probed overnight at 4°C with the following primary antibodies: anti-myc (1:1000, Invitrogen), anti-APP (WO2, 1:1000, Sigma, MABN10), LR11 (1:500, BD Transduction Laboratories), anti-solSORLA (1:1000, IgG5387, Jacobsen et al. 2001), and anti-actin (1:5000, Sigma, A2066). The next day, membranes were washed and incubated with HRP-conjugated secondary antibodies (anti-mouse, anti-rabbit; 1:1500,) for 1 h at room temperature. Proteins were detected with SuperSignal West Femto maximum sensitivity substrate (Thermo Fischer Scientific) using an iBright image analysis system (Thermo Fischer).

[0248] result: We prepared cDNAs encoding human SORLA lacking exon 23, which encodes the first CR domain of the SORLA CR cluster. Transfected cells showed indistinguishable expression levels in lysates (Fig. 7A, left panel). The amount of SORLA (sSORLA) shed from the cell surface into the medium was indistinguishable in cells transfected with SORLA-wt or SORLA-delta-exon 23 (Fig. 7A, right panel), indicating that the SORLA mutant lacking the CR domain corresponding to exon 23 appears to undergo trafficking and maturation similar to wt SORLA and is functional. This is due to the fact that SORLA needs to interact with its respective ligand in order to undergo trafficking and maturation to endosomes. This interaction appears to be functional, since shed SORLA lacking the CR domain corresponding to exon 23 could be detected at levels similar to full-length SORLA.

[0249] Conclusion: We conclude that SORLA lacking its first CR domain can be expressed and selected in cells in a manner indistinguishable from wild-type SORLA.

[0250] Example 3: Retained functionality of SORLA proteins lacking one CR domain (corresponding to the skipped exon 23) compared to mutant proteins corresponding to pathogenic SORL1 variants located in the same CR domain the purpose: The aim is to compare the activity of CR deletion proteins or CR mutant proteins against SORLA, exemplified by the most N-terminal CR domain.

[0251] Materials and Methods: N2a cells were transfected with plasmids encoding either full-length SORLA, full-length SORLA with the mutation D1105H, full-length SORLA with the mutation R1080C, or SORLA with a CR1 deletion (by removal of sequences encoded by exon 23). Western blot analysis of lysates and conditioned media from transfected cells was performed with the commercially available antibody LR11 (anti-SORLA antibody, e.g., Sigma Aldrich, SAB2500979). Further details regarding the materials and methods used are provided in Example 2.

[0252] result: We prepared a cDNA encoding human SORLA lacking exon 23, which encodes the first CR domain of the SORLA CR cluster. Using standard site-directed mutagenesis, we prepared mutant constructs corresponding to the mutations D1105H or R1080C. The mutation R1080C has an odd number of cysteines and represents a group of identified mutations considered pathogenic. The mutation D1105H not only affects a residue important for forming the Asx turn in the CR domain, but is also considered to be as strongly pathogenic as mutations affecting the calcium cage.

[0253] Transfected cells showed indistinguishable expression levels in lysates (Fig. 7B, left panel). Cells transfected with SORLA-wt or SORLA-delta-exon 23 showed indistinguishable amounts of SORLA (sSORLA) shed from the cell surface into the medium, whereas it was greatly reduced in cells expressing pathogenic SORL1 variants (represented here by mutations D1105H and R1080C) (Fig. 7B, lower panel).

[0254] Conclusion: We conclude that SORLA harboring pathogenic variants in CR1 (exon 23) shows strongly reduced shedding and thus generation of strongly reduced sSORLA, whereas SORLA lacking its first CR domain is shedding in a manner indistinguishable from wild-type SORLA.

[0255] We further conclude that sSORLA production is impaired in cells expressing pathogenic SORL1 variants, and monitoring sSORLA is a highly efficient method to assess the efficiency of ASO-induced exon skipping. This further implies that patients treated with ASOs to correct SORL1 variants have increased sSORLA in their cerebrospinal fluid (CSF), since only SORLA variants lacking the CR domain corresponding to the mutated exon become excreted, whereas full-length SORLA with CR domain mutations is likely to be retained in the ER and cannot undergo endosomal processing. This data supports the exon skipping strategy disclosed herein to cure SORL1-related AD.

[0256] Example 4: Application of ASO-mediated exon skipping of exon 23 to AD treatment the purpose: The aim was to demonstrate that SORLA, which lacks the CR domain encoded by exon 23, is able to protect APP from (endosomal) processing.

