Delivery of proteins to Bri2 BRICHOS domains in CNS neurons

Abstract

Disclosed is an isolated protein comprising (i) a first protein moiety selected from the group consisting of: a protein comprising an amino acid sequence having at least 70% identity with residues 113-231 (SEQ ID NO: 2) of Bri2 from human; and a protein comprising an amino acid sequence having at least 70% identity to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); and (ii) a second protein or polypeptide moiety preferably containing at least 50 amino acid residues wherein the second protein or polypeptide moiety is selected from the group consisting of a protein drug, a polypeptide drug, a protein tag, a fluorescent protein, an antibody, an enzyme and / or a neurotrophic factor; wherein the isolated protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 (SEQ ID NO: 3) of Bri2 from human; and wherein the isolated protein does not comprise an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); the isolated protein is useful as a medicament.

Description

Technical Field

[0001] The present invention is in the field of medicine. More particularly, the present invention relates to the use of substances and agents for improving the medical treatment of Alzheimer's disease in mammals, such as humans. The present invention also relates to substances and agents for transporting proteins across the blood-brain barrier in mammals, including humans. The present invention also relates to the delivery of proteins to CNS neurons and the treatment of neurological diseases. Background Art

[0002] A growing number of neurodegenerative diseases, such as Alzheimer's disease and familial British or Danish dementia, are associated with protein misfolding and aggregation. These diseases are characterized by protein deposition in the brain parenchyma and cerebral arteries and occur in both hereditary and sporadic forms. Although these diseases have distinct clinical manifestations, they share common pathological features, such as neuronal loss, protein aggregation, and the presence of tau protein tangles. Biochemically, the proteins involved have a propensity to form β-pleated sheets and readily aggregate into amyloid fibrils. Alzheimer's disease and familial British or Danish dementia exhibit similar neuropathological features. Amyloid plaques, neurofibrillary tangles, congophilic amyloid angiopathy, and neurodegeneration are observed. Alzheimer's disease is one of the most common causes of dementia in humans. It is a chronic, fatal disease associated with neuronal degeneration in the brains of affected individuals and is characterized by the presence of amyloid plaques composed of extracellular deposits of amyloid β-peptide (Aβ-peptide). Neuronal atrophy caused by Aβ aggregation leads to a deficiency of acetylcholine and other signaling substances. It is known that Aβ-peptides with 40-42 amino acid residues are produced by processing the amyloid precursor protein (APP, 695-770 amino acid residues), a type I membrane protein normally expressed by neurons in the central nervous system, but the reasons for this processing are not fully understood. The released Aβ peptide contains a portion of the APP transmembrane region (Aβ residues 29-40 / 42) and contains a discordant helix, i.e., a helix composed of amino acids with a high propensity to form β-strands. When Aβ is removed from its stabilizing membrane environment, it is prone to misfolding and aggregation.

[0003] Bri2 (SEQ ID NO: 1, also known as integral membrane protein 2B, ITM2B) is a ubiquitously expressed 266-residue type II membrane protein ( Figure 1), whose function and folding structure are still unclear. Bri2 is proteolytically cleaved at three locations; furin cleavage in the C-terminal region generates a 23-residue peptide (ABri23), processing of the extracellular domain by ADAM10 results in the release of the BRICHOS domain from the membrane-bound N-terminal portion, and intramembrane cleavage by SPPL2a / 2b releases the intracellular domain. Familial British dementia and familial Danish dementia are caused by mutations in the Bri2 gene that result in the loss of a stop codon, which in turn leads to two different 11-residue extensions in the C-terminal portion and, after furin cleavage, produces 34-residue peptides (ABri and ADan, respectively) instead of the normally released ABri23. Longer peptides tend to aggregate into amyloid fibrils and deposit in brain tissue or cerebral blood vessels, accompanied by neuronal loss and dementia.

[0004] Recent studies have shown that Bri2 and Aβ colocalize in amyloid plaques in the brain parenchyma and blood vessels, suggesting that the two proteins interact at some stage during the process of misfolding and aggregation. Using transfected cell lines, it was found that Bri2 interacts with APP and regulates APP processing by increasing the fragments generated by β-secretase. The production of fusion proteins containing Bri2 and Aβ40 showed that Bri proteins can affect Aβ aggregation properties, and using transgenic mouse models, it was proposed that Aβ23 interacts with Aβ42 and prevents its aggregation (Kim et al. J. Neurosci. [Journal of Neuroscience] 28: 6030-6036 (2008); WO2009 / 009396). It has also been proposed that Aβ production can be reduced or prevented by proteins containing the first 102 amino acid residues of Bri2 (WO 2006 / 138355).

[0005] The BRICHOS domain is a naturally occurring chaperone found in 10 different human proprotein families with anti-amyloid properties, one of which (proSP-C) is associated with amyloid lung disease and one (Bri2 / ITM2b) is previously associated with the amyloid-related dementias Familial British Dementia and Familial Danish Dementia. Recombinant human (rh) BRICHOS domains from proSP-C and Bri2 delay Aβ40 and Aβ42 fibril formation in vitro and in vivo and reduce the neurotoxicity associated with Aβ42 fibril formation (WO 2011 / 62655).

[0006] The blood-brain barrier (BBB) ​​functions to maintain the delicate homeostasis required for normal neuronal function. The BBB also acts as a barrier to substances and agents targeted to the brain, with larger molecules unable to spontaneously cross the BBB. However, the BBB provides an efficient route for transporting compositions such as agents, drugs, and biopharmaceuticals (e.g., proteins) to the central nervous system (CNS). Only small, lipophilic molecules have been shown to be able to passively cross the BBB. Recent studies have shown that isolated recombinant Bri2 BRICHOS protein (Bri2BRICHOS-AU1), tagged with AU1 for immunodetection, can be detected in the brain parenchyma following peripheral (intravenous) administration to wild-type mice, whereas recombinant proSP-C BRICHOS protein can only pass through the cerebrospinal fluid (CSF). This suggests the existence of an unknown mechanism for the transport of Bri2 BRICHOS across the BBB (Mikitsh et al. Perspect Medicin Chem. 6:11-24 (2014); Sanchez-Covarrubias et al. Curr Pharm Des. 20:1422-49 (2014); Tambaro et al. J Biol Chem. 294:2606-2615 (2019)).

[0007] A variety of methods have been developed for delivering compositions through the BBB. Focused ultrasound is combined with intravenous lipid microbubbles (MBs) to promote local reversible opening of the BBB, thereby allowing macromolecules including proteins to be transported to the brain parenchyma around the area targeted by ultrasound (Mikitsh et al. Perspect Medicin Chem. [Perspective Medical Chemistry] 6: 11-24 (2014); Brasnjevic et al. Prog Neurobiol. [Progress in Neurobiology] 87: 212-51 (2009); Konofagou et al. Theranostics. [Theranostics] 2: 1223-37 (2012); Sierra et al. J Cereb Blood Flow Metab. [Journal of Brain Blood Flow and Metabolism] 37: 1236-1250 (2017)). These methods require specialized ultrasound equipment. It is also believed that ultrasound treatment may cause vascular damage under certain conditions.

[0008] et al., Scientific Reports, 10:21765 (2020) disclose the functionalization of amyloid fibrils using fusion proteins containing the Bri2 BRICHOS domain, which reportedly facilitates the synthesis of amyloid proteins functionally modified with different proteins.

[0009] Current treatments for Alzheimer's disease primarily target symptoms and include cholinergic replacement therapy, such as small inhibitors that inhibit acetylcholinesterase, which interact with soluble Aβ oligomers, and so-called β-sheet disruptors, which prevent the elongation of already formed β-sheet structures. Furthermore, there is interest in being able to lower the dose of therapeutic drugs to reduce the side effects associated with combining sonication with other treatments. Summary of the Invention

[0010] It is an object of the present invention to provide new therapeutic options for the treatment of Alzheimer's disease in mammals, including humans.

[0011] Another object of the present invention is to provide robust and convenient methods and means for effectively delivering proteins (such as therapeutic agents) across the blood-brain barrier and into the CNS. In particular, an object of the present invention is to achieve delivery of proteins (such as therapeutic agents) into CNS neurons.

[0012] One object of the present invention is to provide a simple and effective method and means for effectively delivering Bri2 BRICHOS and variants thereof across the blood-brain barrier in the treatment of Alzheimer's disease.

[0013] Another object of the present invention is to reduce the tendency of fibrillation-prone proteins to aggregate into amyloid fibrils, or even to prevent the aggregation of fibrillation-prone proteins into amyloid fibrils.

[0014] Yet another object is to reduce the formation of amyloid plaques, which consist of extracellular deposits of proteins that are prone to fibrillation in the brain of mammals.

[0015] The object of the present invention is to influence the distribution of isolated recombinant Bri2 BRICHOS and variants thereof such that an increased therapeutically effective amount of isolated recombinant Bri2 BRICHOS reaches the brain to effectively combat Aβ42 neurotoxicity.

[0016] Another object of the present invention is to provide proteins and methods for improved treatment, in vivo diagnosis and prognosis, and / or imaging of Alzheimer's disease and other neurological diseases in mammals, including humans.

[0017] Another object of the present invention is to provide novel proteins and methods for treating Parkinson's disease in mammals, including humans.

[0018] The present invention is generally based on the recognition that when administered in combination with lipid microbubbles and / or nanodroplets, isolated recombinant protein Bri2 BRICHOS and variants thereof can be effectively delivered across the blood-brain barrier without the need for a sonication step of any tissue of a mammal. Surprisingly, co-administration with lipid microbubbles and / or nanodroplets achieved improved uptake of Bri2 BRICHOS and variants thereof in the absence of sonication, compared to (a) the absence of lipid microbubbles and / or nanodroplets, and (b) the presence of sonication. The method does not include any step of treating any tissue of a mammal with optical, audio, or ultrasonic waves to cause cavitation of the microbubbles and / or nanodroplets in the tissue.

