Improvement of blood brain barrier integrity

Abstract

The present disclosure relates generally to the use of agents, such as oligonucleotides, to improve, increase, restore or maintain the integrity of the blood-brain barrier; and / or to reduce deposition or accumulation and / or to promote clearance of beta-amyloid. The present disclosure also relates to agents, such as oligonucleotides, that increase, promote or restore levels or amounts of VE-cadherin in cells and that can be used in the methods of the present disclosure. The present disclosure also relates to the use of such agents for the treatment of diseases and conditions associated with blood-brain barrier impairment, including diseases and conditions associated with neuroinflammation and neurodegeneration, and diseases and conditions associated with beta-amyloid deposition, including Alzheimer’s disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), and traumatic brain injury (TBI).

Description

Technical Field

[0001] This disclosure generally relates to the use of pharmaceutical agents, such as oligonucleotides, to improve, increase, restore, or maintain the integrity of the blood-brain barrier; and / or reduce the deposition or accumulation of β-amyloid protein and / or promote its clearance. This disclosure also relates to pharmaceutical agents, such as oligonucleotides, that increase, promote, or restore the level or amount of VE-cadherin in cells and are therefore suitable for use in the methods of this disclosure. This disclosure further relates to the use of such pharmaceutical agents for the treatment of diseases and conditions associated with blood-brain barrier damage, including diseases and conditions associated with neuroinflammation and neurodegeneration, and diseases and conditions associated with β-amyloid protein deposition, including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), and traumatic brain injury (TBI).

[0002] Related applications This application claims priority to Australian Provisional Patent Application No. 2023903693 entitled “Improvement of Blood-Brain Barrier Integrity”, filed on November 16, 2023, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] The blood-brain barrier (BBB) ​​is a highly specialized endothelial cell system that plays a crucial role in protecting the brain from infectious or toxic substances in the blood and in controlling the inflow and outflow of biological substances essential for brain metabolism and neuronal function.

[0004] Effective brain-brain barrier (BBB) ​​function arises from complex and unique interactions among brain endothelial cells (ECs), pericytes, vascular smooth muscle cells, astrocytes, neurons, microglia, and the basement membrane (collectively known as neurovascular units (NVUs)). BBB ECs differ from peripheral ECs in several ways, including the absence of fenestrations, lack of pinocytosis, and the expression of active transport mechanisms to regulate the transport of essential molecules while blocking the passage of unwanted substances. BBB ECs are connected by tight junctions and adhesion junctions that differ from their peripheral counterparts in the types and levels of molecules forming these connections, and it is these junctions between ECs that are primarily responsible for such tight control of cellular permeability. Key components of tight junctions in brain ECs include tight junctionin-1, tight junctionin-3, tight junctionin-5, and tight junctionin-12, closure proteins, lipolysis-stimulating proteins (LSRs), and junction adhesion molecules (JAMs)-A, JAM-B, and JAM-C, with tight junctionin-5 being the most abundant and generally considered the most critical for tight junction function. Closed bands (ZO)-1, ZO-2, and ZO-3 bind to motifs on the intracellular domains of tight junction proteins and closure proteins, as well as the actin cytoskeleton, providing structural integrity for tight junctions. Adhesion proteins such as VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) are involved in the formation of adhesion junctions (see, for example, Knox et al., Molecular Psychiatry (2022) 27:2659-2673; Lochhead et al. (2020) Front. Physiol, Greene et al. (2019) FluidsBarriers CNS 16, 3). The specialization and unique properties of ECs in the BBB, combined with the activity of other cellular and component components of the NVU (e.g., the extensive pericellular coverage surrounding the EC), result in extremely low permeability and a significantly higher transendothelial resistance (TEER; ~1500-200 Ω / cm) than observed in peripheral ECs. 2 (Stamatovic et al., Curr Neuropharmacol. 2008 Sep; 6(3): 179-192.)

[0005] The integrity of the blood-brain barrier (BBB) ​​is crucial for brain homeostasis. Conversely, loss of BBB integrity is associated with many serious diseases, including neurodegenerative and / or neuroinflammatory diseases, such as dementias like Alzheimer's disease (AD), and traumatic brain injury (TBI), and may play a role in the pathology of these diseases. For example, elevated blood pressure and / or pulse pressure can lead to blood-brain barrier dysfunction and may be associated with dementia (Levin et al. 2020. Frontiers Neuroscience, art 669). However, the role of the BBB in the pathogenesis of diseases such as AD, cerebral amyloid angiopathy (CAA), and Lewy body dementia (LBD), and particularly the causal or correlational relationships, as well as the relative importance of any one or more specialized molecules, cells, or structures in the BBB in the occurrence or progression of these diseases, remains unclear.

[0006] β-amyloid protein, and plaques formed from and containing β-amyloid protein, are associated with diseases such as AD, CAA, LBD, and TBI, and are thought to cause at least some of the symptoms associated with those diseases.

[0007] Alzheimer's disease (AD) is an age-related neurodegenerative brain disorder characterized by the formation of extracellular β-amyloid plaques and intracellular neurofibrillary tangles in the brain, neuroinflammation, and loss of neurons and synapses, leading to memory loss, cognitive impairment, and impaired reasoning or judgment. Alzheimer's disease is the most common form of dementia, and it is estimated that nearly 7 million Americans may have the disease.

[0008] CAA is a type of cerebrovascular disorder characterized by the accumulation of β-amyloid protein in the pia mater and small to medium-sized cerebral vessels. β-amyloid deposition is believed to lead to vascular weakening, which can potentially cause intracerebral hemorrhage (ICH). CAA patients may also present with amyloid-associated imaging abnormalities (ARIA).

[0009] Despite decades of research and the development of numerous candidate treatments, few effective agents exist for restoring BBB integrity or preventing or reducing β-amyloid. Therefore, new treatments are needed for this purpose. Summary of the Invention

[0010] This disclosure is partly based on the unexpected discovery that oligonucleotides that inhibit miR-27a from binding to VE-cadherin mRNA and thereby increase or restore VE-cadherin levels effectively restore or maintain the integrity of the blood-brain barrier (BBB) ​​and cerebral vascular system, reduce brain cell senescence, and inhibit and / or reduce β-amyloid deposition in the brain.

[0011] As described above, the BBB is a highly specialized endocrine system that interacts with pericytes, vascular smooth muscle cells, astrocytes, neurons, microglia, and the basement membrane to form a biological barrier at the blood-to-brain interface, effectively separating the brain from the rest of the body. BBB endocrine systems differ considerably from peripheral endocrine systems in several ways, including the nature and characteristics of the connections between endocrine systems. At least in part due to the types and / or expression levels of molecules forming tight junctions and adhesion junctions, particularly tight junction proteins such as tight junctionin-5, BBB endocrine systems exhibit reduced permeability and increased TEER compared to peripheral endocrine systems. The function of the BBB is further supported by other cells and structures, such as pericytes and astrocytes, resulting in a virtually impermeable barrier, unlike the barriers seen in the peripheral vascular system.

[0012] Although VE-cadherin is just one of many molecules and cells that function within the highly complex structure known as the brain vascular bundle (BBB), the inventors have unexpectedly demonstrated that targeting this single molecule to increase its expression is sufficient to restore or maintain the integrity of the BBB and cerebral vascular system in a mouse model of Alzheimer's disease. Furthermore, as surprisingly demonstrated in this paper, targeting VE-cadherin with these molecules can inhibit and / or reduce brain cell aging, and also inhibit and / or reduce β-amyloid deposition, formation, and / or accumulation. In some instances, inhibition of β-amyloid deposition occurs within the cerebral vascular system. In further instances, inhibition of β-amyloid deposition can occur outside the vascular system, such as in the brain parenchyma.

[0013] Therefore, this article provides methods for improving, restoring, or maintaining the integrity of the brain brain (BBB) ​​in subjects; methods for inhibiting and / or reducing β-amyloid deposition, accumulation, and / or plaque formation in the brains of subjects; and methods for reducing or inhibiting brain cell senescence in subjects. Methods for treating diseases and conditions associated with loss of BBB integrity (e.g., neuroinflammatory diseases, neurodegenerative diseases, TBI, hypertension, and / or elevated pulse pressure) are also provided. Methods for treating diseases and conditions associated with β-amyloid deposition (including AD, CAA, LBD, and TBI) are also provided.

[0014] In one aspect, a method for improving, increasing, restoring, or maintaining the integrity of a subject's blood-brain barrier (BBB) ​​is provided, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substituted SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA.

[0015] In some instances, the subject has a disease or condition associated with loss of BBB integrity, or has been identified as having the potential to develop a disease or condition associated with loss of BBB integrity. In one implementation, the disease or condition associated with loss of BBB integrity is a neuroinflammatory or neurodegenerative disease, such as dementia, multiple sclerosis (MS), motor neuron disease (MND), Parkinson's disease, and Huntington's disease. Dementia may be selected from Alzheimer's disease, Lewy body dementia (LBD), Parkinson's disease dementia (PDD), vascular dementia, and frontotemporal dementia (FTD). In another implementation, the disease or condition associated with loss of BBB integrity is traumatic brain injury (TBI). In some instances, the subject has dementia, and administration of oligonucleotides results in cognitive improvement or a slower rate of cognitive decline. In other instances, the condition associated with loss of BBB integrity is hypertension or elevated pulse pressure.

[0016] A method for reducing or inhibiting the deposition or accumulation of β-amyloid protein in the brain of a subject, or increasing or promoting the clearance of β-amyloid protein in the brain of a subject, is also provided, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNAs comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA. In some instances, the subject has been diagnosed with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), or Lewy body dementia (LBD), or has been identified as having the potential to develop AD, CAA, or LBD.

[0017] A method for treating a subject suffering from a disease or condition associated with loss of BBB integrity is also provided, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA.

[0018] In some instances, diseases or conditions associated with loss of BBB integrity are neuroinflammatory or neurodegenerative diseases, such as dementia, multiple sclerosis (MS), motor neuron disease (MND), Parkinson's disease, and Huntington's disease. Dementia can be categorized as Alzheimer's disease, Lewy body dementia (LBD), Parkinson's disease dementia (PDD), vascular dementia, and frontotemporal dementia (FTD). In other instances, conditions associated with loss of BBB integrity are traumatic brain injury (TBI), hypertension, or elevated pulse pressure.

[0019] The invention also provides a method for treating a subject with AD, CAA, LBD, or TBI, or a subject who may develop AD, CAA, or LBD, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA.

[0020] In the methods described above and herein, the oligonucleotide may comprise a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:2 or containing one, two, or three substitutions of SEQ ID NO:2, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA. In some instances, the miR-27a miRNA is hsa-miR-27a comprising the nucleotide sequence shown in SEQ ID NO:13.