[0257] Materials and Methods N2a cells were transfected with APP alone (control) or in combination with either SORLA-wt (full length) or SORLA-delta-exon 23 (deletion of exon 23 by a cDNA cloning strategy). Lysates and conditioned media from cells were analyzed by Western blot using antibodies against APP, SORLA or actin (in lysate samples), or shedding APPα (sAPPα) or SORLA (sSORLA) (in media samples). Further details regarding the materials and methods used are described in Example 2.

[0258] result: Experiment 1: In this setup, N2a cells do not express any or negligible amounts of endogenous SORLA, i.e., endogenous SORLA is undetectable compared to exogenous SORLA (overexpressed by transfection) by Western blot. Lysates (data not shown) and conditioned media from transfected cells were analyzed by Western blot using antibodies for the extracellular domain of SORLA (5387) or APP (anti-myc for cell morphology or WO2 for shed sAPPa). Cells without exogenous SORLA overexpression (control) show a strong signal for sAPP in the medium. In contrast, medium from cells transfected with either full-length SORLA or a SORLA construct with an exon 23 deletion shows significantly reduced levels of sAPP (FIG. 8A).

[0259] Test 2: Again, we observed that levels of shed SORLA (sSORLA) were similar between SORLA-WT and SORLA-ΔEx23, indicating that deletion of CR1 (encoded by exon 23) has no observable effect on receptor biology. We also again observed that the two SORLA mutant (full-length and SORLA-delta-exon 23) proteins had indistinguishable effects on reducing sAPPα production by decreasing APP proteolysis ( Fig. 8, B to C ). Blots from three independent replicates were quantified and data presented as the average of duplicate samples with levels relative to cells without exogenous SORLA (Figure 8D). This quantification further demonstrates that SORLA ΔEx23 is as effective as WT in reducing efflux APPa (sAPPa).

[0260] Conclusion: SORLA deleted for CR1 (encoded by exon 23) was as effective at inhibiting APP processing as the full-length SORLA receptor, demonstrating that targeted deletion of CR1 by ASO to induce skipping of the mutated exon 23 leads to a functional receptor.

[0261] Example 5: Retained functionality of modified SORLA proteins lacking CR domain 11 the purpose The goal was to determine whether CR domains, exemplified by CR domain 11 (corresponding to the deleted exon 33), are dispensable for SORLA.

[0262] Materials and Methods: N2a cells were transfected with either the SORL1-WT or SORL1-ΔEx33 constructs, and lysates and conditioned medium from the cells were analyzed by Western blot using antibodies against SORLA or actin (in lysate samples) or extruded SORLA (sSORLA) (in media samples). Further details regarding the materials and methods used are provided in Example 2.

[0263] result: We observed that the levels of excreted SORLA (sSORLA) were similar between SORLA-WT and SORLA-ΔEx33, indicating that the deletion of CR11 (encoded by exon 33) has no observable effect on the biology of the SORLA receptor, i.e., even SORLA lacking CR11 is processed and excreted similarly to SORLA-WT (Fig. 9 ; media samples).

[0264] Conclusion: We conclude that SORLA lacking CR domain 11 can be expressed and selected in cells in a manner indistinguishable from wild-type SORLA. Our data indicate that deletion of specific CR domains, which may contain pathogenic mutations, exemplified here by CR11, does not impair SORLA processing and function, and thus exon skipping of the respective CR domains is a feasible therapeutic strategy.

[0265] Example 6: Extension of the technique to additional CR domain exons the purpose: We wanted to determine which exons of SORL1 are suitable for an ASO-induced exon-skipping therapeutic strategy.

[0266] Materials and Methods: Examples 3-5 show that specific SORLA exons may be skipped while retaining SORLA function (e.g., cellular processing, excretion, effects on APP processing). We investigated the deletion of additional exons and performed a systematic deletion of exons 23-33 (recombinantly produced constructs). We generated plasmids encoding SORLA deleted in a single CR domain (except for CR7, which was included in the tandem deletion of CRF7+8; ΔEx29+30). HEK293 cells were transfected with constructs engineered to generate SORLA proteins deleted for individual CR domains, i.e., CR1 (ΔEx23), CR2 (ΔEx24), CR3 (ΔEx25), CR4 (ΔEx26), CR5 (ΔEx27), CR6 (ΔEx28), CR7+8 (ΔEx29+30), CR8 (ΔEx30), CR9 (ΔE31), CR10 (ΔE32) or CR11 (ΔE33). Lysates were prepared from cells harvested 72 hours post-transfection, proteins were separated by 26-lane SDS-PAGE NuPAGE system and analyzed by Western blot analysis with polyclonal SORLA serum from rabbits raised against the extracellular fragment of SORLA (sol-SORLA). Further details regarding the materials and methods used are provided in Example 2.