[0019] Furthermore, the present invention is based on the recognition that microbubbles and / or nanodroplets alone, without sonication, can be used to enhance the delivery of proteins comprising Bri2 BRICHOS and variants thereof as a first protein moiety coupled to another (non-Bri2) second protein or polypeptide moiety across the blood-brain barrier, thereby, for example, facilitating the treatment and / or diagnosis of Alzheimer's disease and other neurological diseases involving the second protein or polypeptide moiety.

[0020] One aspect of the present invention is based on the recognition that proteins comprising isolated recombinant protein Bri2 BRICHOS and variants thereof (including Bri2 BRICHOS R221E) can be effectively delivered to CNS neurons across the blood-brain barrier. The method does not require any steps for administering lipid microbubbles and / or nanodroplets. The method does not require any steps for treating any tissue of a mammal with optics, audio, or ultrasound. Bri2BRICHOS R221E is described in WO 2021 / 140140 A1, which is incorporated herein by reference in its entirety.

[0021] For these and other purposes that will be apparent from the following description and the appended claims, the present invention provides, according to a first aspect, an isolated recombinant protein comprising Bri2 BRICHOS and variants thereof for use in a method for treating Alzheimer's disease. According to a second aspect, there is provided a method for treating Alzheimer's disease, the method comprising administering the isolated recombinant protein comprising Bri2 BRICHOS and variants thereof and lipid microbubbles and / or nanodroplets without any sonication.

[0022] According to a third aspect, a protein is provided comprising a first protein portion that is a Bri2 BRICHOS and variants thereof and a second protein or polypeptide portion, and a combination of these with lipid microvesicles and / or nanodroplets. According to a fourth aspect, the combination can be used in a method for transporting proteins across the blood-brain barrier in mammals without any ultrasound treatment.

[0023] The third aspect of the protein comprising a first protein portion (which is Bri2 BRICHOS and variants thereof) and a second protein or polypeptide portion is conducive to increasing the transport of the second protein or polypeptide portion across the blood-brain barrier. The first portion (Bri2BRICHOS) contributes to transport and can be achieved without lipid microvesicles and / or nanodroplets. Then, a particularly useful variant of the first protein portion is Bri2 BRICHOS R221E, because it has been shown herein to improve transport compared to wild-type Bri2 BRICHOS. Transport can be further improved with lipid microvesicles and / or nanodroplets. If lipid microvesicles and / or nanodroplets are included, the method does not require any of the following steps: treating any tissue of a mammal with optics, audio, or ultrasound to cause cavitation of the microvesicles and / or nanodroplets in the tissue.

[0024] According to another aspect, a protein for treating Parkinson's disease is provided, the treatment comprising administering Bri2BRICHOS and variants thereof 。 Also provided is a protein comprising a first protein portion (which is Bri2BRICHOS and variants thereof) and a second protein or polypeptide portion, wherein the second protein or polypeptide portion itself is effective in treating Parkinson's disease. The combination can also be used to treat Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of Bri2 (SEQ ID NO: 1) and processing sites is shown.

[0026] Figure 2 An alignment of several mammalian Bri2 BRICHOS amino acid sequences (SEQ ID NOs: 5-10) is shown.

[0027] Figure 3 The study design in the Examples is shown.

[0028] Figure 4 It is shown that Bri2 BRICHOS, but not proSP-C BRICHOS, were detected in the non-FUS-targeted brain hemisphere following intravenous injection with lipid microbubbles and / or nanodroplets.

[0029] Figure 5 It is shown that Bri2 BRICHOS were detected in both hemispheres after intravenous injection together with lipid microbubbles and / or nanodroplets.

[0030] Figure 6 Shown are intracellular immunostaining of Bri2BRICHOS-AU1 in the cortex and hippocampus following intravenous injection with lipid microbubbles and / or nanodroplets.

[0031] Figure 7 Shown is Western blot detection of Bri2 BRICHOS-AU1 in both brain hemispheres following systemic injection with lipid microbubbles and / or nanodroplets.

[0032] Figure 8 Shown is the finding of Rh Bri2BRICHOS-mCherry in the striatum after systemic injection in combination with lipid microbubbles and / or nanodroplets.

[0033] Figure 9 Shows the effect of repeated injections of Bri2 BRICHOS R221E on the App NL-G-F and App NL-F Detection of Bri2 BRICHOS in mouse brain.

[0034] Figure 10 The permeability of different proteins and macromolecules to hCMEC / D3 cell monolayers is shown.

[0035] Figure 11 Shown are staining of the cerebral cortex of mice injected with NT-Bri2 BRICHOS (A) or PBS (B) with anti-NT antibodies.

[0036] Figure 12 Demonstrated Rh Bri2 BRICHOS-mediated uptake into mouse primary neurons.

[0037] Figure 13 TEM images of α-syn fibrils after fibrillation at 70 μM are shown: (a) molar equivalent in the absence of BRICHOS, (b) molar equivalent in the presence of 100% BRICHOS, and (c) fibril diameters of α-syn fibrils incubated without or with BRICHOS.

[0038] Figure 14 demonstrated that BRICHOS monomers prevent the neurotoxic effects of sonicated α-syn fibrils.

[0039] Supplementary Sequence Listing

[0040]

[0041]

[0042] DETAILED DESCRIPTION

[0043] Bri2 (SEQ ID NO: 1), also known as integral membrane protein 2B (ITM2B), contains an evolutionarily conserved BRICHOS domain (SEQ ID NO: 5) spanning residues 137-231. BRICHOS domains are found in more than 10 different protein families that are functionally unrelated and expressed in different tissues. The name BRICHOS refers to the identification of domains in Bri, which is associated with chondrosarcoma, chondromodulin-1, and pro-pulmonary surfactant protein C (proSP-C), which is associated with respiratory diseases.

[0044] Proteins containing the BRICHOS domain of mammalian Bri2 (ITM2B) and structurally similar proteins have the ability to reduce amyloid fibril formation and aggregation of Aβ peptides and ABri / ADan peptides.

[0045] The blood-brain barrier (BBB) ​​plays a crucial role in maintaining the delicate homeostasis required for normal neuronal function. The BBB also acts as a barrier to substances and pharmaceutical agents targeting the brain, with larger molecules unable to spontaneously cross the BBB. Nevertheless, the BBB provides an effective pathway for transporting pharmaceutical compositions as proteins to the central nervous system (CNS). Only small lipophilic molecules have been shown to passively cross the BBB.

[0046] The present invention is generally based on the recognition that isolated Bri2 BRICHOS protein, by itself or coupled to a second protein or polypeptide moiety, can be effectively delivered to the brain in combination with lipid microvesicles and / or nanodroplets, and therefore will be useful in treating Alzheimer's disease.

[0047] Furthermore, even in the absence of lipid microbubbles and nanodroplets, isolated Bri2 BRICHOS protein coupled to a second (cargo) protein or polypeptide moiety can be efficiently delivered to the brain.

[0048] The lipid microvesicles and / or nanodroplets and the isolated protein can be administered in combination. As used herein, the terms "combination" and / or "combination" mean that the isolated protein and lipid microvesicles and / or nanodroplets can be administered separately and independently of each other in any order. The term can also mean that the recombinant protein and lipid microvesicles and / or nanodroplets can be administered separately and simultaneously. In addition, the term can mean that the isolated protein and lipid microvesicles and / or nanodroplets can be administered in the same pharmaceutical composition.

[0049] The isolated protein according to the present invention can be incorporated into a pharmaceutical composition. Such a composition typically comprises the isolated protein according to the present invention and a suitable pharmaceutically acceptable carrier. As used herein, "suitable pharmaceutical carrier" includes solvents, dispersion media, coatings, isotonicity agents, and absorption delaying agents that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0050] In prior art methods, ultrasound is an important local stimulus for triggering drug release at target tissue and may consist of pressure waves with a frequency of 20 kHz or higher. Like light and audio waves, ultrasound waves are focused, reflected and refracted by a medium.

[0051] Microbubbles and / or nanodroplets are combined with ultrasound treatment and are commonly used for medical diagnosis and for the non-invasive delivery of pharmaceutical compositions and genes to different tissues. Focused ultrasound combined with intravenous administration of microbubbles and / or nanodroplets is a technology that has been shown in multiple in vivo models, which effectively delivers small molecules and macromolecules across the BBB and reaches specific targeted brain regions in a minimally invasive manner. The technology involves administering lipid-based microbubbles and / or nanodroplets together with the pharmaceutical composition to be delivered. Ultrasound waves cavitate microbubbles and / or nanodroplets in the capillaries in the brain. Under these low pressures, microbubbles and / or nanodroplets show stable cavitation, resulting in an increase in the opening of the BBB. This causes the tight junctions between endothelial cells to relax temporarily, resulting in a temporary local opening of the BBB. As a result, the macromolecules present in the circulation are delivered to the brain parenchyma (Galan-Acosta et al. Mol Cell Neurosci [Molecular and Cellular Neuroscience], 103498 (2020)).

[0052] Here, we have designed a favorable method for effectively delivering the composition across tissues such as the blood-brain barrier and into neurons in the CNS. The method combines the administration of isolated proteins with the administration of lipid microbubbles and / or nanodroplets. Importantly, the present method does not involve any steps of ultrasonic treatment. This is advantageous because it does not require any ultrasonic equipment. In addition, side effects associated with ultrasonic treatment, such as vascular damage, can be avoided. Surprisingly, compared with (a) the absence of lipid microbubbles and / or nanodroplets, and (b) the presence of ultrasonic treatment, in the absence of ultrasonic treatment, the co-administration of lipid microbubbles and / or nanodroplets achieves improved uptake of Bri2 BRICHOS and its variants.