[0021] In some embodiments, the oligonucleotide comprises a continuous sequence complementary to a sequence of at least or about 7 bases, at least or about 8 bases, at least or about 9 bases, at least or about 10 bases, at least or about 11 bases, at least or about 12 bases, at least or about 13 bases, at least or about 14 bases, at least or about 15 bases, at least or about 16 bases, at least or about 17 bases, at least or about 18 bases, at least or about 19 bases, at least or about 20 bases, at least or about 22 bases, at least or about 25 bases, at least or about 30 bases, or at least or about 35 bases.

[0022] In one instance, the oligonucleotide binds to positions 22-27 of SEQ ID NO:2. In some instances, the base pairing between the oligonucleotide and SEQ ID NO:2 includes positions 8-28, 8-27, 9-27, 10-27, 11-27, 12-27, 13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27, 20-27, 21-27, 9-28, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28, 17-28, 18-28, 19-28, 20-28, or 21-28 of SEQ ID NO:2.

[0023] In certain instances, the oligonucleotide comprises the sequence shown in any one of SEQ ID NO:3 to 6.

[0024] The oligonucleotide may also contain one or more modified nucleobases, such as LNA, UNA, or 2'-O-methyl. In certain instances, the oligonucleotide contains the sequence shown in any one of SEQ ID NO:7 to 11.

[0025] In some instances, the treatment resulted in a reduction or inhibition of β-amyloid deposition or accumulation in the subject's brain; and / or a promotion or increase in β-amyloid clearance in the subject's brain, for example, in the cerebral vascular system and / or brain parenchyma. In some instances, the reduction or inhibition of β-amyloid deposition or accumulation, and / or the promotion or increase in β-amyloid clearance, was in the pia mater vessels of the brain. In further instances, β-amyloid was in the form of β-amyloid plaques.

[0026] The methods disclosed herein may further include the administration of further therapies, such as therapies including other active agents, devices, physical therapy, cognitive therapy, and / or occupational therapy. In one particular instance, the further therapies include active agents or devices that lower blood pressure (e.g., treat hypertension) and / or pulse pressure.

[0027] In specific instances of the method, the administration of oligonucleotides and optional further therapies resulted in cognitive improvement or a slower rate of cognitive decline.

[0028] Also provided is the use of oligonucleotides in the preparation of medicaments for reducing or inhibiting the deposition or accumulation of β-amyloid plaques in the brain of a subject, or for increasing or promoting the clearance of β-amyloid plaques in the brain of a subject, wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNAs comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA. In some instances, the subject has been diagnosed with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), or traumatic brain injury (TBI), or has been identified as having the potential to develop AD, CAA, or LBD.

[0029] Other aspects relate to the use of oligonucleotides in the preparation of medicaments for treating subjects with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), or traumatic brain injury (TBI), wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNAs comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA.

[0030] Other aspects relate to the use of oligonucleotides in the preparation of medicaments for restoring or maintaining the integrity of a subject's blood-brain barrier (BBB), wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:1 or containing one, two, or three substitutions of SEQ ID NO:1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNAs comprising a seed region containing the sequence UCACAG or UCAGAGU to said RNA. In some instances, the subject suffers from a disease or condition associated with loss of BBB integrity, or has been identified as having the potential to develop a disease or condition associated with loss of BBB integrity, such as neuroinflammatory diseases and / or neurodegenerative diseases, such as those selected from dementia, multiple sclerosis (MS), motor neuron disease (MND), Parkinson's disease, and Huntington's disease; or TBI, hypertension, and / or elevated pulse pressure. In some instances, dementia is selected from Alzheimer's disease, Lewy body dementia (LBD), Parkinson's disease dementia (PDD), vascular dementia, and frontotemporal dementia (FTD). Attached Figure Description

[0031] This document describes embodiments of the present disclosure by way of non-limiting example only, with reference to the following figures.

[0032] Figure 1 These are photographs of brain tissue sections from 4-month-old APPswe / PS1dE9 mice that were administered CD5-2 or control Blockmir and Biotin before euthanasia. Biotin leakage was detected in the control mice (grey, red box), while mice receiving CD5-2 showed little or no biotin leakage.

[0033] Figure 2 These are photographs of brain tissue sections from 7-month-old APPswe / PS1dE9 mice that were administered CD5-2 or the control blockmir prior to euthanasia. Albumin leakage was observed in the control mice, while the CD5-2-treated mice showed a relatively reduced amount of albumin leakage.

[0034] Figure 3 These are photographs of brain tissue sections from 6-month-old APPswe / PS1dE9 mice that were administered CD5-2 or control blockmir prior to euthanasia. Significant β-amyloid deposition was observed in the leptomeningeal vessels (LV) and brain parenchyma in mice receiving control blockmir, while mice receiving CD5-2 showed significantly reduced β-amyloid deposition, particularly in the LV.

[0035] Figure 4 These are photographs of brain tissue sections from wild-type mice or from 6-month-old APPswe / PS1dE9 mice that were euthanized and administered CD5-2 or the control blockmir. Albumin leakage (green, arrow) and reduced VE-cadherin expression (red) were observed in APPswe / PS1dE9 mice receiving the control blockmir. Administration of CD5-2 to APPswe / PS1dE9 mice restored VE-cadherin expression and reduced albumin leakage to levels observed in WT mice.

[0036] Figure 5 This diagram illustrates the activity of blockmir CD5-2, CD5-4, and CD5-9, particularly VE-cadherin expression (relative to GADPH) in endothelial cell isolates, as shown by Western blotting. A. Relative VE-cadherin expression in cells transfected with control blockmir, CD5-2, and CD5-4. B. Relative VE-cadherin expression in cells transfected with control blockmir, CD5-2, and CD5-9. C. Schematic diagram of blockmir's structure.

[0037] Figure 6This is a graphical representation of VE-cadherin and tight junction protein-5 expression in the endothelial cell junctions of the pia mater of APPswe / PS1dE9 mice administered CD5-2 or control blockmir. Brain tissue sections were stained and analyzed by confocal microscopy, showing the mean intensity per pia mater area. Mean values ​​from two independent experiments (n=2–3 mice / group). A. VE-cadherin (VEC). B. Tight junction protein-5.

[0038] Figure 7 This image shows albumin leakage in the pia mater of 6-month-old APPswe / PS1dE9 mice administered CD5-2 or the control blockmir. A single mouse received the disordered blockmir control, and two littermates received CD5-2. Brain tissue sections were stained for CD31 (red) or albumin (green) and analyzed by confocal microscopy. A. Representative section from a mouse administered the disordered control blockmir. Arrows indicate albumin leakage. B. Representative section from a mouse administered CD5-2. Arrows indicate albumin leakage. C. Graphical representation showing the amount of albumin leakage per cortical vascular area.

[0039] Figure 8 This is a graphical representation of vascular density in APPswe / PS1dE9 mice administered CD5-2 or control blockmir. Brain tissue sections were stained against CD31 and analyzed by confocal microscopy, and the area of ​​CD31-positive vessels was measured. The values ​​are averages from two independent experiments (n=2–3 mice / group).

[0040] Figure 9 Amyloid deposition in the pia mater and parenchyma of APPswe / PS1dE9 mice administered CD5-2 or the control randomized blockmir. A single mouse received the randomized blockmir control, and two littermates received CD5-2. Brain tissue sections were stained for β-amyloid (green) and DAPI (blue) and analyzed by confocal microscopy. A. Representative stained sections from mice administered the randomized control blockmir and those administered CD5-2. White dashed lines indicate pia mater vessels and the location of penetrating vessels. B. Paired comparisons of amyloid counts in the mouse cortex (shown as the total number of deposited amyloids relative to the cortical region perimeter). N=4 per group.

[0041] Figure 10This image shows the senescence of brain cells in 7-month-old APPswe / PS1dE9 mice pre-administered with CD5-2 or the control blockmir. Brain tissue sections are stained for p21 (red), lamin B1 (green), and DAPI (blue) and analyzed by confocal microscopy. A. Representative sections from mice administered CD5-2 and mice administered the disordered control blockmir. DAPI and p21 staining. B. Representative stained sections similar to those in A). p21 and lamin B1 staining. C. Enlarged view of dashed box (a). D. Enlarged view of dashed box (b). Senescent leptomeningeal cells and neurons exhibit high p21 expression localized to the nucleus (DAPI) and loss of the lamin B1 nuclear membrane. White arrows indicate senescent leptomeningeal vascular-associated cells. Yellow arrows indicate senescent neuronal populations.

[0042] Some of the accompanying drawings and text contain color representations or entities. Color illustrations may be obtained upon request from the applicant or from the appropriate patent office. If obtained from the patent office, a fee may be charged. Detailed Implementation

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise stated, all patents, patent applications, publications, databases, websites, and other published materials mentioned throughout this disclosure are incorporated herein by reference in their entirety. Where multiple definitions exist for a term, the definition in this section shall prevail. When URLs or other such identifiers or addresses are referenced, it should be understood that such identifiers may change and specific information on the Internet may appear and disappear, but equivalent information can be found by searching the Internet. References to identifiers demonstrate the availability and public dissemination of such information.

[0044] As used herein, the singular forms “a,” “an,” and “the” also include the plural aspect (i.e., at least one or more) unless the context clearly specifies otherwise. Thus, for example, reference to “a polypeptide” includes a single polypeptide as well as two or more polypeptides.

[0045] In the context of this specification, the term “about” should be understood as a range of numerical values ​​that a person skilled in the art would consider equivalent to the value in the context of achieving the same function or result.

[0046] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the said integer or step or group of steps, but not to exclude any other integer or step or group of steps.

[0047] As used herein, the term "oligonucleotide" refers to a single-stranded sequence of ribonucleotide or deoxyribonucleotide bases, known analogs of natural nucleotides, or mixtures thereof. "Oligonucleotide" encompasses nucleic acid-based molecules, including DNA, RNA, PNA, LNA, UNA, or any combination thereof. Oligonucleotides primarily consisting of ribonucleotide bases (natural or non-natural) may be called RNA oligonucleotides. Oligonucleotides are typically short sequences (e.g., less than 50 nucleotides in length) that can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences.

[0048] An "antisense oligonucleotide" is an oligonucleotide that is complementary to a specific DNA or RNA sequence. Generally, in the context of this invention, an antisense oligonucleotide is an RNA oligonucleotide that is complementary to a specific mRNA or miRNA. The antisense oligonucleotide binds to and partially or completely silences or inhibits the activity of its complementary miRNA. Not all bases in an antisense oligonucleotide need to be complementary to the "target" or miRNA sequence; the oligonucleotide only needs to contain enough complementary bases to enable it to recognize the target. The oligonucleotide may also contain additional bases. The antisense oligonucleotide sequence can be an unmodified ribonucleotide sequence or can be chemically modified or conjugated using various methods as described herein.