[0267] result: The Western blot in Figure 10 shows the results of systematic deletion of exons 23-33, which correspond to CR domains 1-11 of SORLA, respectively. Blots of SORLA are known to show a doublet band upon expression of SORLA in HEK cells, with the upper band (which runs slower in the gel due to its larger molecular size) representing mature SORLA and the lower band (which runs faster in the gel due to its smaller molecular size). This can be seen, for example, in the doublet band shown for full-length (FL) SORLA in lanes 1 and 2. Each of the constructs resulted in the expression of SORLA receptors with specific CR domain deletions. Interestingly, some deletions showed surprising results suggesting that potentially not all CR domains can be deleted without disrupting receptor function, i.e., the SORLA double banding pattern is disrupted in CR4 (ΔE26) and CR9 (ΔEx31) (*). We will analyze this further in follow-up experiments. Similar results were obtained when the experiment was repeated. Issues with the deletion of exon 29 are discussed below in Example 7. Skipping exon 29 results in an unintended stop codon at the novel exon-exon boundary between exons 28 and 30. Therefore, targeting variants in exon 29 requires simultaneous deletion of the adjacent CR domains.

[0268] Conclusion: We conclude that some exons of SORL1 are suitable for the ASO-induced exon skipping therapy disclosed herein for AD for carriers of pathogenic SORL1 variants in these exons. Our results further indicate that certain CR domains, like other CR domains, may be more suitable for deletion via exon skipping, for example.

[0269] Example 7: Attempted deletion of exon 29: Double deletion ASO treatment the purpose: Skipping exon 29 generates an unintended stop codon at the novel exon-exon boundary between exons 28 and 30. Therefore, targeting mutations in exon 29 requires concomitant deletion of the adjacent CR domain.

[0270] Materials and Methods: We establish exon skipping of exon 29 and the adjacent CR domain of SORLA, e.g., exon 28 or exon 30. Deletion of two exons requires two ASOs targeting target sites in exons 28 and 29 or exons 29 and 30, respectively. Further details regarding the materials and methods used are described in Example 2.

[0271] result: We prepare cDNAs for SORLA with a deletion of exons 28+29 and SORLA with a deletion of exons 29+30.

[0272] Conclusion: The feasibility of ASO-mediated exon skipping of more than one exon will be demonstrated.

[0273] Example 8: ASO rescue experiments the purpose: We provide evidence that ASOs targeting mutated SORL1 exons 23 / 27 / 33 increase the activity of the treated allele.

[0274] Materials and Methods: EBV is used to immortalize a cell line derived from the patient (i.e. lymphoblasts). In parallel, control cells from healthy recipients are obtained and immortalized in parallel. Cells of both origins are treated with ASO for 48 hours or left untreated for controls. Cell lysates and conditioned media are analyzed using Western blot and an antibody against SORLA.

[0275] result: Untreated cells from healthy carriers show two distinct bands in lysates corresponding to mature and immature full-length SORLA, whereas untreated cells from patients with pathogenic variants in the CR domain of SORLA show mostly only the immature receptor variant. Both ASO-treated cells show a strong signal for the mature protein in lysates, and WB analysis of media samples shows a similar pattern with high levels of sSORLA coming from cells that have intracellular mature SORLA protein.

[0276] Conclusion: We conclude that ASOs can correct the production of pathogenic misfolded SORLA protein (with 11 CR domains, one of which is pathogenically mutated), that ASO treatment leads to skipping of the exon harboring the pathogenic mutation, and that these cells express a functional protein (with 10 CR domains with the mutated CR domain removed) as evidenced by the presence of mature SORLA in lysates and sSORLA in the medium.

[0277] Example 9: Optimization of ASO23.2 and ASO23.3 the purpose: The objective is to optimize the lead ASO targeting exon 23 of SORL1, for example, based on the results described in Example 1.