[0053] The method does not include any step of treating any mammalian tissue with optical, audio or ultrasonic waves to cause cavitation of microbubbles and / or nanodroplets in the tissue.

[0054] For these and other purposes that will be apparent from the following description, the present invention provides, according to a first aspect, an isolated recombinant protein for use in a method of treating Alzheimer's disease in a mammal in need thereof, the protein being selected from the group consisting of an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4); the mammal including a human, the method comprising the steps of:

[0055] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0056] administering the isolated recombinant protein to the mammal; wherein the isolated recombinant protein is not contained within the microvesicles and / or nanodroplets; and

[0057] The method does not include any step of sonicating any tissue of the mammal.

[0058] Our experiments show that lipid microbubbles and / or nanodroplets alone, i.e., without sonication, can mediate the uniform transfer of Bri2 BRICHOS across the BBB, and that when Bri2 BRICHOS and lipid microbubbles and / or nanodroplets are administered in combination, Bri2 BRICHOS are delivered to the brain parenchyma and efficiently taken up by neurons in the cortex and hippocampus.

[0059] Surprisingly, it was found that when administered in combination with microbubbles and / or nanodroplets, the Bri2 BRICHOS protein was delivered to the wild-type mouse brain. This was particularly surprising considering various methods that have been previously developed in which focused ultrasound combined with intravenous lipid microbubbles and / or nanodroplets are used to deliver drugs and other compositions across the BBB by allowing the transport of macromolecules, including proteins, into the brain parenchyma surrounding the ultrasound-targeted area.

[0060] The present invention is based on the surprising insight disclosed herein that microbubbles and / or nanodroplets, by themselves, without being combined with any sonication step of any mammalian tissue, have the ability to mediate increased transfer of isolated recombinant proteins across the BBB. Microbubbles and / or nanodroplets increase the uptake of Bri2 BRICHOS across the BBB and into neurons.

[0061] An advantage of this aspect of the invention is that instantaneous and localized opening of the BBB can be achieved without the use of any additional processing steps, such as sonication and the additional equipment associated with such additional processing. Thus, an advantageous method is provided for effectively delivering a composition across tissues such as the blood-brain barrier and into neurons in the CNS. As shown herein, another advantage is that, as described in WO 2011 / 162655, the uptake of an isolated recombinant protein into the brain parenchyma is highly enhanced when administered in combination with lipid microbubbles and / or nanodroplets compared to administration of the isolated recombinant protein alone.

[0062] As used throughout the specification and in the appended claims, the term "% similarity" is calculated as for "% identity," except that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met, and Cys are similar; the basic residues Lys, Arg, and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gln, Asn, Ser, Thr, and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

[0063] Throughout this specification, alternative embodiments achieve corresponding percentages of similarity instead of specified percentages of identity. Other alternative embodiments achieve specified percentages of identity and another, higher percentage of similarity, which is selected from the preferred percentages of identity for each sequence. For example, an isolated recombinant protein sequence may have a similarity of 70% to another protein sequence; or it may have a 70% identity to another sequence; or it may have a 70% identity to another sequence and, in addition, a 90% similarity to another sequence.

[0064] For the avoidance of doubt, an amino acid sequence having at least a given identity to residues 113-231 of Bri2 from human, or any one of the BRICHOS domains of Bri2, consists of greater than or equal to 70, such as greater than or equal to 80, for example greater than or equal to 90 amino acid residues. A preferred size range is 70-100 amino acid residues, such as 80-100 amino acid residues, for example 90-100 amino acid residues.

[0065] It should be noted that the BRICHOS domain of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10) is highly conserved, see Figure 2 Without wishing to be bound by any particular theory, it is expected that the BRICHOS domain has the desired activity against Aβ and ABri / ADan peptides. Preferably, the isolated recombinant protein according to the present invention is selected from the group consisting of proteins comprising an amino acid sequence that is at least 80%, preferably at least 90%, such as at least 95% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10). In a preferred embodiment, the isolated recombinant protein according to the present invention contains all amino acid residues that are conserved in the BRICHOS domain of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10), i.e., all amino acid residues of SEQ ID NO: 5, except residues 42, 76 and 82 (corresponding to residues 178, 212 and 218 of full-length Bri2, i.e., SEQ ID NO: 1). In a specific embodiment, the isolated recombinant protein according to the present invention is selected from the group consisting of a protein comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10), i.e., it contains one of these BRICHOS domains, preferably the human BRICHOS domain (SEQ ID NO: 5).

[0066] In a preferred embodiment, the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is selected from the group consisting of Glu and Asp. In a specific embodiment, the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is Glu. We mutated rh Bri2 BRICHOS so that the monomers are stable relative to larger oligomers, and rh Bri2BRICHOS R221E (SEQ ID NO: 13) selectively reduces the formation of Aβ42 oligomers and alleviates Aβ42-induced neurotoxicity in hippocampal slice preparations. Rh Bri2 BRICHOS R221E can pass through the BBB of mice and shows a higher tendency to pass than the wild-type protein. This result is consistent with previous observations that monomeric wild-type rh Bri2 BRICHOS passes through the BBB more efficiently than larger oligomers.

[0067] Contrary to previous teachings, the isolated recombinant protein according to the present invention does not comprise an amino acid sequence that is at least 70% identical to residues 1-89 of Bri2 from humans (SEQ ID NO: 3). In certain embodiments, the isolated recombinant protein according to the present invention does not comprise an amino acid sequence that is at least 50% identical to residues 1-89 of Bri2 from humans (SEQ ID NO: 3). This means that the isolated recombinant protein according to the present invention comprises a core amino acid sequence that shows a high degree of similarity or identity to residues 113-231 of Bri2 from humans (SEQ ID NO: 2) and / or the mammalian Bri2 BRICHOS domain (SEQ ID NO: 5-10), and optionally one or more other amino acid sequences that may not show a high degree of similarity or identity to residues 1-89 of Bri2 from humans (SEQ ID NO: 3).

[0068] For the avoidance of doubt, amino acid sequences shorter than 10 amino acid residues are not considered relevant insofar as they are excluded from the isolated recombinant protein according to the invention. Thus, the isolated recombinant protein according to the invention does not comprise an amino acid sequence consisting of more than or equal to 10 amino acid residues having at least the given identity with residues 1-89 of Bri2 from human (SEQ ID NO: 3).

[0069] In addition, the isolated recombinant protein according to the present invention does not comprise an amino acid sequence that is at least 70% identical to residues 244-266 of Bri2 from humans, i.e., human ABri23 (SEQ ID NO: 4). In certain embodiments, the isolated recombinant protein according to the present invention does not comprise an amino acid sequence that is at least 50% identical to residues of human ABri23 (SEQ ID NO: 4). As described above, this means that the isolated recombinant protein according to the present invention comprises a core amino acid sequence that shows a high degree of similarity or identity to residues 113-231 of Bri2 from humans (SEQ ID NO: 2) and / or the mammalian Bri2 BRICHOS domain (SEQ ID NO: 5-10), and optionally one or more other amino acid sequences that may not show a high degree of similarity or identity to human ABri23 (SEQ ID NO: 4).

[0070] For the avoidance of doubt, amino acid sequences shorter than 10 amino acid residues are not considered relevant insofar as they are excluded from the isolated recombinant protein according to the invention. Thus, the isolated recombinant protein according to the invention does not comprise an amino acid sequence consisting of more than or equal to 10 amino acid residues having at least the given identity with human ABri23 (SEQ ID NO: 4).

[0071] In a preferred embodiment, the isolated recombinant protein used according to the present invention is selected from the group consisting of: a protein comprising an amino acid sequence that is at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 70% identical to the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

[0072] In a preferred embodiment, the isolated recombinant protein for use in a method of treating Alzheimer's disease in a mammal in need thereof is selected from the group consisting of an amino acid sequence that is at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence that is at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence that is at least 70% identical to human ABri23 (SEQ ID NO: 4); the mammal including a human, the method consisting of the following steps:

[0073] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0074] administering the isolated recombinant protein to the mammal;

[0075] The isolated recombinant protein is not contained in the microvesicles and / or nanodroplets.

[0076] In certain embodiments, the combination of the isolated recombinant protein and lipid microvesicles and / or nanodroplets is for use in a method of treating Alzheimer's disease in a mammal (including a human) in need thereof, the method comprising the steps of:

[0077] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0078] administering the isolated recombinant protein to the mammal;

[0079] wherein the isolated recombinant protein is not contained within the microvesicles and / or nanodroplets; and

[0080] The method does not include any step of sonicating any tissue of the mammal.

[0081] In a specific embodiment, the combination of the isolated recombinant protein and lipid microvesicles and / or nanodroplets is for use in a method of treating Alzheimer's disease in a mammal (including a human) in need thereof, the method consisting of the following steps:

[0082] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0083] administering the isolated recombinant protein to the mammal;

[0084] The isolated recombinant protein is not contained in the microvesicles and / or nanodroplets.

[0085] Pharmaceutical compositions comprising the isolated recombinant protein are useful as medicaments, particularly for treating disorders such as Alzheimer's disease in mammals (including humans).

[0086] A pharmaceutical composition is formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral (e.g., intravenous, intraarterial), intraperitoneal, intramuscular, intradermal, and intranasal.

[0087] Administration of the microbubbles and / or nanodroplets and the isolated recombinant protein may comprise injection.