[0049] As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of a known analogue of a deoxyribonucleotide, ribonucleotide base, or a natural nucleotide, or a mixture thereof. "Polynucleotide" includes nucleic acid-based molecules, including DNA, RNA, PNA, LNA, UNA, or any combination thereof. Unless otherwise stated, this term includes references to a specified sequence and its complementary sequence. Polynucleotides can be chemically modified by various means known to those skilled in the art. Therefore, "polynucleotide" includes nucleic acid-based molecules, including DNA, RNA, PNA, LNA, UNA, or any combination thereof.

[0050] As used herein with respect to oligonucleotides and polynucleotides, the term "nucleotide" refers to a single nucleobase or monomeric unit within an oligonucleotide or polynucleotide. The terms "nucleotide" and "monomer" are used interchangeably herein. The nucleobase may be part of a DNA, RNA, INA, LNA, UNA (or any combination of two or more thereof) oligonucleotide or polynucleotide. In some embodiments, the nucleobase may be a universal base. Modified nucleobases are also contemplated in this invention, as described below.

[0051] As used herein, the term "variant" refers to substantially similar sequences. Typically, polypeptide sequence variants also share common qualitative biological activities, such as receptor-binding activity. Furthermore, these polypeptide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The terms "sequence identity" or "percentage of sequence identity" can be determined by comparing two best-aligned sequences or subsequences across a comparison window or span, wherein the polynucleotide sequence portion of the comparison window may optionally include additions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.

[0052] As used herein, the term "complementary" refers to the ability of two single-stranded nucleotide sequences to pair bases, typically according to the Watson-Crick base pairing rule, i.e., between G and C and between A and T or U. In some embodiments, G also pairs with U, and vice versa, to form so-called wobble base pairs. In another embodiment, the base inosine (I) may be included within the oligonucleotide of the invention. I pairs with A, C, and U bases. In yet another embodiment, universal bases may be used. Universal bases typically pair with G, C, A, U, and T bases. Typically, universal bases do not form hydrogen bonds with opposing bases on the other strand. In yet another embodiment, a complementary sequence refers to a continuous sequence containing only Watson-Crick base pairs. For the two nucleotide molecules to be complementary, they do not need to exhibit 100% complementarity in the base pairing regions, but must have sufficient complementarity for base pairing to occur. Therefore, a certain degree of mismatch between sequences is permissible, and the sequences can still be complementary. As used herein, the terms “capable of pairing with… bases” and “complementary to…” are used interchangeably.

[0053] As used herein, the term "substitution" refers to the replacement of a nucleobase at a specific position within an oligonucleotide or polynucleotide by another nucleobase. Substitution can occur, for example, due to a single nucleotide polymorphism in the target RNA. The term "substitution" also encompasses the deletion and addition of nucleobases.

[0054] As used herein, the term "blockmir" refers to a sterically blocking oligonucleotide that binds to an RNA target, thereby blocking the binding of one or more miRNAs to the target and affecting the activity of the target. Blockmir is constructed to prevent the recruitment of cellular RNAi mechanisms or RNase H. RNAi mechanisms refer to cellular components essential for siRNA and miRNA activity or the RNAi pathway. The major component of RNAi mechanisms is the RNA-induced silencing complex (RISC complex). Blockmir is described in, for example, WO2008 / 061537, WO 2012 / 069059, and WO 2014 / 053014, the disclosures of which are incorporated herein by reference.

[0055] In the context of this specification, the term "activity" as it relates to a polynucleotide (e.g., DNA, mRNA, or miRNA), protein, or polypeptide means any one or more cellular functions, actions, effects, or influences exhibited by the polynucleotide, protein, or polypeptide. For example, in the context of mRNA, activity would generally refer to the expression of mRNA, i.e., its translation into a protein or peptide. Thus, the regulation of target mRNA activity by oligonucleotides as described herein may include the degradation and / or translational regulation of mRNA. Regulation of mRNA activity may also include influencing intracellular transport of mRNA. In the context of the oligonucleotides of this invention, "activity" generally means the ability of an oligonucleotide to inhibit the interaction between miR-27a and VE-cadherin, thereby increasing VE-cadherin expression.

[0056] As used herein, the term "inhibiting" and its variations, such as "inhibition" and "inhibits," do not necessarily imply complete inhibition of a specified event, activity, or function. Rather, inhibition can be of a degree and / or duration sufficient to produce the desired effect. Inhibition can be prevention, delay, reduction, or otherwise obstruction of an event, activity, or function. Such inhibition can be of a magnitude and / or a temporal nature. In the specific context, the terms "inhibition," "reduction," and "prevention," and their variations, are used interchangeably. The inhibition of β-amyloid plaque formation, deposition, or accumulation by the oligonucleotides of the present invention can be direct or indirect, and can be of a magnitude and / or a temporal nature.

[0057] As used herein, the terms “promoting” and “inducing” and their variations, such as “promotion” and “inducement”, do not necessarily imply complete promotion or induction of a specified event, activity, or function. Rather, promotion or induction can be of a degree and / or time sufficient to produce the desired effect. The promotion or induction of β-amyloid plaque clearance by the oligonucleotides of the present invention can be direct or indirect, and can be of an amplitude and / or temporal nature.

[0058] As used herein, the term "effective amount" includes, within its meaning, a non-toxic but sufficient quantity or dose of a drug or compound to provide the desired effect. The exact amount or dose required will vary depending on the subject and factors such as the type of substance being treated, the subject's age and general condition, the severity of the condition being treated, the specific drug being administered, and the method of administration. Therefore, it is impossible to specify an exact "effective amount." However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experimental methods.

[0059] As used herein, the terms “treating,” “treatment,” “preventing,” and “prevention” refer to any and all uses of remedying a condition or symptom in any way, preventing the development of a condition or disease, or otherwise preventing, hindering, delaying, or reversing the progression of a condition or disease or other undesirable symptom. Therefore, the terms “treating” and “preventing,” etc., should be considered in their broadest context. For example, treatment does not necessarily mean that the patient is treated until fully recovered. In conditions that present or are characterized by multiple symptoms, treatment or prevention does not necessarily have to remedy, prevent, hinder, delay, or reverse all of the stated symptoms, but may prevent, hinder, delay, or reverse one or more of the stated symptoms. In the case of some disorders, the method of the present invention involves “treating” the disorder in terms of reducing or improving the occurrence of highly undesirable events associated with the disorder or the irreversible consequences of the disorder’s progression, but may not itself prevent the initial occurrence of the event or outcome. Therefore, treatment includes improving the symptoms of a particular disorder or preventing or otherwise reducing the risk of developing a particular disorder. Indications for successful “treatment” include any objective or subjective parameter, such as relief; remission; improved memory or slower memory decline; a condition that is more tolerable to the patient; a slower rate of degeneration or decline or disease progression; making the final point of deterioration less debilitating; or improvement in the subject’s physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of physical examination, neurological examination, and / or psychiatric evaluation. In some instances of this disclosure, when the treated disease is dementia, treatment may result in an improvement or reduction in cognitive decline (or the rate of cognitive decline) and / or improvement in cognitive function.

[0060] As used herein, references to diseases or conditions associated with loss of BBB integrity refer to diseases or conditions characterized by (i.e., evidence of) loss of BBB integrity. Loss of BBB integrity is typically assessed by measuring BBB permeability. This can be performed in animal models of the disease, using ex vivo analysis, and / or in vivo analysis in patients suspected of having or likely to develop diseases or conditions associated with loss of BBB integrity in a clinical setting. Loss of BBB integrity can contribute to the pathology, clinical outcomes, and / or symptoms of a disease and may include neuroinflammation, neurodegeneration (e.g., loss of neuronal function), plaque deposition, cognitive decline or impairment, muscle weakness, and / or decreased mobility. Diseases or conditions associated with loss of BBB integrity include neuroinflammatory diseases, neurodegenerative diseases, TBI, hypertension, and / or elevated pulse pressure.

[0061] The phrase “improvement, restoration, or maintenance of the integrity of the blood-brain barrier (BBB)” (or its grammatical variations) means improving, restoring, or maintaining the BBB’s function as a barrier at the blood-brain interface, for example, preventing macromolecules from entering the brain. In some instances, the BBB is improved by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more compared to a reference (e.g., an earlier time point or control BBB). In some instances, BBB integrity is restored to what was observed at an earlier time point or to a control or reference (e.g., a normal or healthy control or reference) (e.g., restored to at least 80%, 85%, 90%, or 95%). In other instances, the BBB is maintained, i.e., not lost or reduced. BBB integrity is typically assessed by measuring BBB permeability using methods known in the art (e.g., those described herein), where permeability is negatively correlated with BBB integrity.

[0062] As used herein, references to diseases or conditions associated with β-amyloid deposition refer to diseases or conditions characterized by β-amyloid deposition (i.e., evidence of it). Typically, β-amyloid is deposited in the brain (e.g., in the cerebrovascular system and / or parenchyma). β-amyloid deposition can be directly assessed using various imaging techniques or indirectly using biomarkers (e.g., blood or CSF β-amyloid levels). Diseases or conditions associated with β-amyloid deposition include AD, CAA, LBD, and TBI.

[0063] As used in this article, "Alzheimer's disease" or AD refers to a disease characterized by progressive cognitive impairment. The symptoms of Alzheimer's disease typically worsen over time as the disease progresses, generally progressing through three stages: "mild" (early form of Alzheimer's), "moderate" (intermediate form), and "severe" (late form). In mild Alzheimer's disease, symptoms may include, for example, memory loss, misplacing or losing items, difficulty remembering names or recalling words, increased difficulty planning or organizing, longer time spent completing normal daily tasks, and recurring problems. In moderate Alzheimer's disease (which is often the longest stage of the disease for many patients), damage occurs in areas of the brain that control language, reasoning, sensory processing, and conscious thought. At this stage, symptoms may include, for example, forgetting events or a person's personal history, having difficulty recognizing family and friends, inability to learn new information, difficulty performing multi-step tasks, impulsive behavior, altered sleep patterns, hallucinations, delusions, and paranoia. In severe Alzheimer's disease, memory and cognitive skills continue to deteriorate, patients often lose the ability to respond to their environment, engage in conversation and / or control their movements, and require a high level of assistance with daily activities and personal care.

[0064] In some implementations, the patient has "late-onset" Alzheimer's disease, which refers to a form of Alzheimer's disease in which the patient develops clinical symptoms of the disease after approximately age 65. In some implementations, the patient has "early-onset" Alzheimer's disease, which refers to a form of Alzheimer's disease in which the patient develops clinical symptoms of the disease before age 65. In some implementations, patients with early-onset Alzheimer's disease develop clinical symptoms in their 30s, 40s, or 50s. In some implementations, the patient has familial Alzheimer's disease (FAD), a genetic form of Alzheimer's disease caused by an autosomal dominant mutation affecting APP processing. The mention of Alzheimer's disease also includes early Alzheimer's disease.