[0278] Materials and Methods: Based on our described observations that ASO23.2 and ASO23.3 showed the most efficient exclusion of exon 23 of SORL1 in our preliminary studies using transfected HEK293 cells (Example 1), we prepare new variants of these two ASOs with the aim of optimizing their efficiency. First, we trim the sequence by moving one base at a time 5' and 3' to the target sequence and by trying to extend the length of the ASO or shorten the sequence. Then, we optimize each ASO with respect to backbone chemistry, exploring, for example, phosphorodiamidate morpholine oligomers (PMOs), peptide nucleic acids (PNAs), and locked nucleic acids (LNAs) (reviewed in Dhuri 2020 et al.). To analyze exon skipping efficiency, human cell lines (HEK293 and SH-SY5Y) are transfected and RNA is harvested 48 hours post-transfection using standard RNA extraction protocols. After preparation of cDNA, RT-PCR analysis is performed using primers specific for the exons flanking Ex23 to then quantify the levels of transcripts where Ex23 has been successfully excluded.

[0279] result: The inventors identify one or more ASOs optimized for application in human cells to skip Ex23 of SORL1.

[0280] Conclusion: One or more of the identified ASOs will be used in trials to test their clinical efficacy.

[0281] Example 10: Preparation of SORL1-associated Alzheimer's disease (SAAD) cell model (iPSC) the purpose: We aimed to generate induced pluripotent stem cell (iPSC) lines carrying an engineered mutant form of Ex23 of SORL1.

[0282] Materials and Methods: Amyloid beta (Aβ) peptides, a pathological hallmark of Alzheimer's disease, are determined using a mesoscale discovery assay, and endosome size is determined using immunocytochemistry applying a Rab5 antibody and quantification of Rab5 positive structures from confocal images using an ImageJ plugin.

[0283] result: Based on our studies in N2a cells carrying SORL1 mutations D1105H and R1080C (e.g., Example 3), if we provide evidence that these are pathogenic mutations, we will introduce these two mutations individually according to standard protocols for guide RNA and CRIPS-Cas9 methodology. Introduction of the mutations will be verified using sequencing and clones expressing the selected mutant proteins. Each cell line will be used to generate human neurons according to published differentiation protocols and then "phenotyped" by assays for measurement of amyloid beta secretion and endosomal swelling according to standard protocols.

[0284] Conclusion: We will generate two iPSC models, each carrying a pathogenic SORL1 variant present in Ex23, and establish the disease phenotype based on Rab5-positive endosomal structures and endosomal processing of amyloid precursor protein to amyloid beta peptide. These cell models will be used for efficacy experiments.

[0285] Example 11: Preparation of lymphoblastic disease model the purpose: We aimed to generate lymphoblastoid cells derived from AD patients and carriers of the Ex23 pathogenic variant of SORL1.

[0286] Materials and Methods: SORLA maturation is determined using WB analysis of cell lysates comparing cells from carriers of pathogenic variants with control cells.

[0287] result: Pathogenic variants in SORL1 result in defective maturation of the protein. Western blot analysis of lymphoblast lysates from control individuals with wild-type SORL1 shows both mature and immature SORLA protein, whereas lysates of cells isolated from carriers of pathogenic SORL1 variants contain relatively more immature SORLA protein and less mature SORLA protein. Furthermore, because shedding to generate the shed sSORLA fragment occurs only for mature SORLA, the levels of sSORLA in the medium from these cells indicate lower levels of sSORLA from lymphoblasts derived from SORL1 variant carriers compared with cells from wild-type SORL1 humans.

[0288] Conclusion: We generate patient-derived lymphoblastoid cell lines from individuals harboring pathogenic SORL1 variants at Ex23 and establish a disease phenotype based on SORLA maturation and secretion.

[0289] Example 12: Establishing the efficacy levels required to rescue the AD phenotype in iPSC disease models the purpose: The goal is to determine the level of Ex23 skipping required to revert the cellular disease phenotype so that it is indistinguishable from control cells with wild-type SORL1.

[0290] Materials and Methods: We prepare neurons from iPSC cells harboring a pathogenic mutation in Ex23 of SORL1 and syngeneic control cells, then treat the cells with our flagship ASO(s) that induce skipping of Ex23 of SORL1. We titrate our ASO(s) using concentrations ranging from 2 nM to 500 nM, as well as perform treatments over multiple variable time courses.

[0291] result: In cells with mutant SORL1, the disease phenotype is gradually rescued, with the greatest effect seen in cells with the highest degree of exon skipping obtained. The level of Aβ secretion and the size of Rab5-positive endosomes will be used as parameters of the disease phenotype. The degree of exon skipping will be assessed using a qPCR assay to detect Ex23 deletion transcripts.