[0088] Sterile injectable solutions can be prepared by the following: according to the recombinant protein of the separation of the present invention with the required amount, as required, with the combination of the composition of more than one enumeration or these compositions mixed in a suitable solvent, then carry out filter sterilization. Generally speaking, dispersions can be prepared by mixing the recombinant protein of the separation of the present invention in the sterile vehicle containing dispersion medium and other required components. With regard to the sterile powder for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which methods produce powders according to the recombinant protein of the separation of the present invention and from any other desired composition of its previous sterile filtered solution.

[0089] In general, a sterile injectable solution of lipid microbubbles and / or nanodroplets can be prepared as described in, for example, Feshitan, JA et al., J. Colloid Interface Sci. [Journal of Colloid and Interface Science] 329 (2), 316-324 (2009). In short, gas perfluorobutane (PFB) can be used to form microbubbles and / or nanodroplets. PFB can act as a gas core that can be introduced to activate lipid microbubbles and / or nanodroplets, and then separate the microbubbles and / or nanodroplets. The microbubbles and / or nanodroplets can be coated with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and polyoxyethylene-40 stearate (PEG40S) DSPC and PEG40S. The dried lipid film obtained can be hydrated with filtered PBS and mixed into a final lipid suspension.

[0090] The lipid mixture can be ultrasonically processed so that lipid aggregates are dispersed into small unilamellar liposomes. PFB gas can be introduced by making it flow through the surface of lipid suspension. Subsequently, the suspension at gas-liquid interface can be applied with higher power ultrasonic processing to produce microbubbles and / or nanodroplets. After separation, microbubbles and / or nanodroplets according to the present invention can be incorporated into sterile vehicles, for example, microbubbles and / or nanodroplets suspension can be collected in 30mL syringes, washing and size fractionation can be achieved by centrifugation, so that all microbubbles and / or nanodroplets in the suspension are collected in the cake leaning against the syringe plunger. Remaining suspension (swimming below (infranatant)) (it may contain residual lipids and the vesicle that do not form a part for microbubble shell) can be recycled to produce next batch of microbubbles and / or nanodroplets. All gained filter cakes can be merged, and be resuspended in PBS to improve total yield.

[0091] In certain embodiments, the isolated recombinant protein and the lipid microvesicles and / or nanodroplets are administered intravenously.

[0092] When the isolated protein according to the present invention is applied to an animal (e.g., a human) to treat Alzheimer's disease, a physician, veterinarian, or researcher can, for example, first prescribe a relatively low dosage and then increase the dosage until an appropriate response is obtained. In addition, it should be understood that the specific dosage level for any particular animal subject will depend on a variety of factors, including the activity of the specific isolated recombinant protein used, the subject's age, body weight, general health, sex, diet, time of administration, route of administration, and excretion rate, any drug combination, and the expression or activity level to be regulated.

[0093] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.

[0094] The dosage may vary within the stated range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays in which, for example, the rate of fibril formation or the rate of cell death is observed. A dose can be formulated in animal models to achieve a circulating plasma concentration range determined in cell culture that includes the IC 50 (ie, the concentration of the test compound that achieves a half-maximal inhibition of symptoms.) Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0095] As defined herein, the therapeutically effective amount (i.e., effective dose) of the isolated recombinant protein according to the present invention ranges from about 1 to 50 mg / kg body weight. In one embodiment, the isolated recombinant protein is administered in a therapeutically effective amount of at least 1 mg / kg, such as at least 5 mg / kg, more preferably at least 10 mg / kg.

[0096] In another embodiment, a therapeutically effective amount of the isolated recombinant protein is administered that is less than 50 mg / kg, such as less than 30 mg / kg, more preferably less than 20 mg / kg.

[0097] The recombinant protein of separation can be administered to the experimenter in the extended time, for example, in the experimenter's lifetime. The dosage of 1mg / kg to 50mg / kg body weight is generally suitable. By administering the recombinant protein of separation in combination with lipid microvesicles and / or nano-droplets according to the present invention, it is possible to administer the recombinant protein of separation significantly lower than the dosage previously described in WO 2011 / 162655. The administration of lipid microvesicles and / or nano-droplets causes the local and reversible opening of the BBB, thereby allowing the recombinant protein of effective transportation separation to enter the brain substance and increase the intake of the recombinant protein of separation to enter the brain substance. This aspect of the present invention is also advantageous, because the risk of the side effect related to the administration of the therapeutic composition of the preventive and therapeutic method for treating the experimenter suffering from Alzheimer's disease or being in the risk of suffering from Alzheimer's disease (or being susceptible to Alzheimer's disease) is minimized.

[0098] In some cases, lipid microvesicles and / or nano droplets and isolated recombinant proteins can be used once a week, for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, even more preferably for about 4, 5 or 6 weeks. Also long-term administration of compounds is possible. Technicians will appreciate that certain factors can affect the dosage and time course required for effective treatment of the experimenter, including but not limited to the severity of the disease or obstacle, previous treatment, the general health status and / or age of the experimenter, and other diseases of existence. In addition, the lipid microvesicles and / or nano droplets and isolated recombinant protein treatment experimenter with a therapeutically effective amount can comprise a single treatment, or preferably can comprise a series of treatments.

[0099] In further embodiments, the isolated protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identical to the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

[0100] In a preferred embodiment, the isolated protein is selected from the group consisting of: residues 113-231 of Bri2 from human (SEQ ID NO: 2); and the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

[0101] According to a related aspect, the invention provides an isolated protein selected from the group consisting of a protein comprising an amino acid sequence that is at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0102] In another embodiment, the isolated protein is selected from the group consisting of a protein comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0103] In certain embodiments, the isolated protein is from the group consisting of any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0104] In certain embodiments, the isolated protein is made up of less than or equal to 200 amino acid residues, such as less than or equal to 150 amino acid or even 100 amino acid residues. In certain embodiments, the isolated protein is made up of more than or equal to 90 amino acid residues, such as more than or equal to 100 amino acid residues.

[0105] A preferred size range for the isolated protein is 80-200 amino acid residues, such as 90-150 amino acid residues, for example 90-100 amino acid residues.

[0106] Microbubbles and / or nanodroplets are small, gas-filled microspheres. They may consist of a gas surrounded by a lipid, lipid polymer, or polymer shell. They may also be similar in size to red blood cells, ranging from 0.5-10 μm.

[0107] In the present invention, the microbubble of described invention can be used for the microbubble of described invention.When applying the acoustic energy field, the microbubble of inflation and / or nano-droplet can oscillate and vibrate.Microbubble and / or nano-droplet with the hydrophilic outer layer that interacts with blood flow and the hydrophobic inner layer that accommodates gas molecules are thermodynamically the most stable.Air, sulfur hexafluoride and perfluorocarbon gas can be used as the component inside microbubble.In order to improve the stability and persistence in blood, high molecular weight and the gas with low solubility in blood are the attractive candidate of microbubble gas core.Microbubble and / or nano-droplet can be used for drug delivery, and they can not only be used as drug medium, can also be used as the means of other impenetrable barriers (particularly blood-brain barrier) of infiltration.

[0108] In further embodiments of the present invention, the microbubbles and / or said nanodroplets are coated with lipids.

[0109] As disclosed herein, microbubbles and / or nanodroplets can comprise 1,2-distearyl alcohol-sn-glycero-3-phosphocholine (DSPC), 1,2-distearyl alcohol-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol) 2000), and perfluorobutane gas cores. Microbubbles and / or nanodroplets can, for example, comprise sulfur hexafluoride, polyethylene glycol (PEG, Macrogol), distearoylphosphatidylcholine (DSPC), sodium 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol, and palmitic acid.

[0110] In a preferred embodiment of the invention, individual microbubbles and / or nanodroplets according to the invention have a diameter in the range of 1-8 μm, such as 2-6 μm, such as 4-5 μm.

[0111] The skilled person will appreciate that the embodiments discussed above with respect to the first aspect of the present disclosure are equally relevant and applicable to the second, third, and other aspects disclosed herein. This is particularly applicable to embodiments related to isolated recombinant proteins and lipid microvesicles and / or nanodroplets, the combination of the present invention enabling the isolated protein of the recombinant to be effectively and evenly transferred across the BBB and into the brain parenchyma, as well as to be effectively taken up by neurons in the cortex and hippocampus, and to embodiments related to modes of administration and routes of administration. For the sake of brevity, this will not be repeated or simply mentioned.

[0112] In a second aspect of the present invention, there is provided a method for treating Alzheimer's disease in a mammal (including a human) in need thereof, the method comprising the following steps:

[0113] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0114] administering to the mammal an isolated recombinant protein selected from the group consisting of an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4);

[0115] wherein the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; and wherein the method does not comprise any step of sonicating any tissue of the mammal.

[0116] In an embodiment of the second aspect, there is provided a method for treating Alzheimer's disease in a mammal (including a human) in need thereof, the method comprising the following steps:

[0117] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal

[0118] administering to the mammal an isolated recombinant protein selected from the group consisting of an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4);

[0119] The isolated recombinant protein is not contained in the microvesicles and / or nanodroplets.

[0120] As a further aspect of the present invention, there is provided an isolated recombinant protein consisting of residues 113-231 of human Bri2 (SEQ ID NO: 2).

[0121] The present invention provides both preventative and therapeutic methods for treating a subject at risk of Alzheimer's disease (or susceptible to Alzheimer's disease). As used herein, the term "treatment" is defined as applying or administering to a patient the recombinant protein and lipid microvesicles and / or nanodroplets separated according to the present invention, or applying or administering to a tissue or cell line from a patient suffering from Alzheimer's disease, with symptoms of disease or a constitution susceptible to disease the recombinant protein and lipid microvesicles and / or nanodroplets separated according to the present invention for the purpose of curing, healing, alleviating, mitigating, changing, remedying, improving, improving or influencing a disease, a symptom of disease or a constitution susceptible to disease. In a specific embodiment, treatment is selected from the group consisting of preventative treatment, palliative treatment and curative treatment.