[0065] Cerebral amyloid angiopathy, or CAA, is a type of cerebrovascular disease characterized by the accumulation of β-amyloid protein in the pia mater and small to medium-sized cerebral blood vessels. β-amyloid deposition is thought to lead to vascular weakening, which can result in intracerebral hemorrhage (ICH). Subjects with CAA may also present with cognitive impairment, sporadic microbleeds, hemosiderin deposition, inflammatory leukoencephalopathy, Alzheimer's disease, or transient neurological symptoms. Although a definitive diagnosis of CAA can only be made through post-mortem brain examination, a "probable" diagnosis of CAA is made during the subject's lifetime through imaging or tissue sampling. For the purposes of this disclosure, a diagnosis of "probable" CAA is considered a diagnosis of CAA. Evidence for CAA includes lobar, cortico-subcortical, or cortical hemorrhage; pathological evidence of CAA; multiple hemorrhages confined to cortical, lobular, or cortico-subcortical regions; and / or single lobar, cortical, or cortico-subcortical hemorrhages and focal or disseminated superficial hemorrhages.

[0066] Traumatic brain injury, or TBI, refers to brain injury caused by external force. TBI can be classified based on severity (e.g., mild, moderate, or severe), mechanism (closed or penetrating head injury), or other characteristics (e.g., occurring in a specific location (e.g., focal) or over a wide area (e.g., diffuse)). TBI can lead to temporary or permanent impairment of cognitive, physical, and psychosocial functioning. TBI is not a single pathophysiological event but a complex disease process with structural damage and functional deficits caused by both primary and secondary injury mechanisms. Primary injury is the result of immediate mechanical destruction of brain tissue upon exposure to external force and includes contusions, vascular injury (hemorrhagic disease), and axonal shearing, in which the axons of neurons are stretched and torn. Secondary injury develops gradually from minutes to months to years after the primary injury, caused by a cascade of various metabolic, cellular, and molecular events (including amyloid plaque formation) that ultimately lead to brain cell death, tissue damage, and atrophy. TBI is considered a major risk factor for dementia (including Alzheimer's disease).

[0067] As used herein, “APP” refers to “amyloid precursor protein”. The protein encoded by the APP gene is a type I membrane protein having E1 and E2 domains. Cleavage of the APP protein produces a β-amyloid (Aβ) fragment and the APP intracellular domain (AICD). Human APP gene and protein sequences (including splice and isotype variants) are well known and described, for example, in NCB GenBank accessions AH005295.2 and NM_000484.3. In some embodiments, the APP gene or protein is a variant (e.g., a polymorphic variant, splice variant, or truncated protein) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a naturally occurring APP gene or protein (e.g., any described above) or a fragment thereof.

[0068] The terms “β-amyloid protein” or “β-amyloid peptide” or Aβ are used interchangeably and refer to peptides of approximately 27 to 49 amino acids produced by enzymatic processing of APP. The β-amyloid population is heterogeneous, although the 40-amino acid peptide (Aβ40) is the most abundant (approximately 80-90%), followed by the 42-amino acid peptide (Aβ42, approximately 5-10%). The longer forms of Aβ, and especially Aβ42, are more hydrophobic and fibrillation-forming, and are the predominant class of deposits in the brain. In contrast, Aβ40 is more common in cerebral amyloid angiopathy (CAA), although Aβ42 is also frequently present. “β-amyloid plaques” refer to insoluble aggregates of β-amyloid protein, and particularly aggregates of β-amyloid fibrils. β-amyloid protein and β-amyloid plaques in the brain can be detected and measured by any known method, including staining tissue with anti-Aβ antibodies and brain imaging techniques such as positron emission tomography (PET). In other cases, brain β-amyloid protein is detected indirectly by detecting markers of brain β-amyloid protein. Non-limiting examples include detecting β-amyloid protein (and particularly Aβ40 and / or Aβ42) in cerebrospinal fluid (CSF) or plasma samples (see, for example, Li et al., Neurology. 2022 Feb 15; 98(7): e688-e699).

[0069] As used herein, the term "subject" refers to a mammal and includes humans, primates, livestock (e.g., sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats), and captive wild animals (e.g., foxes, kangaroos, deer). Preferably, the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human. "Subject" and "patient" are used interchangeably herein.

[0070] It will be understood that the terms and related definitions above are for illustrative purposes only and are not intended to be restrictive.

[0071] Table 1. Sequences A single underscore indicates locked nucleic acid (LNA) monomers; a double underscore indicates 2'-O-methylRNA monomers; bold indicates phosphate thioesters; and italics indicates UNA monomers.

[0072] Oligonucleotides Oligonucleotides suitable for the uses and methods described herein are provided. Such oligonucleotides include those that inhibit the interaction between the 3' untranslated region (3'UTR) of a VE-cadherin (CDH5) mRNA molecule containing the sequence shown in SEQ ID NO:1 and a repressive miRNA. This oligonucleotide binds to the 3'UTR to inhibit the interaction between the miRNA containing the seed sequence UCACAG or UCACAGU and the VE-cadherin 3'UTR. In a specific instance, the miRNA is miR-27a. The nucleotide sequence of mature human miR-27a (hsa-miR-27a) is provided in SEQ ID NO:13. Variants of this miRNA are also considered herein. Variants include nucleotide sequences substantially similar to the sequence of miR-27a. For example, variant miRNAs may contain sequences showing at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:13. Compared to the expression levels observed in the absence of oligonucleotides, the oligonucleotides function to increase VE-cadherin expression by inhibiting the interaction between miR27a and VE-cadherin. In some instances, VE-cadherin expression is increased by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more (see, for example, International Patent Publication No. WO2014053014 and the examples below).

[0073] Oligonucleotides typically comprise a sequence complementary to a sequence of at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 22, at least about 25, at least about 30, and at least about 35 consecutive bases selected from the sequence shown in SEQ ID NO:2 or containing 1, 2, or 3 substitutions.

[0074] In one embodiment, the oligonucleotide may comprise a continuous sequence complementary to a sequence selected from the sequence shown in SEQ ID NO:2 or a SEQ ID NO:2 sequence containing 1, 2, or 3 substitutions, comprising no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, and 35 consecutive bases.

[0075] In another embodiment, the oligonucleotide may comprise a continuous sequence complementary to a sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, and 35 consecutive bases selected from the sequence shown in SEQ ID NO:2 or the sequence containing 1, 2, or 3 substitutions.

[0076] Typically, oligonucleotides bind to positions 22-27 of SEQ ID NO:2. This region represents the complementary sequence to the miR-27a seed sequence and is the target site ('anti-seed' region) for miR-27a binding to the 3'UTR of VE-cadherin mRNA. Base pairings between the oligonucleotide and SEQ ID NO:2 may include positions 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 9-32, 9-31, 9-30, 9-29, 9-28, 9-27, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 11-32, 11-31, 11-30, 11-29, 11-28, and 11-27. 12-27, 13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27, 20-27, 21-27, 9-28, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28, 17-28, 18-28, 19-28, 20-28 or 21-28.

[0077] In one embodiment, base pairing between the oligonucleotide and the sequence SEQ ID NO:2 terminates at position 27 of SEQ ID NO:2. In other embodiments, base pairing may terminate at position 28, 29, 30, 31, 32, or 33 of SEQ ID NO:2. In another embodiment, base pairing between the oligonucleotide and the sequence SEQ ID NO:2 begins at position 22 of SEQ ID NO:2. In other embodiments, base pairing may begin at position 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 of SEQ ID NO:2.

[0078] Those skilled in the art will understand that oligonucleotides can be of any suitable length, depending on their exact function or purpose. In some instances, oligonucleotides are no longer than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases. Typically, oligonucleotides are between 8 and 25 bases in length. Even more commonly, oligonucleotides are between 10 and 20 bases in length. In some instances, the oligonucleotide is at least 8 bases long and no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases; at least 9 bases long and no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases; at least 10 bases long and no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases; at least 11 bases long and no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases; or at least 12 bases long and no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

[0079] To achieve strong binding to its target RNA, the length of the oligonucleotide can be increased. In some cases, using shorter oligonucleotides can improve delivery into the cell. Furthermore, in other cases, the position of the oligonucleotide relative to the antiseed sequence of the target RNA can be adjusted. For example, the positions of bases complementary to positions 22-27 of the target RNA in SEQ ID NO:2 can be adjusted such that they are positioned, for example, at the 5' end, the 3' end, or the middle or near the middle of the oligonucleotide. Typically, the positions of bases complementary to positions 22-27 are placed within the oligonucleotide such that they originate at position 1, position 2, position 3, position 4, position 5, or position 6, or upstream of position 2, position 3, position 4, position 5, or position 6, or downstream of position 1, position 2, position 3, position 4, position 5, or position 6, wherein the positions are counted starting from the 5' end of the oligonucleotide.

[0080] In some implementations, the target RNA sequence, such as the sequence of SEQ ID NO:2, may contain one, two, or three substitutions. Alternatively, the sequence may not contain substitutions. When substitutions are present, they may be located in complementary regions between the oligonucleotide and the target RNA. Substitutions may be single nucleotide polymorphisms (SNPs), which can enhance or degrade miRNA regulation of a given target RNA. SNPs can create novel miRNA target sites, thereby causing aberrant miRNA regulation of a given target RNA. RNA editing can also induce substitutions.

[0081] Oligonucleotides may be able to activate RNase H. RNase H cleaves the RNA portion of the RNA-DNA double helix, and the structural requirements for RNase H activation are well known to those skilled in the art. Similarly, the oligonucleotides of the present invention may be able to recruit cellular RNAi mechanisms and direct these mechanisms to target RNA. This can lead to cleavage of the target RNA or repression of its translation.

[0082] In certain embodiments of the invention, the oligonucleotides cannot recruit either the RNAi mechanism or RNase H. Therefore, the oligonucleotides of the present invention typically block the activity of the RNAi mechanism at a specific target RNA. The oligonucleotides do this by isolating the target sequence (miRNA binding site) of the target RNA, causing the RNAi mechanism to not recognize the target sequence. Oligonucleotides of the present invention with this activity may also be called blockmirs because they block the regulatory activity of a given miRNA at a specific miRNA binding site on the target RNA. To achieve the ability of the oligonucleotides of the present invention to prevent the recruitment or activation of RNase H, the oligonucleotides typically do not contain five or more consecutive DNA nucleobases.

[0083] Oligonucleotides may contain a variety of sequence and structural modifications depending on their intended use and function, as will be further described below. Those skilled in the art will understand that the sequence and structural modifications described herein are merely exemplary, and the scope of the invention should not be limited by reference to those modifications, but rather other modifications known to those skilled in the art may be employed, provided that the oligonucleotide retains the desired function or activity.