[0292] Conclusion: These experiments will enable us to correlate the effect on disease phenotype with the level of induced SORL1 exon skipping, and we will determine the efficacy required for ASOs that induce skipping of Ex23 of mutated SORL1 to revert the cellular phenotype of iPSC models to one resembling non-diseased cells.

[0293] Example 13: Establishing the efficacy level required to rescue the AD phenotype in lymphoblastic disease models the purpose: The goal is to determine the level of Ex23 skipping required to revert the cellular disease phenotype so that it is indistinguishable from control cells with wild-type SORL1.

[0294] Materials and Methods: We will treat lymphoblastoid cells (from carriers and non-carriers) with our flagship ASO(s) that induce Ex23 skipping of SORL1. We will titrate our ASO(s) using concentrations ranging from 2 nM to 500 nM, as well as perform treatments over multiple variable time courses. In cells with mutant SORL1, the disease phenotype will be gradually rescued, with the greatest effect seen for cells with the highest degree of exon skipping obtained. The levels of SORLA maturation and sSORLA secretion will be used as parameters of the disease phenotype. The degree of exon skipping will be assessed using a qPCR assay to detect Ex23 deletion transcripts.

[0295] result: These experiments will allow correlation of effects on disease phenotype with the level of induced SORL1 exon skipping.

[0296] Conclusion: We identify the efficacy required for ASOs inducing skipping of mutated SORL1 Ex23 to revert the cell phenotype in a lymphoblast model to one resembling non-diseased cells.

[0297] [Table 6] TIFF2024524332000008.tif223162TIFF2024524332000009.tif227162TIFF202 4524332000010.tif229162TIFF2024524332000011.tif221162TIFF20245243320 00012.tif224162TIFF2024524332000013.tif233162TIFF2024524332000014.t if227162TIFF2024524332000015.tif224162TIFF2024524332000016.tif104162

[0298] SEQ ID NO: 49 to SEQ ID NO: 75: PCR primers used for amplification of the SORL1 fragment (Example 2, Table 5). The following is an explanatory example of the nomenclature used in the above table: - "Forward-SORL1E1 ΔE23-Δ28, and ΔE30-ΔE33" means that this sequence is a forward primer for SORL1-ΔE23 (E1-22 PCR product), SORL1-ΔE24 (E1-23), SORL1-ΔE25 (E1-24), SORL1-ΔE26 (E1-25), SORL1-ΔE27 (E1-26), SORL1-ΔE28 (E1-27), SORL1-ΔE30 (E1-29), SORL1-ΔE31 (E1-30), SORL1-ΔE32 (E1-31), SORL1-ΔE33 (E1-32). - "Reverse-SORL1-ΔE23-E1-22" means that this sequence is the reverse primer (5'-3') for SORL1-ΔE23 (E1-22 PCR product).

[0299] References Dhuri 2020, Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development, J.Clin.Med.2020,Jun.26;9(6):2004.doi:10.3390 / jcm9062004. Holstege 2020,Exome sequencing identifies novel AD-associated genes.24,2020,July.24,doi.org / 10.1101 / 2020.07.22.2015925 Jacobsen 2001,Activation and functional characterization of the mosaic receptor SorLA / LR11,J.Biol.Chem.2001,Jun.22;276(25):22788-96.doi:10.1074 / jbc.M100857200.Epub.2001,Apr.9. Piva 2009,SpliceAid:a database of experimental RNA target motifs bound by splicing proteins in humans,Bioinformatics.2009,May.1;25(9):1211-3.doi:10.1093 / bioinformatics / btp124.Epub.2009,Mar.4.

Claims

1. An antisense oligonucleotide (ASO) that binds to a target site on the pre-mRNA of SORL1 and / or is complementary to the target site, wherein the nucleotide sequence of the target site is contained in a nucleotide sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, or contained in a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, the antisense oligonucleotide.

2. The oligonucleotide that binds to the target site and / or is complementary to the target site causes exon skipping of an exon encoding the complementary type repeat (CR) domain of SORLA, and the exon is exon 23 having the sequence of SEQ ID NO: 1, exon 24 having the sequence of SEQ ID NO: 2, exon 25 having the sequence of SEQ ID NO: 3, exon 26 having the sequence of SEQ ID NO: 4, exon 27 having the sequence of SEQ ID NO: 5, exon 28 having the sequence of SEQ ID NO: 6, exon 29 having the sequence of SEQ ID NO: 7, exon 30 having the sequence of SEQ ID NO: 8, exon 31 having the sequence of SEQ ID NO: 9, exon 32 having the sequence of SEQ ID NO: 10, and exon 33 having the sequence of SEQ ID NO: 11, or a nucleotide sequence having at least 80% sequence identity or homology to any one of SEQ ID NOs: 1 to 11, The oligonucleotide according to claim 1, comprising or consisting of.

3. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises a backbone containing phosphorothioate (PS).

4. The oligonucleotide according to claim 1, wherein the oligonucleotide further comprises a modification at at least one nucleotide position or at each nucleotide position.

5. The oligonucleotide according to claim 1, wherein the modification is a modification of the nucleic acid backbone, the nucleic acid base, the ribose sugar, and / or 2'-ribose substitution.

6. The oligonucleotide contains a 2'-O-methoxyethyl sugar modification, and / or The oligonucleotide contains a 2'-O-methyl ribose modification, The oligonucleotide according to claim 1.

7. The target site is a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, or composed of, or containing, a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, the oligonucleotide according to claim 1.

8. The oligonucleotide is a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, or composed of, or containing, a nucleotide sequence having at least 80% sequence identity or homology to a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, the oligonucleotide according to claim 1.

9. A composition comprising the oligonucleotide according to any one of claims 1 to 8.

10. The composition according to claim 9, wherein the composition is a pharmaceutical composition.

11. The composition according to claim 9, comprising one or more of the oligonucleotides.

12. A pharmaceutical composition comprising the antisense oligonucleotide (ASO) according to any one of claims 1 to 8 for use in medicine.

13. A pharmaceutical composition comprising the antisense oligonucleotide (ASO) according to any one of claims 1 to 8 for use in the prevention, treatment and / or alleviation of Alzheimer's disease (AD) or a disease or disorder associated with Alzheimer's disease.

14. A pharmaceutical composition for use according to claim 13, wherein an effective amount of said oligonucleotide is administered to the eye, spinal cord, cerebrospinal fluid, brain and / or liver.

15. A pharmaceutical composition comprising the antisense oligonucleotide (ASO) according to any one of claims 1 to 8 for use in a method of intervening in exon skipping in a SORL1 transcript in a cell, tissue or organ, wherein said exon is selected from the group consisting of exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32 and exon 33 of SORL1.

16. The pharmaceutical composition according to claim 15, wherein one ASO is used or more than one ASO is used.

17. The pharmaceutical composition according to claim 15, which intervenes in exon skipping of one exon or intervenes in exon skipping of more than one exon.

18. A method for determining the efficiency of ASO-mediated SORL1 exon skipping in a subject, said method comprising the following steps: a) Analyzing the level of secreted SORLA in a first sample containing cerebrospinal fluid from a patient obtained before treatment with the ASO; b) Analyzing the level of secreted SORLA in a second sample containing cerebrospinal fluid from the same patient as in a) obtained after treatment with the ASO; c) Comparing the levels of secreted SORLA in said samples of a) and b); comprising whereby, if the level of secreted SORLA in b) is higher than that in a), it is determined as exon skipping.

19. A method for examining whether a patient identified as having a mutation in SORL1 will benefit from treatment by ASO-mediated exon skipping, said method comprising the following steps: a) Identifying a mutation in any one of exons 23 to 33 of SORL1; b) introducing the SORL1 mutation identified in the patient into a cell line; c) selecting one or more ASOs targeting the exon carrying the identified mutation; d) contacting a first aliquot of cells with a medium containing the selected one or more ASOs and contacting a second aliquot of cells with a medium not containing the ASOs; e) analyzing the levels of secreted SORLA in the first and second aliquots; f) comparing the levels of secreted SORLA in the first and second aliquots; comprising: whereby, if the level of secreted SORLA in the first aliquot is higher than that in the second aliquot, it is determined that the patient will benefit from treatment with one or more ASOs. **Claim 20** A method for generating an ASO suitable for treating patients with Alzheimer's disease (AD), wherein the patient has a mutation in an exon encoding the complementary type repeat (CR) domain of SORLA, and the method comprises the following steps: a) identifying the ASO according to any one of claims 1 to 8, for example, by a computer; b) determining whether the target site of the ASO contains the mutation or does not contain the mutation; comprising: whereby, it is determined that an ASO binding to a target site not containing the mutation is suitable for treating AD patients.

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