[0122] In one aspect, the present invention provides a method for preventing diseases or conditions associated with fibrillation caused by Aβ peptides and / or ABri / ADan peptides (i.e., reducing the risk of infection, or reducing the rate at which symptoms associated with the disease or condition appear) by administering to a subject an isolated recombinant protein and lipid microvesicle and / or nanodroplet that reduces polypeptide aggregation according to the present invention. Subjects at risk of Alzheimer's disease can be identified by any one or combination of appropriate diagnostic or prognostic assays known in the art. Preventive agents can be administered before the manifestation of characteristic symptoms of the disease, thereby preventing or alternatively delaying the progression of the disease.

[0123] The isolated recombinant protein and lipid microvesicles and / or nanodroplets according to the present invention can be administered to a patient in a therapeutically effective dose to prevent, treat or improve disorders involving fibrillation associated with Alzheimer's disease. A therapeutically effective dose refers to an amount of the compound sufficient to cause alleviation of the symptoms of the disorder. The toxicity and therapeutic efficacy of such a compound can be determined by standard pharmaceutical procedures as described above.

[0124] According to various aspects of the present invention, the Bri2 BRICHOS protein and its variants can be used for the delivery of proteins or polypeptides such as therapeutic agents, antibodies and protein tags by providing unique advantages such as improving the therapeutic potential of drugs and drug targeting, in vivo diagnosis and prognosis, and in vivo imaging. Thus, the protein can contain at least one additional protein or polypeptide portion, wherein the protein portion can be a biopolymer, oligomer and oligopeptide, such as a peptide and polypeptide.

[0125] As a third aspect of the present invention, there is provided an isolated protein comprising

[0126] (i) a first protein portion selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); and

[0127] (ii) a second protein or polypeptide portion, preferably comprising at least 50 amino acid residues;

[0128] wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and

[0129] wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to human ABri23 (SEQ ID NO: 4).

[0130] When used in combination with microbubbles and / or nanodroplets, proteins comprising a first protein portion are unique in their in vivo therapeutic applications by providing tissue-specific targeting and / or release of drugs for treatment and therapy, in vivo diagnosis and prognosis, and in vivo imaging.

[0131] The Bri2 BRICHOS domain provides the ability to transport a second protein or polypeptide moiety across the blood-brain barrier in mammals without any sonication. An isolated protein comprising the Bri2 BRICHOS domain and a second protein or polypeptide moiety (such as a protein drug, a polypeptide drug, a protein tag, a fluorescent protein, an antibody, an enzyme, and / or a neurotrophic factor) is advantageous because it facilitates and enhances the treatment of Alzheimer's disease and other neurological diseases.

[0132] In an embodiment, the first protein portion of the isolated protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0133] In another embodiment, the first protein portion is selected from the group consisting of a protein comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0134] In one embodiment of the present invention, the first protein portion of the isolated protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% identical to any one of residues 113-231 of Bri2 from human (SEQ ID NO: 2) and the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

[0135] In further embodiments, the first protein portion is selected from the group consisting of: residues 113-231 from human Bri2 (SEQ ID NO: 2); and the BRICHOS domain from human Bri2 (SEQ ID NO: 5).

[0136] In one embodiment, the amino acid residue corresponding to position 221 in SEQ ID NO: 1 in the first protein portion is selected from the group consisting of Glu and Asp, preferably Glu. We mutated rh Bri2 BRICHOS so that the monomer is stable relative to the larger oligomer, and rh Bri2 BRICHOS R221E (SEQ ID NO: 13-14) selectively reduces the generation of Aβ42 oligomers and alleviates Aβ42-induced neurotoxicity in hippocampal slice preparations. Rh Bri2 BRICHOS R221E can pass through the BBB of mice and shows a higher passing tendency than the wild-type protein. This result is consistent with previous observations that monomeric wild-type rh Bri2 BRICHOS passes through the BBB more effectively than larger oligomers. The improved passing tendency of the R221D / E variant is particularly advantageous when it is desired to be effectively delivered to the brain without the need for co-administration of lipid microbubbles and nanodroplets.

[0137] In one embodiment, the first protein portion consists of less than or equal to 200 amino acid residues, such as less than or equal to 150 amino acid residues. In one embodiment, the first protein portion consists of more than or equal to 90 amino acid residues.

[0138] In general, delivering large therapeutic molecules to the brain to treat central nervous system (CNS) diseases is a major challenge in drug development. The BBB functions to restrict the movement of substances from circulating blood to the CNS. Consequently, only approximately 0.1% of circulating antibodies cross an intact BBB, severely limiting the therapeutic utility of antibody therapies for CNS disorders. Furthermore, the BBB excludes 100% of large molecule neurotherapeutics and over 98% of all small molecule drugs from the brain.

[0139] In one embodiment, the isolated protein does not comprise a cleavage site between the first protein portion and the Bri2-BRICHOS sequence and the second protein or polypeptide portion. In another embodiment, the isolated protein contains a cleavage site between the first protein portion and the Bri2-BRICHOS sequence and the second protein or polypeptide portion, such as the cleavage site in Bri2, which in the native protein is naturally cleaved by a proprotein convertase to release the Abri peptide.

[0140] The present invention provides an isolated protein, wherein the second protein or polypeptide portion of the isolated protein contains 50 to 2000 amino acid residues, such as 50 to 1000 amino acid residues, such as 50 to 500 amino acid residues, such as 50 to 100 amino acid residues. In another object of the present invention, the size of the second protein or polypeptide portion of the isolated protein is 5-200 kDa, such as 5-100 kDa, such as 5-50 kDa, such as 5-10 kDa.

[0141] The inventors have surprisingly found that an isolated protein comprising Bri2 BRICHOS and a second (cargo) protein or polypeptide portion can cross the BBB despite its larger size.

[0142] In some embodiments of the invention, a first protein moiety of an isolated protein is linked directly or indirectly to the amino terminus or carboxyl terminus of a second protein or polypeptide moiety.

[0143] In another embodiment of the invention, the second protein or polypeptide portion of the isolated protein constitutes the amino terminus and / or carboxyl terminus of the isolated protein.

[0144] As mentioned above, the BBB often hinders the brain delivery of large therapeutic molecules (such as protein drugs, peptide drugs, protein tags, fluorescent proteins, antibodies, enzymes and / or neurotrophic factors).

[0145] Protein and peptide drugs can be used to replace proteins that are abnormal or deficient in specific diseases. They can also increase the body's supply of beneficial proteins to help reduce the impact of disease treatment. Common and widely used examples of protein drugs are insulin, interferon alpha, and interleukin-2.

[0146] A protein tag is a peptide sequence that is grafted onto a protein, such as a gene grafted onto a recombinant protein. Examples of protein tags include solubilization tags, epitope tags, and fluorescent tags.

[0147] Solubility tags are used, particularly for recombinant proteins expressed in chaperone-deficient species such as Escherichia coli (E. coli), to aid in the correct folding of the protein and prevent its precipitation.

[0148] Epitope tags are short peptide sequences that are chosen because they reliably generate high-affinity antibodies in many different species.

[0149] Fluorescent tags and proteins are used to provide visual readouts of proteins. They can be used to label components in cells, tissues, or organs so that they can be studied using fluorescence spectroscopy, fluorescence microscopy, and other imaging techniques. GFP and its variants are the most commonly used fluorescent tags. On the other hand, mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs), belonging to a group of fluorescent protein chromophores used as tools for visualizing genes and analyzing their function in experiments.

[0150] The second protein portion can also increase the stability of the isolated protein and the first protein portion, and can itself be used as a therapeutic agent, for example to increase the half-life in vivo. Thus, the frequency of treatment can be reduced. One possibility is that the second protein portion is an Fc portion, i.e., a crystallizable fragment region (Fc region) from the tail region of an antibody, which interacts with cell surface receptors called Fc receptors and some proteins of the complement system.

[0151] In another embodiment, the second protein or polypeptide portion of the isolated protein is selected from the group consisting of a protein drug, a polypeptide drug, a protein tag, a fluorescent protein, an antibody, an enzyme and / or a neurotrophic factor.

[0152] Antibodies, also known as immunoglobulins (Ig), are large, Y-shaped proteins that the immune system uses to identify and neutralize foreign substances, such as pathogenic bacteria and viruses. Exploiting their binding mechanism, antibodies are widely used in therapy, where they are used to treat diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and many forms of cancer. Monoclonal antibodies that have been studied and / or used to treat Alzheimer's disease are aducanumab, gantenerumab, 3D6 (bapineuzumab), and m266 (solanezumab).

[0153] In preferred embodiments, the second protein or polypeptide portion of the isolated protein is an antibody, such as a monoclonal antibody. Preferred monoclonal antibodies are selected from (i) aducanumab, gantnerumab, 3D6 (bapineumab), m266 (solanesin), donetumab, and lencanerumab or (ii) aducanumab, gantnerumab, 3D6 (bapineumab), and m266 (solanesin). A preferred monoclonal antibody is donetumab. Another preferred monoclonal antibody is lencanerumab.

[0154] Neurotrophic factors (NTFs) are a family of biomolecules, almost all of which are peptides or small proteins that support the growth, survival, and differentiation of developing and mature neurons. Neurotrophic factors also promote the initial growth and development of neurons in the central and peripheral nervous systems, and they are able to regenerate damaged neurons in test tubes and animal models. Some neurotrophic factors are also released by target tissues to guide the growth of developing axons. In research, neurotrophic factors are often used in combination with other techniques, and the neurotrophic factors can be fixed to a scaffold structure. In neurodrug delivery systems, they are loosely fixed so that they can be selectively released at a specified time and in a specified amount.