[0084] By way of example only, oligonucleotide sequences can be modified by adding one or more thiophosphates (e.g., monothiophosphate or dithiophosphate) between residues in the sequence, or by including one or more morpholine rings in the backbone. Alternative non-phosphate linkages between residues include phosphonates, hydroxylamines, hydroxyhydrazines, amides and carbamates, methylphosphonates, thiophosphates, phosphoramides, or boron derivatives. The nucleotide residues present in the oligonucleotide can be naturally occurring nucleotides or modified nucleotides. Suitable modified nucleotides include 2'-O-methylnucleotides, 2'-O-fluoronucleotides, 2'-O-methoxyethylnucleotides, universal nucleotide bases such as 5-nitroindole; LNA, UNA, PNA, and INA nucleotide bases, 2'-deoxy-2'-fluoroarabinose (FANA), and arabinose (ANA). The naturally occurring ribose portion of the ribonucleotide can be replaced, for example, with a hexose, a polycyclic heteroalkyl ring, or a cyclohexenyl group. Alternatively, or additionally, the oligonucleotide sequence may be conjugated to one or more suitable chemical moieties at one or both ends. For example, an oligonucleotide may be conjugated to cholesterol via a suitable linker, such as hydroxyproline at the 3' end. As a further example, an oligonucleotide may be conjugated to N-acetylgalactosamine (GalNAc).

[0085] Specific modifications of interest include those that increase the affinity of the oligonucleotide for its complementary sequence, i.e., those that increase the melting temperature of the oligonucleotide pairing with the complementary sequence bases, or those that increase the biostability of the oligonucleotide. Such modifications include 2'-O-fluorine, 2'-O-methyl, and 2'-O-methoxyethyl groups. The use of LNA, UNA, PNA, and INA monomers is also commonly employed. For shorter oligonucleotides, a higher percentage of affinity-increasing modifications is typically present. If the oligonucleotide is less than 12 or 10 nucleotides in length, it can be composed entirely of affinity-increasing units, such as LNA monomers, UNA monomers, or 2'-O-methyl RNA nucleotides.

[0086] In a particular embodiment, the fraction of monomers modified at the bases or sugars in the oligonucleotide relative to monomers not modified at the bases or sugars may be less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, greater than 99%, greater than 95%, greater than 90%, greater than 85%, greater than 75%, greater than 70%, greater than 65%, greater than 60%, greater than 50%, greater than 45%, greater than 40%, greater than 35%, greater than 30%, greater than 25%, greater than 20%, greater than 15%, greater than 10%, and greater than 5% or greater than 1%.

[0087] Lipids and / or peptides can also be conjugated to oligonucleotides. Such conjugation can improve bioavailability and prevent oligonucleotides from activating RNase H and / or recruiting RNAi mechanisms. Conjugation of larger volume portions typically occurs at the central portion of the oligonucleotide, for example, anywhere among the five central monomers. Alternatively, at one of the bases complementary to positions 1-6 of SEQ ID NO:1 or positions 22-27 of SEQ ID NO:2. In yet another embodiment, the portion can be conjugated at the 5' or 3' end of the oligonucleotide. An exemplary hydrophobic portion is a cholesterol portion that can be conjugated to the oligonucleotide to prevent the oligonucleotide from recruiting RNAi mechanisms and improve the bioavailability of the oligonucleotide. For example, the cholesterol portion can be conjugated to one or more nucleobases complementary to positions 22-27 of the SEQ ID NO:2 sequence, conjugated at the 3' end of the oligonucleotide, or conjugated at the 5' end of the oligonucleotide.

[0088] Different modifications can be placed at different positions within the oligonucleotide to prevent the oligonucleotide from activating RNase H and / or recruiting RNAi mechanisms.

[0089] In one specific embodiment, phosphate thioester nucleotide linkages can link monomers in oligonucleotides to improve the biostability of the oligonucleotides. All linkages of the oligonucleotides can be phosphate thioester linkages. In another embodiment, the fraction of phosphate thioester linkages can be less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 65%, greater than 60%, and greater than 50%. In some instances, all nucleobases except the 3' nucleobase are linked via phosphate thioester linkages.

[0090] In one embodiment, the oligonucleotide may not contain any RNA nucleobases. This helps prevent the oligonucleotide from recruiting RNAi mechanisms, increasing the biological stability of the oligonucleotide. For example, the oligonucleotide may consist of LNA and DNA nucleobases, and these may be linked by phosphate thioester linkages as outlined above. In an alternative embodiment, the oligonucleotide does not contain any DNA nucleobases. In an alternative embodiment, the oligonucleotide does not contain any morpholino and / or LNA nucleobases.

[0091] In one embodiment, the oligonucleotide may comprise a mixture of DNA and RNA nucleobases to prevent the oligonucleotide from activating RNase H and recruiting RNAi mechanisms. For example, the DNA and RNA nucleobases may alternate along the length of the oligonucleotide, or alternatively, one or more DNA nucleobases may be positioned adjacent to each other, and one or more RNA nucleobases may be positioned adjacent to each other.

[0092] In another specific embodiment, the oligonucleotide comprises a mixture of LNA monomers and 2'-O-methyl RNA nucleotides. As described above, the LNA and 2'-O-methyl RNA nucleotides may alternate along the length of the oligonucleotide, or alternatively, one or more LNA nucleotides may be positioned adjacent to each other, and one or more 2'-O-methyl RNA nucleotides may be positioned adjacent to each other.

[0093] In some embodiments, the number of nucleotides in the oligonucleotide that increase the affinity of the oligonucleotide for the complementary sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides. In some embodiments, the number of nucleotides in the oligonucleotide that increase the affinity of the oligonucleotide for the complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 nucleotides.

[0094] In a particular embodiment, the nucleobases that increase the affinity of the oligonucleotide for the complementary sequence may be located on the flanks of the oligonucleotide, i.e., at or near either of the 5' and 3' ends, or at or near the center of the oligonucleotide. The nucleobases that increase the affinity of the oligonucleotide for the complementary sequence may also be evenly distributed along the length of the oligonucleotide.

[0095] Table 2 lists exemplary oligonucleotide sequences. Therefore, this document provides oligonucleotides comprising the sequence shown in SEQ ID NO:3, 4, 5, or 6, less than 50 nucleotides in length, and optionally further comprising at least one modified nucleotide (e.g., an LNA nucleotide and / or a 2'-O-methyl nucleotide). Such oligonucleotides inhibit the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. In another example, the oligonucleotide comprises the sequence shown in SEQ ID NO:7, less than 50 nucleotides in length (e.g., less than 40, 35, 30, 35, or 30 nucleotides), and inhibits the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. In another example, the oligonucleotide comprises the sequence shown in SEQ ID NO:8, is less than 50 nucleotides in length (e.g., less than 40, 35, 30, 35, or 30 nucleotides), and inhibits the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. In another example, the oligonucleotide comprises the sequence shown in SEQ ID NO:9, is less than 50 nucleotides in length (e.g., less than 40, 35, 30, 35, or 30 nucleotides), and inhibits the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. In another example, the oligonucleotide comprises the sequence shown in SEQ ID NO:10, is less than 50 nucleotides in length (e.g., less than 40, 35, 30, 35, or 30 nucleotides), and inhibits the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. In another example, the oligonucleotide comprises the sequence shown in SEQ ID NO:11, is less than 50 nucleotides in length (e.g., less than 40, 35, 30, 35, or 30 nucleotides), and inhibits the binding of miR-27a to RNA molecules comprising SEQ ID NO:1, 2, 14, or 15. The aforementioned oligonucleotide can increase the expression and / or activity of VE-cadherin in cells and reduce vascular leakage or permeability. Therefore, in methods involving contacting cells with the aforementioned oligonucleotide (e.g., in methods involving administering the oligonucleotide to a subject), the expression and / or activity of VE-cadherin increases and / or vascular leakage or permeability decreases (e.g., compared to when cells are not contacted with the oligonucleotide or when the subject has not been administered the oligonucleotide).By virtue of its ability to increase VE-cadherin levels and reduce vascular leakage or permeability, the aforementioned oligonucleotides are suitable for use in methods of treating related diseases and conditions, including but not limited to those described herein and those described in WO2014053014 (e.g., edema, cardiovascular disease, myocardial infarction, peripheral vascular disease, ischemia, stroke, cancer, atherosclerosis, psoriasis, diabetes, autoimmune diseases such as rheumatoid arthritis, thrombocytopenia, altitude sickness, barotrauma, iatrogenic disorders, bacterial infections, viral infections, and ocular conditions associated with vascular leakage such as nonproliferative and proliferative retinopathy, macular edema, glaucoma, and macular degeneration).

[0096] Table 2. Oligonucleotide sequences A single underscore indicates an LNA monomer; a double underscore indicates a 2'-O-methylRNA monomer; and bold indicates an UNA monomer.

[0097] Composition Oligonucleotides can be formulated as pharmaceutical compositions that may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. Such compositions can be administered via any convenient or suitable route, such as parenteral (e.g., subcutaneous, intra-arterial, intravenous, intramuscular), oral (including sublingual), nasal, or local routes. In cases where direct delivery of an appropriate concentration of oligonucleotides to the site of treatment within the body is required, administration may be regional rather than systemic. Regional administration provides the ability to deliver very high local concentrations of oligonucleotides to the desired site and is therefore suitable for achieving the desired therapeutic or preventative effect while avoiding exposure of other organs of the body to the compound, and potentially reducing side effects.

[0098] The oligonucleotides of the present invention can be packaged and delivered in suitable delivery media that can be used to target or deliver oligonucleotides or to further protect or stabilize them. As examples, the delivery media may comprise liposomes or other similar compositions such as micelles (e.g., polymeric micelles), lipoprotein-based drug carriers, microparticles, nanoparticles, liposome nanoparticles (LNPs), lipid nanoparticles, or dendritic macromolecules.

[0099] Liposomes can be derived from phospholipids or other lipid substances and are formed from monolayers or multilayers of hydrated liquid crystals dispersed in an aqueous medium. Specific examples of liposomes used for administering or delivering compositions to target cells are DODMA, synthetic cholesterol, DSPC, PEG-cDMA, DLinDMA, or any other non-toxic, physiologically acceptable, and metabolizable lipids capable of forming liposomes. Compositions in liposome form may contain stabilizers, preservatives, and / or excipients. Methods for preparing liposomes are well known in the art, see, for example, Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), p. 33 ff., the contents of which are incorporated herein by reference. Biodegradable microparticles or nanoparticles formed from, for example, polylactide (PLA), polylactide-co-glycolic acid (PLGA), and ε-caprolactone can be used.

[0100] Other means of packaging and / or delivering oligonucleotides and optionally one or more other pharmaceutical agents to facilitate delivery to the brain will also be known to those skilled in the art. By way of example only, delivery platforms may include RNA-lipid complex technologies comprising cationic lipids, fusion-promoting or stabilizing colipids, and polyethylene glycol-modified lipids.

[0101] Examples of pharmaceutically acceptable carriers or diluents are softened or distilled water; saline solutions; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, arachis oil, or coconut oil; silicone oils, including polysiloxanes such as methyl polysiloxane, phenyl polysiloxane, and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropyl methylcellulose; lower alkanols such as ethanol or isopropanol; lower aryl alcohols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol, or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum arabic or gum arabic; and petrolatum. Typically, one or more carriers will account for 10% to 99.9% by weight of the composition.

[0102] Suitable drug forms for injection include sterile aqueous solutions (in the case of water solubility) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Formulations must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, the inclusion of isotonic agents, such as sugars or sodium chloride, will be preferred. Prolonged absorption of injectable compositions can be achieved by using agents with delayed absorption in the composition, such as aluminum monostearate and gelatin.

[0103] Sterile injectable solutions are prepared by incorporating the desired amount of the active compound, along with various other ingredients listed above, into a suitable solvent, followed by filtration and sterilization. Dispersions are typically prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0104] When the active agents are properly protected, they can be administered orally, for example with an inert diluent or with an absorbable edible carrier, or they can be encapsulated in hard or soft-shell gelatin capsules, or they can be compressed into tablets, or they can be directly incorporated into food. For oral therapeutic administration, the active compound can be blended with excipients and used in the form of ingestible tablets, buccal lozenges, tablets, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and formulations should contain at least 1% by weight of the active compound. The percentage of the composition and formulation can, of course, vary and can conveniently be between about 5% and about 80% per unit weight. The amount of active compound in such therapeutically useful compositions ensures that a suitable dosage will be obtained. Preferred compositions or formulations according to the invention are prepared such that an oral unit dosage form contains between about 0.1 µg and 2000 mg of the active substance.

[0105] Tablets, lozenges, pills, capsules, etc., may also contain the following components: binders such as gum arabic, arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin may be added, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the materials of the types mentioned above. Various other materials may be present as coatings or otherwise alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavoring. Of course, any materials used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic at the amounts used. In addition, oligonucleotides may be incorporated into sustained-release formulations and preparations.

[0106] Treatment This document provides methods for improving, increasing, restoring, or maintaining BBB integrity in subjects (e.g., subjects with reduced or impaired BBB integrity and / or subjects suffering from diseases or conditions associated with BBB damage or loss of BBB integrity); methods for reducing or inhibiting the deposition or accumulation of β-amyloid protein in the brain of subjects with this need; methods for increasing or promoting the clearance of β-amyloid protein in the brain of subjects with this need; and / or methods for inhibiting or reducing cellular (e.g., brain cells) senescence in subjects with this need. The method includes the step of administering an effective amount of the oligonucleotide described herein to the subject with this need.

[0107] Typically, the subject has been diagnosed with a disease or condition associated with BBB damage or loss of BBB integrity, or has been identified as likely to develop such a disease or condition. Diseases and conditions associated with BBB damage or loss of BBB integrity include neuroinflammatory and / or neurodegenerative diseases, including but not limited to dementia, multiple sclerosis (MS), motor neuron diseases (MND) (e.g., amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), and pseudobulbar palsy), Parkinson's disease, and Huntington's disease. Dementia may be associated with or characterized by plaque deposition, or may have little or no association with or evidence of β-amyloid deposition. Non-limiting examples of dementia include Alzheimer's disease, Lewy body dementia (LBD), Parkinson's disease dementia (PDD), vascular dementia, and frontotemporal dementia (FTD). Other conditions associated with loss of BBB integrity include traumatic brain injury (TBI), hypertension (including chronic hypertension), and elevated pulse pressure (e.g., at least 40 mm Hg, 45 mm Hg, 50 mm Hg, 55 mm Hg, 60 mm Hg, or 65 mm Hg), which are also associated with dementia. Therefore, in some instances, subjects had TBI and dementia, or hypertension and dementia, or elevated blood pressure and dementia.

[0108] Subjects with or at risk of developing neuroinflammatory and / or neurodegenerative diseases include those with one or more biomarkers indicating the severity of one or more diseases or those indicating susceptibility to developing neuroinflammatory and / or neurodegenerative diseases. Such biomarkers are known in the art and may be used by clinicians optionally in conjunction with other clinical signs of disease (e.g., mobility, cognition, etc.).

[0109] For example, when the disease is dementia (including Alzheimer's disease, FTD, LBD, PDD, and vascular dementia), biomarkers can be selected from mutations in genes encoding amyloid precursor protein (APP) and presenilin 1 and 2; mutations in the ε4, 2, and 3 alleles (APOE-ε4, APOE-ε2, APOE-ε3) of the apolipoprotein E (APOE) gene; and mutations in one or more of the trigger receptor 2 (TREM2), MAPT, GRN (also known as PGRN), TARDBP, VCP, and CHMP2B genes expressed on myeloid cells. Elevated serum and / or CSF levels of α-synuclein, S100A9 and S100B, chromogranin, circulating DNA, heat shock proteins, and amyloid protein can also be measured. In some cases, high blood pressure (or hypertension) is a risk factor for developing dementia, particularly vascular dementia.

[0110] In another example, the biomarkers for MND may be selected from one or more of, for example, SOD1, TDP-43, FUS, C9ORF72, ALS2, ALS4, ALS8, NEK1, UBQLN2, VCP, SETX, ANG, PFN1, MATR3, CHCHHD10, TUBA4A, TBK1, C21orf2, and OPTN, or their expression products. In some MND embodiments, the presence of cytoplasmic deposition of TDP-43-positive inclusion bodies and / or elevated serum and / or CSF levels of neurofilaments may also be measured.

[0111] In other embodiments, subjects at risk of neuroinflammatory or neurodegenerative diseases may also be identified by measuring elevated levels of one or more disease-associated pro-inflammatory cytokines such as TNF, IL-1-α, IL-6, IFN-β, IL-1β, IL-8, IL-18, C-reactive protein (CRP), IL-17, chemokines, CD14+ hypermonocytes, and peripheral blood mononuclear cells (PBMCs) mRNA transcripts. In one embodiment, the disease is MND, and the cytokines are selected from one or more of IL-6 or IL-17. In one embodiment, the disease is dementia, and the cytokines are selected from IL-1, IL-6, and TNF-α.

[0112] In some instances, the subject has a disease associated with β-amyloid deposition, such as AD, CAA, LBD, or TBI, or has been identified as potentially developing AD, CAA, or LBD. Therefore, methods are also provided for treating subjects with AD, CAA, LBD, or TBI, or for treating subjects potentially developing AD, CAA, or LBD, wherein the method includes the step of administering an effective amount of the oligonucleotide described herein to the subject. In some instances, TBI is mild, moderate, or severe TBI, or moderate-to-severe TBI.

[0113] In some instances, the subject has been diagnosed with Alzheimer's disease (AD), including early-onset AD, late-onset AD, familial AD, early-onset AD, and / or late-onset AD. In other instances, the subject has been determined (e.g., by a clinician) to be likely to develop AD, for example, having a moderate or high probability of developing AD, such as a 20%, 30%, 40%, 50%, or higher probability. In other instances, the subject has been diagnosed with atrial fibrillation (CAA) (including possible CAA), or has been determined to be likely to develop CAA, for example, having a moderate or high probability of developing CAA, such as a 20%, 30%, 40%, 50%, or higher probability.

[0114] According to the present invention, any suitable amount or dose of the oligonucleotide of the invention can be administered to a subject in need. The therapeutically effective amount for any specific subject may depend on a variety of factors, including: the disease being treated and its severity; the activity of the oligonucleotide used; the composition used; the subject's age, weight, general health, sex, and diet; the time of administration; the route of administration; the rate of molecular or pharmaceutical isolation; the duration of treatment; drugs used in combination with or concurrently with the treatment; and other relevant factors known in medicine. Those skilled in the art will be able to determine the effective, non-toxic amount of the protein conjugate used through routine experiments.

[0115] Oligonucleotides may be administered alone or in combination with other therapies. Therapies may include other active agents, devices, physical therapy, cognitive therapy, occupational therapy, or any other therapy that maintains or improves BBB integrity and / or treats diseases or conditions, including neuroinflammatory diseases, neurodegenerative diseases, hypertension, and / or elevated pulse pressure.

[0116] In one instance, additional therapies include the use of active agents and / or devices for lowering blood pressure and / or pulse pressure, including lowering blood pressure and / or pulse pressure in cerebral blood vessels. Non-limiting examples of such agents include angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), and calcium channel blockers. Non-limiting examples of such devices include those described in International Patent Publication No. WO2021119737. These devices are placed around the outer wall of a blood vessel (e.g., the carotid artery, such as the common carotid artery or internal carotid artery) to alter the geometry of the vessel in order to, for example, change the transmission dynamics of arterial blood pressure to the cerebral microvascular system and absorb excess energy from arterial pulsations. In some instances, the application of active agents and / or devices for lowering blood pressure and / or pulse pressure results in a reduction of blood pressure and / or pulse pressure to normal levels or ranges, such as systolic blood pressure less than 120, 125, 130, 135 or 140 mm Hg, diastolic blood pressure less than 80, 85 or 90 mm Hg, and / or pulse pressure less than 40, 45, 50, 55 or 60 mm Hg.

[0117] In other examples, additional therapies include active agents used to treat dementia, and include, for example, cholinesterase inhibitors such as donepezil, rivastigmine or galantamine, memantine, and lecanemab. In further examples, active agents are used to treat MND and include, for example, riluzole, agents that block the interaction between CD40 and CD40 ligands, including antibodies that specifically bind to CD40 and / or CD40 ligands, and anti-inflammatory agents. In further examples, active agents are used to treat Parkinson's disease and include levodopa, carbidopa-levodopa, dopamine agonists, monoamine oxidase B (MAO B) inhibitors, catechol O-methyltransferase (COMT) inhibitors, amantadine, and adenosine receptor antagonists. Other active agents that can be administered to treat MS include, for example, ofamumab, teriflunomide, dimethyl fumarate, diloxicame fumarate, monomethyl fumarate, fingolimod, sinimod, ozamod, and pencimod.

[0118] Those skilled in the art will recognize that dose escalation studies will be conducted when determining an appropriate and effective dose range for human administration based on the mouse studies illustrated herein. Therefore, those skilled in the art will understand that the doses and dose ranges described above are merely exemplary based on the doses administered in the mouse studies illustrated herein, and the actual doses or dose ranges to be used in humans may vary depending on the results of such dose escalation studies. Based on the data illustrated herein, an appropriate and effective dose or dose range to be administered to humans can be determined through routine optimization without undue burden or experimentation.