[0155] In an embodiment, the second protein or polypeptide portion of the isolated protein is a neurotrophic factor selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), ephrins, epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and / or interleukins.

[0156] Enzymes are proteins that act as biological catalysts and accelerate chemical reactions. Many chemical conversions that maintain life and accelerate all biochemical processes in the body require enzymes. These characteristics distinguish them from other types of drugs. Due to these characteristics, enzymes are widely used in medicine, whether used alone or in combination with other therapies, with the purpose of safely treating various diseases. Examples of therapeutic enzymes used in the treatment of various disorders and different therapies are α-L-iduronidase, iduronic acid sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase and / or lysosomal acid lipase. Additional examples of therapeutic enzymes that can constitute the second protein include enzymes that are known to be genetically mutated and therefore functionally defective in lysosomal storage diseases (LSDs).

[0157] In another embodiment, the second protein or polypeptide portion of the isolated protein is selected from the group consisting of α-L-iduronidase, iduronic acid sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase and / or lysosomal acid lipase.

[0158] Fusion proteins are proteins created by joining two or more genes that originally encoded separate proteins. Translation of these fused genes produces a single protein with functional properties derived from each of the original proteins. Recombinant fusion proteins are artificially produced using recombinant DNA technology and are used in biological research or therapy.

[0159] In some embodiments of the invention, the isolated protein is a recombinant fusion protein.

[0160] Chemical ligation is the process of chemically linking two or more protein molecules, such as chemically linking a first protein moiety to a second protein or polypeptide moiety. Chemical ligation enables the attachment of moieties (such as proteins, polypeptides, protein drugs, labels, and fluorescent molecules) to another protein molecule for transport to and targeting of cells and tissues, providing target-specific therapies and treatments, and / or improved in vivo diagnosis and prognosis, imaging, and aiding detection of one or more molecules.

[0161] In another embodiment of the invention, the first protein portion of the isolated protein is chemically linked to the second protein or polypeptide portion.

[0162] As a further aspect of the present invention, there is provided a combination of an isolated protein and a plurality of lipid microvesicles and / or nanodroplets as disclosed above, wherein the isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0163] In one embodiment, a kit is provided, comprising an isolated protein as disclosed above and a plurality of lipid microvesicles and / or nanodroplets, wherein the isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0164] Brain-penetrating molecules, recombinant proteins, and / or biologics are generally unsuccessful unless they cross the BBB. Therefore, BBB drug delivery is a limiting factor in the future development of novel brain therapeutics.

[0165] The inventors have devised a convenient method for effectively delivering compositions across tissues such as the BBB and into neurons in the CNS.

[0166] For these and other purposes, according to another aspect of the present invention, there is provided a method for transporting an isolated protein disclosed herein across the blood-brain barrier in a mammal (including a human) in need thereof, the method comprising the following steps:

[0167] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal; and

[0168] The isolated protein is administered to the mammal.

[0169] In certain embodiments, the method does not include any step of ultrasonicating any tissue of the mammal. According to yet another aspect, the present invention provides an isolated protein disclosed herein for use in a method of transporting the isolated protein across the blood-brain barrier in a mammal (including a human) in need thereof, the method comprising the steps of:

[0170] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal;

[0171] administering the isolated protein to the mammal;

[0172] The isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0173] In a certain embodiment, the method does not include any step of sonicating any tissue of the mammal.

[0174] The method does not include any step of treating any mammalian tissue with optical, audio or ultrasonic waves to cause cavitation of microbubbles and / or nanodroplets in the tissue.

[0175] According to another aspect, a method for transporting an isolated protein disclosed herein across the blood-brain barrier in a mammal (including a human) in need thereof comprises or consists of the following steps:

[0176] The isolated protein is administered to the mammal.

[0177] In certain embodiments, there is provided an isolated protein disclosed herein for use in a method of treatment involving transporting the isolated protein across the blood-brain barrier in a mammal (including a human) in need thereof, the method of treatment consisting of the following steps:

[0178] administering a plurality of lipid microbubbles and / or nanodroplets to the mammal; and

[0179] administering the isolated protein to the mammal;

[0180] The isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0181] It was unexpectedly found that after 2 hours of application together with lipid microvesicles and / or nanodroplets, rh Bri2 BRICHOS fused with short peptide tags or globular proteins was detected in brain parenchyma. The analysis of brain parenchyma carried out by immunohistochemistry and western blotting clearly shows that after applying rh Bri2 BRICHOS-AU1 and microvesicles and / or nanodroplets, the distribution of the protein delivered in two hemispheres is similar, wherein there is strong staining in cortex, hippocampus and choroid plexus. It was observed that the rh Bri2BRICHOS-AU1 delivered together with microvesicles and / or nanodroplets is internalized by cells, which is consistent with the result obtained from the rh Bri2BRICHOS-AU1 mediated by FUS (Galan-Acosta et al., Mol Cell Neurosci. [molecular and cellular neuroscience] 2020: 103498). Bri2 is not only expressed in CNS, but also expressed in peripheral tissues, and it is foreseeable that there is a transport system allowing crosstalk between two sites.

[0182] Consistent with the notion that drug transport into the cerebrospinal fluid (CSF) cannot be extrapolated to crossing the BBB, it can be noted that another BRICHOS protein, rh proSP-C BRICHOS, was detected in the CSF after intravenous injection in mice, but not in brain homogenates. This suggests that passage through the blood-CSF barrier does not guarantee transport across the BBB or into the brain parenchyma (Tambaro et al. J Biol Chem. 294: 2606-2615 (2019)). The permeability of the BBB varies between different brain regions, for example, in areas close to the choroid plexus and in the periventricular region, permeability is higher than in other areas of the brain.

[0183] Furthermore, quantitative analysis of the total rh Bri2 BRICHOS domain reaching the brain parenchyma by sandwich ELISA revealed that rh Bri2 BRICHOS-AU1 was abundant in both hemispheres. According to this analysis, 1% of the fully injected rh Bri2 BRICHOS domain administered with lipid microbubbles and / or nanodroplets was detected in the brain 2 hours after injection, which is 2-10 times the passage observed when rh Bri2 BRICHOS-AU1 was administered without microbubbles and / or nanodroplets (Tambaro et al., J Biol Chem. 2019;294(8):2606-15) or with microbubbles and / or nanodroplets plus FUS (Galan-Acosta et al., Mol Cell Neurosci. 2020:103498). For the rh Bri2 BRICHOS-mCherry fusion protein, the extent of the microvesicle- and / or nanodroplet-mediated increase in BBB crossing was not quantified, but the results suggest that the effect of the fusion protein was detectable in the brain only when the fusion protein was administered with microvesicles and / or nanodroplets.

[0184] In summary, the current data indicate that microbubbles and / or nanodroplets significantly increase rh Bri2 BRICHOS's passage across the BBB, even when fused to a large 30 kDa globular protein. Incorporation or association of drugs with lipid nanoparticles can increase bioavailability, but in the present invention, rh Bri2 BRICHOS protein and microbubbles and / or nanodroplets are administered independently, making it unlikely that rh Bri2 BRICHOS crosses the BBB along with the microbubbles and / or nanodroplets. Without wishing to be bound by any particular theory, the inventors suspect that the increased passage of rh Bri2 BRICHOS or rhBri2 BRICHOS-mCherry across the BBB in the presence of microbubbles and / or nanodroplets is mediated by an increased half-life of rh Bri2 BRICHOS in the circulation. Other means of increasing the half-life of rh Bri2 BRICHOS in the circulation would likely increase its ability to cross the BBB, and fusion of rh Bri2 BRICHOS to other proteins (including, for example, antibodies, enzymes, or neurotrophic factors) might increase their ability to enter the brain parenchyma, as functional mCherry has now been shown to be transported into the brain of wild-type mice.

[0185] The present findings indicate that in the presence of microbubbles and / or nanodroplets, rh Bri2 BRICHOS crosses the BBB into the brain parenchyma to a greater extent (1% of the injected dose) than that observed with peripherally administered antibodies (approximately 0.1% of the injected dose) (Bard et al. Nat Med. 2000;6(8):916-919 and Zuchero et al. 2016;89(1):70-82). Rh Bri2 BRICHOS BBB crossing is also significantly more effective than affibodies designed to improve CNS uptake, which were only detected in small amounts in the cerebrospinal fluid after injection (Meister et al. Int J Mol Sci. 2020;21(8)).

[0186] As described above, proteins comprising isolated recombinant protein Bri2 BRICHOS and variants thereof (including Bri2BRICHOSR221E) can be effectively delivered across the blood-brain barrier to CNS neurons. The method does not require any steps for administering lipid microbubbles and / or nanodroplets, and the method does not require any steps for treating any tissue of a mammal with light waves, audio waves, or ultrasound. Therefore, even in the absence of lipid microbubbles and nanodroplets, isolated Bri2 BRICHOS proteins coupled to a second protein or polypeptide portion can be effectively delivered to the brain.

[0187] We demonstrate that cargo proteins fused to rh Bri2 BRICHOS can cross a human BBB model constructed from brain microvascular endothelial cells and astrocytes as efficiently as proteins known to cross the BBB. In contrast, 4 kDa dextran or the fusion partner alone do not cross, and rh Bri2 BRICHOS does not cause nonspecific leakage or cytotoxicity. Intravenously injected fusion proteins containing Bri2 BRICHOS cross the mouse BBB and colocalize with early endosomes in brain and cerebellar neurons. Our results demonstrate that fusions with rh Bri2 BRICHOS can deliver multimodal biopharmaceuticals for CNS disorders.