[0119] In some instances, subjects received 1, 2, 3, 4, 5, 6, 7, or more doses of oligonucleotide over a period of time, such as every 1, 2, 3, 4, 6, 7, 8, 9, 10, or more days, weeks, or months. In some instances, subjects received a maintenance dose over a period of time, such as every 1, 2, 3, 4, 6, 7, 8, 9, 10, or more days, weeks, or months.

[0120] Application of the oligonucleotides of the present invention (optionally in conjunction with further therapies) can improve, increase, restore or maintain the integrity of the BBB, resulting in, for example, an improvement of at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or more in BBB integrity compared to before application, and / or limiting any loss of BBB integrity to, for example, less than 30%, 25%, 20%, 15%, 10%, 5% or less over time. BBB integrity can be assessed by measuring BBB permeability using non-invasive imaging techniques such as magnetic resonance imaging, positron emission tomography and perfusion computed tomography (see, for example, Avsenik et al., Radiol Oncol. 2015 June; 49(2): 107–114 and Harris et al., Eur J Nucl MedMol Imaging. 2023; 50(4): 1051–1083).

[0121] In some instances, administration of oligonucleotides (and optional additional therapies) resulted in a reduction of at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% or more in the amount of β-amyloid deposits in the brain compared to pre-administration levels. In other instances, administration of oligonucleotides resulted in an inhibition or slowing of the rate of accumulation of β-amyloid deposits in the brain compared to pre-administration levels or compared to subjects with similar disease and β-amyloid deposit levels who had not received oligonucleotides. In such cases, the rate or amount of β-amyloid deposition may be reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% or more. The reduction in the amount or rate of β-amyloid deposition or accumulation in the brain may occur in the vascular system of the brain, such as in the pia mater vessels or other small to medium-sized vessels of the brain, and / or in the parenchyma of the brain. In addition, β-amyloid deposits may contain β-amyloid monomers and / or β-amyloid aggregates, such as β-amyloid oligomers, β-amyloid fibrils, and / or β-amyloid plaques.

[0122] The level of β-amyloid deposits in the brain can be assessed using any method known in the art. In some instances, β-amyloid deposits are directly visualized and measured using brain imaging techniques such as positron emission tomography. In other instances, biomarkers of brain β-amyloid deposits are used to indirectly measure the level of β-amyloid deposits in the brain. These include, but are not limited to, the detection of β-amyloid (and particularly Aβ40 and / or Aβ42) in cerebrospinal fluid (CSF) or plasma samples (see, for example, Li et al., Neurology. 2022 Feb 15; 98(7): e688–e699).

[0123] Administration of the oligonucleotides of this disclosure can lead to improvement or relief of one or more symptoms of a disease or condition associated with loss of BBB integrity and / or β-amyloid deposition. In specific instances, when the disease is dementia (including dementia caused by hypertension and / or pulse pressure), administration of the oligonucleotides of this disclosure results in improvement of cognitive function or a slowing of the rate of cognitive decline (e.g., improvement or slowing of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). Cognitive assessment tools are well known and include, for example, the 10-point cognitive screening (10-CS), the 6-item cognitive impairment test (6CIT), the 6-item screening scale, the memory impairment screening (MIS), the Mini-Cog, or the Test Your Memory (TYM).

[0124] Reagent test kit Embodiments of the present invention also provide kits for use according to the invention. For example, the kits of the present invention may contain one or more oligonucleotides disclosed herein, and optionally, disordered oligonucleotides as controls. Such kits can be used, for example, in medical or biological research activities, including studies of β-amyloid deposition and plaque formation, accumulation, or clearance. Kits according to the invention may also include other components required for the use of oligonucleotides, such as buffers and / or diluents. The kit typically includes containers for containing the various components and instructions for using the kit components in the methods of the present invention.

[0125] In order to make the present invention readily understood and practiced, specific preferred embodiments will now be described by way of the following non-limiting examples.

[0126] References to any prior publications (or information derived therefrom) or to any known matter in this specification are not, and should not be construed as, an acknowledgment or endorsement or, in any way, an implication that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge in the field of effort covered by this specification. Example

[0127] Example 1: General Method Oligonucleotides Blockmir was synthesized by RiboTask, Denmark. The sequences of these oligonucleotides are provided in Table 3. The oligonucleotides were formulated in PBS.

[0128] Table 3. Oligonucleotide sequences A single underscore indicates an LNA monomer; a double underscore indicates a 2'-O-methylRNA monomer; and bold indicates a phosphate thioester.

[0129] AD mouse model The AD transgenic mouse model used in this study was the APPswe / PS1dE9 mouse, one of the most widely used AD transgenic mouse models. These transgenic mice overexpress the human amyloid precursor protein gene (APP) with a Swedish mutation and the human presenilin-1 gene with exon 9 deletion, both of which are familial AD genes. β-amyloid deposition can be seen in these mice as early as 4-6 months of age, with significant β-amyloid deposition appearing by 9 months of age.

[0130] APP / PS1 transgenic mice and wild-type littermates were obtained from Jackson Labs USA and maintained by BioResources Pty Ltd (ABR), NSW, Australia.

[0131] Oligonucleotide delivery CD5-2 or control oligonucleotide, or PBS alone, were administered intravenously at 30 mg / kg to 4-month-old (pre-plaque formation) and 6- or 7-month-old (post-plaque formation) APP / PS1 mice and wild-type controls. Mice were monitored for any adverse reactions within 20 minutes of injection.

[0132] Tissue preparation and analysis Mice were euthanized prior to cardiac perfusion. In cases where biotin was used to detect and measure vascular leakage, mice were injected with 100 mg / kg body weight of unconjugated biotin one hour before euthanasia.

[0133] The total time from euthanasia to cardiac perfusion was 10 minutes per mouse. Mice were perfused through the left ventricle with 10 mL of cold 1X PBS followed by 10 mL of 1% PFA / 1X PBS. The cerebral cortex was collected and fixed by immersion in 4% PFA at 4°C for 5 to 6 hours. In some studies, the brain was sagittally bisected along the midline. One half was fixed in 4% PFA in cold 1X PBS for 4–5 hours, while the other half was fixed overnight in methanol at 4°C.

[0134] Tissue sections of 100 µm thickness were cut using a vibratory microtome (Leica).

[0135] For PFA samples, tissues were blocked overnight with 1% BSA in PBST (PBS containing 1% Triton-X). Sections were stained overnight with antibodies against blood vessels (isolectin-B4), endothelial cells (CD31), VE-cadherin, pericytes (PDGFRb, CD13), amyloid (D54D2), albumin, and biotin leakage. After primary antibody incubation, sections were thoroughly washed with tris-buffered saline (TBST) containing 0.3% Triton-X100 and incubated for 2 hours at room temperature with a 1:500 secondary antibody (Molecular Probes, Invitrogen) preconjugated with Alexa Fluor 555, Alexa Fluor 594, and / or Alexa Fluor 647. Sections were then washed three times with TBST and stained with DAPI for 10 minutes at room temperature. Finally, sections were washed once in PBS and mounted with Prolong Gold anti-fading mounting medium. Albumin detected at the blood vessels at the top and bottom of the Z-stack was excluded because these leaks were considered artifacts of the tissue sections.

[0136] The methanol-fixed brain was cut into 100 µm thick sagittal sections and stained with 1:200 rabbit anti-mouse tight junction protein-5 (Invitrogen #34-1600) and rat anti-mouse CD31 (BD #553370).

[0137] For quantification of stained sections, whole-brain sections were tiled and scanned using a Leica confocal microscope and Leica Application Suite (LAS) software at 20x magnification. The expression of VE-cadherin and tight junction protein-5 in the pia mater vascular system was analyzed using ImageJ software (version 2.14.0 / 1.54f). The pia mater region was delineated, and the expression of VE-cadherin or tight junction protein-5 in the blood vessels was thresholded and the average intensity was measured.

[0138] VE-cadherin expression analysis Human endothelial cells were cultured in a humidified 37°C, 5% CO2 incubator in medium 199 (Sigma-Aldrich, MO, USA) supplemented with 20% fetal bovine serum (FBS), 100 U / ml penicillin, 100 U / ml streptomycin, 15 μg / ml endothelial growth factor (BD Biosciences, MA, USA), and 15 μg / ml heparin (Sigma-Aldrich, MO, USA). Oligonucleotides diluted in 37.5 µl of OptiMEM (ThermoFisherScientific, MA, USA) were transfected into the cells using Hiperfect transfection reagent (Qiagen, Hilden, Germany) and incubated overnight, after which the medium was replaced. The effect of oligonucleotides on cell lysates was assessed by Western blotting analysis 48 hours post-transfection.

[0139] Example 2. Effects of CD5-2 on BBB integrity and β-amyloid deposition In the APPswe / PS1dE9 mouse model of Alzheimer's disease (AD), vascular leakage was used as a biomarker or indicator of BBB integrity. Vascular leakage was first assessed by detecting biotin leakage in the brains of 4-month-old APPswe / PS1dE9 mice injected with biotin prior to euthanasia. One week before euthanasia and brain tissue preparation, mice were administered control blockmir, CD5-2, or PBS alone. Figure 1 As shown, untreated APPswe / PS1dE9 mice exhibited vascular leakage, indicating impaired BBB and vascular system in these pre-plaque mice. In contrast, sections from APPswe / PS1dE9 mice receiving CD5-2 showed little or no biotin, indicating little or no vascular leakage in these mice. As expected, no vascular leakage was observed in wild-type mice (data not shown).

[0140] Albumin and VE-cadherin were also used as biomarkers to assess BBB and vascular integrity. One week prior to euthanasia and brain tissue section preparation, APPswe / PS1dE9 and wild-type mice (7 months old) were administered either control blockmir or CD5-2. Figure 2 As shown, large albumin leakage was observed in control-treated APPswe / PS1dE9 mice. In contrast, APPswe / PS1dE9 mice receiving CD5-2 showed reduced albumin leakage.

[0141] In further studies, 6-month-old APPswe / PS1dE9 and wild-type mice were administered either control blockmir or CD5-2 one week prior to euthanasia and brain tissue section preparation. The sections were then stained to detect signs of β-amyloid deposition. Figure 3 As expected, significant β-amyloid deposition was detected in the leptomeningeal vessels (LV) and brain parenchyma of control-treated APPswe / PS1dE9 mice. Surprisingly, however, CD5-2 treatment in APPswe / PS1dE9 mice significantly reduced β-amyloid deposition in the LV walls. When assessing albumin leakage and VE-cadherin expression in the brain tissue of these 6-month-old mice, reduced VE-cadherin expression and significant albumin leakage were observed in APPswe / PS1dE9 mice receiving control blockmir, while APPswe / PS1dE9 mice receiving CD5-2 showed reduced albumin leakage and restored VE-cadherin levels (similar to wild-type mice). Figure 4 ).

[0142] These data clearly demonstrate that APPswe / PS1dE9 mice already exhibit impaired BBB and vascular integrity as early as 4 months of age, which persists into 6-7 months of age, accompanied by LV and β-amyloid deposition in the brain parenchyma. Surprisingly, administration of CD5-2 was sufficient to restore or improve BBB and vascular integrity in the brain. Furthermore, CD5-2 administration reduced β-amyloid deposition. Therefore, CD5-2 not only restores BBB integrity in APPswe / PS1dE9 mice but also reduces β-amyloid deposition or accumulation and / or promotes β-amyloid clearance.

[0143] Example 2. Effects of CD5-2, CD5-4 and CD5-9 on VE-cadherin expression.

[0144] CD5-2 has previously been shown to increase VE-cadherin expression and reduce vascular leakage (see International Patent Publication No. WO2014053014). Two other related blockmirrors, CD5-4 and CD5-9, were also tested. Figure 5 C) Increased or enhanced ability of VE-cadherin, compared with CD5-2. CD5-4 and CD5-9 were engineered to substitute for and / or improve binding to the VE-cadherin 3' UTR (e.g., cross-species binding), and / or reduce or eliminate potential off-target binding (e.g., with ORC4 and / or FXR1). Figure 5As shown in A and B, both CD5-4 and CD5-9 exhibited comparable or even improved activity compared to CD5-2, significantly increasing VE-cadherin expression in endothelial cells. This suggests that these blockmirras can be used in the same manner as CD5-2, including improving BBB and vascular integrity; and reducing β-amyloid deposition or accumulation and / or promoting β-amyloid clearance. In fact, further studies confirmed that CD5-4 possessed the expected activity in vivo (i.e., as expected based on the activity of CD5-2) (data not shown).

[0145] Example 3. Further evaluation of the effects of CD5-2 on blood vessels.

[0146] Further studies were conducted to evaluate the effects of CD5-2 on the brain. As described above, APPswe / PS1dE9 mice (6-7 months old) were administered CD5-2 or the control Blockmir one week prior to euthanasia and brain tissue preparation. Various analyses were performed on sections to assess the effects of CD5-2 on VE-cadherin and tight junction protein-5 expression, albumin leakage, leptomeningeal vascular density, amyloid deposition, and senescence.

[0147] VE-cadherin and tight junction protein-5 As described above, mice were perfused with cold PBS followed by 1% PFA. The brain was sagittally bisected along the midline. One half was drop-fixed in cold 4% PFA for 4–5 hours, and then the PFA-fixed brain was cut into 100 µm thick sagittal sections and stained for VE-cadherin or albumin. The other half was drop-fixed in methanol at 4°C overnight, and the methanol-fixed brain was cut into 100 µm thick sagittal sections and stained for tight junction protein-5 and CD31. As described above, the expression of VE-cadherin or tight junction protein-5 in the pia mater vascular system was measured by confocal microscopy using a planar scanning method.

[0148] like Figure 6 As shown, CD5-2 not only increased VE-cadherin in EC junctions as expected, but also increased tight junction protein-5.

[0149] albumin leakage Brain sections were prepared as described above and stained with goat anti-mouse albumin and rat anti-mouse CD31. As mentioned above, whole-brain sections were tiled and scanned at 20x magnification using confocal microscopy, and albumin leakage in the Z-stacks was compared between mice treated with the control blockmir (randomized control) and those treated with CD5-2. Albumin leakage was defined as albumin leaking from the entire blood vessels in the middle of the Z-stacks. Leakage at the top or bottom of the brain sections or Z-stacks was considered artifacts and excluded. Once leakage was identified, the number of leaks in each cortex per mouse in each treatment group was counted. This count was then normalized relative to the vascular density of each brain segment.

[0150] like Figure 7 As shown, the application of CD5-2 significantly reduced the amount of albumin leakage.

[0151] pia mater vascular density Using ImageJ software, delineate the pia mater region in the brain using the region of interest. Use CD31 expression (as above); Figure 7 ), measuring the area of ​​CD31-positive vessels to determine the area / density of leptomeningeal vessels. For example... Figure 8 As shown, CD5-2 significantly reduced blood vessel density in APPswe / PS1dE9 mice.

[0152] Amyloid deposition Brain slices were prepared as described above and stained with rabbit anti-human β-amyloid. This antibody recognizes all classes of human β-amyloid peptides, including 1-42 and 1-40. Whole brain slices were tiled and scanned using a Leica confocal microscope at 20x magnification with Leica Application Suite (LAS) software. Cortical regions were delineated using ImageJ software. β-amyloid expression in the cortical regions was thresholded, and the amount of amyloid was analyzed (cutoff value of 5 pixels). The total amount of amyloid deposited relative to the perimeter of the cortical region was measured. The pia mater region was defined by DAPI nuclear staining from the apex of the cortex. Amyloid deposition in this layer was also compared between treatment groups.

[0153] like Figure 9 As shown, CD5-2 reduces amyloid protein deposition in both the pia mater and parenchyma.

[0154] senescence Senescent cells were detected by the loss of p21 and lamin B1. Brain slices prepared as described above were stained for the aging markers p21 and lamin B1. Senescent cells are characterized by high p21 expression and loss of the nuclear envelope of lamin B1. DAPI was used to stain the cell nucleus and identify p21 expression in the nucleus. The pia mater and penetrating vascular regions of the entire sagittal brain slice were imaged at 40x magnification using a Leica confocal microscope. The images were stacked and analyzed using ImageJ software.

[0155] like Figure 10 As shown, CD5-2 reduced the number of senescent cells in APPswe / PS1dE9 mice.

Claims

1. A method for reducing or inhibiting the deposition or accumulation of β-amyloid protein in the brain of a subject, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two, or three substituted SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG to the RNA.

2. A method for increasing or promoting the clearance of β-amyloid protein in the brain of a subject, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two or three substituted SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants or miRNA comprising a seed region containing the sequence UCACAG to the RNA.

3. The method according to claim 1 or 2, wherein the subject has been diagnosed with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), or traumatic brain injury (TBI), or has been determined to have the potential to develop AD, CAA, or LBD.

4. A method of treating a subject with AD, CAA, LBD, or TBI, or a subject who may develop AD, CAA, or LBD, comprising administering to the subject an effective amount of an oligonucleotide comprising a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two, or three substitutions of SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG to the RNA.

5. The method according to any one of claims 1-4, wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO:2 or containing 1, 2 or 3 substituted SEQ ID NO:2, wherein the oligonucleotide inhibits the binding of miR-27a, its variants or miRNA comprising a seed region containing the sequence UCACAG to the RNA.

6. The method according to claim 5, wherein the miR-27a miRNA is hsa-miR-27a containing the nucleotide sequence shown in SEQ ID NO:

13.

7. The method according to any one of claims 1 to 6, wherein the oligonucleotide comprises a continuous sequence complementary to a sequence of at least or about 7 bases, at least or about 8 bases, at least or about 9 bases, at least or about 10 bases, at least or about 11 bases, at least or about 12 bases, at least or about 13 bases, at least or about 14 bases, at least or about 15 bases, at least or about 16 bases, at least or about 17 bases, at least or about 18 bases, at least or about 19 bases, at least or about 20 bases, at least or about 22 bases, at least or about 25 bases, at least or about 30 bases, or at least or about 35 bases.

8. The method according to any one of claims 1 to 7, wherein the oligonucleotide is bound to positions 22-27 of SEQ ID NO:

2.

9. The method according to any one of claims 1 to 8, wherein the base pairing between the oligonucleotide and SEQ ID NO: 2 comprises positions 8-28, 8-27, 9-27, 10-27, 11-27, 12-27, 13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27, 20-27, 21-27, 9-28, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28, 17-28, 18-28, 19-28, 20-28, or 21-28 of SEQ ID NO:

2.

10. The method according to any one of claims 1 to 9, wherein the oligonucleotide comprises the sequence shown in SEQ ID NO:

4.

11. The method according to any one of claims 1 to 10, wherein the oligonucleotide comprises one or more modified nucleobases.

12. The method according to claim 11, wherein the modified nucleobase is an LNA nucleobase, an UNA nucleobase, or a 2' O-methyl nucleobase.

13. The method according to any one of claims 1 to 12, wherein the oligonucleotide comprises the sequence shown in SEQ ID NO:

7.

14. The method of claim 4, wherein the treatment results in a reduction or inhibition of the deposition or accumulation of β-amyloid protein in the brain of the subject; and / or a promotion or increase in the clearance of β-amyloid protein in the brain of the subject.

15. The method according to any one of claims 1, 2 or 14, wherein the reduction or inhibition of the deposition or accumulation of β-amyloid protein, and / or the promotion or increase of the clearance of β-amyloid protein, is in the vascular system of the brain and / or the parenchyma of the brain.

16. The method of claim 15, wherein the reduction or inhibition of β-amyloid deposition or accumulation, and / or the promotion or increase of β-amyloid clearance, is in the pia mater vessels of the brain.

17. The method according to any one of claims 1, 2 and 14-16, wherein the β-amyloid protein is in the form of β-amyloid plaques.

18. The method according to any one of claims 1 to 17, further comprising administering further treatment.

19. The method of claim 18, wherein the further therapy comprises other active agents, devices, physical therapy, cognitive therapy, and / or occupational therapy.

20. The method of claim 18 or 19, wherein the further therapy comprises an active agent or device for lowering blood pressure and / or pulse pressure.

21. The method according to any one of claims 1 to 20, wherein the administration of the oligonucleotide and optional further therapies results in an improvement in the subject's cognition or a slowing of the rate of cognitive decline.

22. Use of an oligonucleotide in the preparation of a medicament for reducing or inhibiting the deposition or accumulation of β-amyloid plaques in the brain of a subject, wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two, or three substitutions of SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG to said RNA.

23. Use of an oligonucleotide in the preparation of a medicament for increasing or promoting the clearance of β-amyloid plaques in the brain of a subject, wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two or three substitutions of SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants or miRNA comprising a seed region containing the sequence UCACAG to said RNA.

24. The use according to claim 22 or 23, wherein the subject has been diagnosed with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), or traumatic brain injury (TBI), or has been determined to have the potential to develop AD, CAA, or LBD.

25. Use of an oligonucleotide in the preparation of a medicament for treating a subject with Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia (LBD), or traumatic brain injury (TBI), wherein the oligonucleotide comprises a continuous sequence complementary to at least eight consecutive bases of an RNA sequence comprising SEQ ID NO: 1 or containing one, two, or three substitutions of SEQ ID NO: 1, wherein the oligonucleotide inhibits the binding of miR-27a, its variants, or miRNA comprising a seed region containing the sequence UCACAG to the RNA.

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