[0188] As demonstrated herein, an isolated protein comprising a first protein portion, which is a Bri2 BRICHOS protein or a variant thereof, and at least one additional protein or polypeptide portion, can be used in a method of transporting the isolated protein across the blood-brain barrier in a mammal, including a human, in need thereof, the method comprising or consisting of the following steps:

[0189] - administering said isolated protein to said mammal.

[0190] Furthermore, an isolated protein comprising a first protein portion which is a Bri2 BRICHOS protein or a variant thereof and at least one additional protein or polypeptide portion may be used in a method of treating a medical condition in a mammal (including a human) in need thereof, the method comprising or consisting of the following steps:

[0191] - administering said isolated protein to said mammal.

[0192] Therefore, the isolated protein can be used as a medicine. The isolated protein can be used to treat a method of a medical condition in a mammal (including a human) in need thereof, the method comprising or consisting of the following steps:

[0193] - administering said isolated protein to said mammal.

[0194] In these methods, an isolated protein comprising a first protein portion (which is a Bri2 BRICHOS protein or a variant thereof) and at least one additional protein or polypeptide portion can be effectively delivered to the brain even in the absence of lipid microbubbles and nanodroplets.

[0195] It is also contemplated that the isolated protein according to the present invention can be administered by gene therapy, such as by transfecting cells in the nervous system (preferably the brain) using an expression vector, plasmid, or virus so that the isolated protein is expressed by these cells in the central nervous system. This can be used to treat Alzheimer's disease. This may also be helpful in treating other neurological diseases.

[0196] The isolated Bri2 BRICHOS protein can also be effectively delivered to the brain and could be used to treat Parkinson's disease.

[0197] Aggregation of the α-synuclein (α-syn) protein is associated with several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy, characterized by the abnormal accumulation of misfolded α-syn in intracellular inclusions called Lewy bodies in neurons. The specific molecular mechanisms underlying disease onset remain unknown, but there is strong evidence that α-syn aggregation into amyloid fibrils plays a crucial role, as mutations in the gene encoding α-syn are known to cause earlier onset or more aggressive forms of PD. Significant efforts have been made to inhibit amyloid formation in PD, but no effective treatment has been developed to date. In particular, methods to inhibit toxic aggregation pathways and species are needed, as the generation of intermediate oligomeric α-syn assemblies has been proposed to contribute to α-syn-associated neurotoxic effects.

[0198] Without wishing to be bound by any particular mechanism, we demonstrate here that BRICHOS effectively delays α-synuclein self-assembly by binding to the fibril surface, thereby specifically inhibiting fibril end elongation, secondary nucleation events, and associated oligomer formation. Our results further demonstrate that BRICHOS directly binds to oligomeric species and mitigates the toxic effects associated with α-synuclein.

[0199] We demonstrated that the BRICHOS domain can prevent α-syn aggregation in a concentration-dependent manner by affecting the secondary nucleation and elongation of WT α-syn. Interestingly, BRICHOS has been reported to have a similar inhibitory mechanism on Aβ42 aggregation (Chen et al., Nat Commun, 8(1):2081, 2017; Chen et al., Communications biology, 3(1):1-12, 2020). Previous studies have demonstrated that BRICHOS binds to Aβ42 fibers along the fibril surface ( et al., Sci Rep [Science Reports], 10(1):21765, 2020). One hypothesized mechanism is that BRICHOS competes with α-syn monomers while binding to secondary nucleation sites along the fibril surface, as shown for Aβ42.

[0200] We demonstrate for the first time the interaction between BRICHOS and α-syn oligomeric species. By binding to α-syn oligomers and fibrils, BRICHOS not only interferes with secondary nucleation but also binds to products commonly associated with toxicity in vitro. This binding ability is crucial for reducing oligomer-induced toxicity.

[0201] While oligomers are the most toxic species, sonicated α-syn fibrils also display toxicity in various in vitro and in vivo models. Here, we demonstrate that BRICHOS protects against neurotoxicity associated with sonicated α-syn fibrils in mouse hippocampal slices. This is explained by the fact that BRICHOS binds to α-syn fibrils and inhibits oligomer formation through secondary nucleation with endogenous α-syn. BRICHOS also interferes with the release of oligomeric species from the ends of existing α-syn fibrils, which exhibit toxic behavior in neuronal cells.

[0202] Based on relevant in vitro models, BRICHOS may be used as a therapeutic candidate for α-synucleinopathies, including Parkinson's disease.

[0203] The Bri2 BRICHOS protein can also be coupled to a second protein or polypeptide (such as glucocerebrosidase, progranulin, prosaposin, cathepsin D, and antibodies) that can be effective in treating Parkinson's disease. Without wishing to be bound by any particular theory, it is believed that the combined action of Bri2 BRICHOS and the second protein / polypeptide will play an important role in the treatment of Parkinson's disease. It is also expected that the high efficiency of this agent will require its less frequent administration.

[0204] Thus, there is provided a method of treating an alpha-synucleinopathy (including Parkinson's disease) in a mammal, including a human, in need thereof, comprising or consisting of the following steps:

[0205] - administering to said mammal an isolated protein as defined herein.

[0206] In addition, a method for delaying the accumulation of alpha-synuclein in a mammal in need thereof, including a human, is provided, the method comprising or consisting of the following steps:

[0207] - administering to said mammal an isolated protein as defined herein.

[0208] The invention will now be further illustrated by the following non-limiting examples.

[0209] Example

[0210] These examples demonstrate that microvesicles can be used to enhance the delivery of Bri2 BRICHOS and their fusion proteins across the BBB and promote the treatment of AD and other neurological diseases.

[0211] Recombinant protein expression and purification

[0212] The Rh Bri2 BRICHOS domain (which corresponds to residues 113-231 of the full-length Bri2 protein) is connected to the AU1 tag or mCherry protein at the C-terminus. Bri2 BRICHOS-AU1 (SEQ ID NO: 11) and Bri2BRICHOS-mCherry (SEQ ID NO: 12) containing BRICHOS proteins were expressed and purified in Escherichia coli. Before being injected into mice, rhBri2 BRICHOS-AU1 was dialyzed against filtered and autoclaved phosphate buffered saline (PBS) (pH 7.4) (6-8 kDa membrane, Spectrum lab). Endotoxins were eliminated by passing the protein through a Pierce High-Capacity endotoxin removal column (Thermo Scientific). The final protein preparation was filtered through a 0.22 μm Millex-GV filter (Millipore Ltd.) and stored at -20 ° C and thawed a few minutes before injection.

[0213] As a control, the proSP-C BRICHOS domain (proSP-C residues 59-197) was generated as described in: Galan-Acosta et al., Mol Cell Neurosci. 2020: 103498.

[0214] animal

[0215] Female and male C57BL / 6J mice, 4–6 months old, weighing 24–30 g, were used. All animals were maintained on a 12-h light–dark cycle and grouped in cages of 5 mice with free access to food and water. All experiments were conducted in accordance with the Animal Experimentation Ethics Committee of the Stockholm South ( Stockholms )(dnr 03049), Linköping Ethics Committee ( Etiska ) (ID855) or were approved and performed in accordance with the guidelines of the Columbia University Institutional Animal Care and Use Committee.

[0216] Experimental design

[0217] Figure 3The study design in the Examples is shown. (A) Mice received two intravenous injections into the tail vein of microvesicles and 1, 10, or 20 mg / kg rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11), or PBS for control. (B) Mice received 20 mg / kg of (i) rh Bri2 BRICHOS-mCherry (SEQ ID NO: 12) and microvesicles, or (ii) rh Bri2 BRICHOS-mCherry alone.

[0218] Two different types of commercially available microbubbles (Lantheus Medical Imaging, Massachusetts, USA) or (Bracco, Milan, Italy) was administered at a dose of 5 μl / g body weight. Figure 3 A), a total of 14 mice were divided into four groups and received intravenous injections of microbubbles and PBS (n=4), microbubbles and 1 (n=4), 10 (n=6), or 20 (n=4) mg / kg body weight of rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11). In the second part of the study ( Figure 3 In B), a total of three mice were intravenously administered 20 mg / kg rhBri2 BRICHOS-mCherry (SEQ ID NO: 12) (n=1) or microbubbles + 20 mg / kg rh Bri2 BRICHOS-mCherry (n=2). Two mice received 10 mg / kg rh Bri2 BRICHOS-AU1 injections. Microbubbles, the remaining mice were administered microbubbles.

[0219] All animals received two intravenous injections in the lateral tail vein using a 29-gauge needle. Prior to administration, the microbubble vial was activated by mechanical stirring for 20 seconds, and the microbubble solution was injected into the tail vein over 60 seconds, followed immediately by a slow injection of rhBri2 BRICHOS protein or PBS control. Before injection, mice were anesthetized with 2%-4% isoflurane (with 2% oxygen) and placed under a heat lamp to dilate the tail vein. Two hours after injection, mice were anesthetized and perfused transcardially with 120 ml of PBS, and one was administered Mice treated with 10 mg / kg rh Bri2BRICHOS-AU1 were perfused with PBS for 5 minutes followed by 4% paraformaldehyde (PFA) for 7 minutes. Brains were extracted from the skull and then snap-frozen in dry ice and stored at -80°C or post-fixed in 4% PFA for 48 hours before embedding in paraffin.

[0220] Antibody

[0221] For immunohistochemistry (IHC), a polyclonal rabbit anti-AU1 (Abcam, catalog number ab3401) primary antibody was used at a dilution of 1:200, and a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (GE Healthcare, catalog number NA934) was diluted to 1:2000. For Western blotting, the primary antibody was a polyclonal rabbit anti-AU1 (Abcam, catalog number ab3401) at a dilution of 1:600, and a fluorescent-labeled secondary anti-rabbit (Li-Cor, catalog number 926-32213) antibody was used at a dilution of 1:10,000. For sandwich ELISA, a goat anti-Bri2 BRICHOS antibody was used for capture at a dilution of 1:250, and a polyclonal rabbit anti-AU1 (Abcam, catalog number ab3401) antibody was used for detection at a dilution of 1:2000.

[0222] A rabbit anti-proSP-C antibody was used as previously reported in Galan-Acosta et al., Mol Cell Neurosci. 2020: 103498.

[0223] Immunohistochemistry analysis (IHC)

[0224] 5 μm thick coronal sections of paraffin-embedded tissue were placed on Superfrost Plus microscope slides (Thermo Scientific) and dried overnight at room temperature (RT) to remove residual water. The sections were deparaffinized by washing in xylene and rehydrated in gradually decreasing ethanol concentrations (from 99% to 70%). The sections were immersed in DIVA decloaker 1X solution (Concord, USA) in a decloaking chamber (Biocare Medical), pressure boiled at 110°C for 30 minutes, or incubated in a water bath heated at 95°C for 30 minutes. The slides were cooled at room temperature (RT) for 20 minutes, then washed with PBS buffer (PBST) containing 0.1% tween 20, and incubated for 5 minutes with peroxidase blocking solution (Dako). The sections were washed with Tris-buffered saline (TBS) and blocked with background penalizer (Biocare) for an additional 10 minutes. Primary antibodies diluted in DAKO (Agilent) antibody diluent were incubated at room temperature (RT) for 45 minutes. The slides were then washed in TBS and incubated with Mach 2 Double stain 2 containing an alkaline phosphatase (AP)-conjugated anti-rabbit secondary antibody for 30 minutes at room temperature (RT). AP staining was detected with permanent red (Biosite). The sections were counterstained with hematoxylin (Mayer), dehydrated with ethanol (from 70% to 99%), clarified in xylene, and mounted with DEPEX mounting medium (Merck).

[0225] Microscopic examination

[0226] The stained sections were visualized using a Nikon Eclipse E800M light microscope with Plan-Apochromate objectives at 10x and 20x magnifications. The rhBri2BRICHOS-mCherry protein in the brain samples was detected using a Nikon fluorescence microscope and recorded using a 20x objective.

[0227] Western blot

[0228] Brain tissue from one mouse treated with rh Bri2 BRICHOS-AU1+ microvesicles and a negative control was homogenized in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v / v) Triton X-100, 0.1% (w / v) SDS, and 10 mM EDTA supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN). The homogenate was centrifuged at 14,000 rpm (20,800 x g) for 30 min at 4°C, and the supernatant was collected and stored at -20°C. Protein concentration was determined by the BCA assay. The homogenate was diluted in homogenization buffer and 1x SDS reducing buffer (containing 2-mercaptoethanol) so that 100 μg of total protein was loaded per sample and well. The samples were heated at 97°C for 10 minutes and separated on 4%-20% precast polyacrylamide gels (Bio-Rad) and blotted on nitrocellulose (General Health Medical) membranes. After blotting, the membranes were blocked with 5% skim milk prepared in 0.1% Tween / TBS for 1 hour at room temperature (RT). Thereafter, the membranes were rinsed with 0.1% Tween / TBS and the primary antibody diluted in 0.1% Tween / TBS was added overnight at 4°C. The membranes were washed three times with 0.1% Tween / TBS and then incubated with the secondary antibody prepared in 0.1% Tween / TBS for 1 hour at room temperature (RT). After washing away the unbound secondary antibody with 0.1% Tween / TBS, images were acquired using a fluorescence imaging system (Ricoll, OdysseyCLx).

[0229] Sandwich ELISA

[0230] 96-well plates (Nunc MicroWell™) were coated with anti-Bri2 BRICHOS capture antibody diluted in coating buffer (50 mM carbonate pH 9.6) and incubated overnight at 4°C. After washing three times in 0.05% Tween / PBS, the plates were blocked with 1% BSA / PBS for 1 hour. After the washing and blocking steps, brain samples (250 μg / ml) and standards diluted in 0.05% Tween / PBS were incubated for 2 hours at room temperature (RT). The plates were washed and rabbit anti-AU1 primary antibody diluted in 0.05% Tween / PBS was added overnight at 4°C. The plates were washed three times and anti-rabbit secondary antibody diluted in 0.05% Tween / PBS was added for 2 hours at room temperature (RT). After washing, tetramethylbenzidine (TMB) (Thermo Fisher) solution was added and incubated in the dark for 30 minutes. The reaction was stopped by adding a stop solution of TMB substrate (Thermo Fisher Scientific), and the absorbance was measured at 450 nm using the value of brain homogenate from untreated mice as a blank.A standard curve was obtained from 0.1 to 64 ng of rh Bri2 BRICHOS-AU1 protein.

[0231] The concentration and total amount of rhBri2 BRICHOS-AU1 per brain hemisphere were estimated by assuming a brain density of 1.04 mg / ml, an average brain hemisphere weight of 225 mg, and an average total protein value of 25.6 mg in brain hemisphere homogenates.

[0232] result

[0233] In the presence of lipid microbubbles, rh Bri2 BRICHOS but not proSP-C BRICHOS cross the BBB in the non-sonicated hemisphere

[0234] The presence of rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11) in the brain parenchyma was investigated. Figure 4The results show that rh Bri2 BRICHOS, but not rh proSP-C BRICHOS, were detected in the non-FUS-targeted hemisphere of the brain following intravenous injection with lipid microbubbles. Immunohistochemical analysis was performed on cortical and hippocampal sections from the contralateral non-FUS-targeted hemisphere (AH) of mice treated with rh proSP-C BRICHOS + microbubbles (AB, EF, IJ) or rh Bri2 BRICHOS-AU1 + microbubbles (CD, GH). (IJ) Controls show no rh Bri2 BRICHOS-AU1 staining in either the contralateral or ipsilateral sides of mice treated with intravenous proSP-C BRICHOS + microbubbles and FUS. Tissues were stained using rabbit anti-proSP-C antibodies (AB, EF) or rabbit anti-AU1 antibodies (CD, GH), followed by AP-conjugated secondary antibodies and visualization with permanent red AP solution. All samples were counterstained with hematoxylin. The scale bar in AH is 100 μm and the scale bar in IJ is 200 μm.

[0235] Thus, 2 hours after intravenous administration of 10 mg / kg Rh Bri2 BRICHOS-AU1 to wild-type mice in the presence of microbubbles, Rh Bri2 BRICHOS-AU1 was found in the cortex and hippocampus of the contralateral hemisphere, which is not FUS-targeted. Figure 4 C- Figure 4 D, Figure 4 G- Figure 4 H). Two hours after intravenous injection of 10 mg / kg rh proSP-C BRICHOS and microbubbles in FUS-treated wild-type mice, no Rh proSP-C BRICHOS were observed in the non-sonicated hemisphere ( Figure 4 A- Figure 4 B. Figure 4 E- Figure 4 F). Immunohistochemical staining of rh Bri2BRICHOS-AU1 in brain sections of mice treated with FUS+microbubbles and administered 10 mg / kg rh proSP-C BRICHOS 2 hours after injection was negative, indicating that the immunoreactivity with anti-AU1 antibody was not an artifact caused by any recombinant protein injection ( Figure 4 I- Figure 4 J).

[0236] Microbubbles increase BBB permeability of rh Bri2 BRICHOS in wild-type mice

[0237] The effect of microbubbles alone (i.e., without any FUS application) on the permeability of rh Bri2 BRICHOS across the BBB was further investigated by administering the rh Bri2 BRICHOS domain with either an AU1 tag (rh Bri2 BRICHOS-AU1; SEQ ID NO: 11) or mCherry protein (rh Bri2 BRICHOS-mCherry; SEQ ID NO: 12) genetically linked to the C-terminus. Microbubbles (5 μl / g body weight) and different doses of rh Bri2 BRICHOS-AU1 were injected into the lateral tail vein of adult wild-type mice, and brain perfusion was performed 2 hours later and collected for analysis. The presence of rh Bri2 BRICHOS-AU1 in the brain parenchyma was investigated by immunohistochemistry and Western blotting, while the amount of rh Bri2 BRICHOS-AU1 was assessed by sandwich ELISA ( Figure 3 A).

[0238] To investigate the effect of microbubbles on the BBB permeability of rh Bri2 BRICHOS fused to a folded globular protein, rh Bri2 BRICHOS-mCherry (total molecular weight 44 kDa) was chosen because it can be detected by the fluorescent properties of folded mCherry (which also remains unchanged when fused to rh Bri2 BRICHOS). and 20 mg / kg rh Bri2 BRICHOS-mCherry (SEQ ID NO: 12) were intravenously injected into two wild-type mice, while control mice received only rh Bri2 BRICHOS-mCherry ( Figure 3 B) Brains were collected 2 hours after injection and analyzed macroscopically and under a fluorescence microscope.

[0239] The inventors unexpectedly discovered that microbubbles alone increase the rate at which rh Bri2 BRICHOS-AU1 crosses the BBB.

[0240] Figure 5Shown are images of Bri2 BRICHOS detected in both hemispheres following intravenous injection with lipid microbubbles. (AG) Images of the cortex, hippocampus, and lateral ventricle from mice treated with microbubbles plus 10 mg / kg rh Bri2 BRICHOS-AU1. (HJ) Images from mice injected with 10 mg / kg rh proSP-C BRICHOS and microbubbles. (KM) Images from control mice that did not receive injections. All sections were first incubated with anti-AU1 antibodies, then with AP-conjugated anti-rabbit antibodies, developed with permanent red AP solution, and counterstained with hematoxylin. Scale bars in panels A and B represent 500 μm and 200 μm, respectively.

[0241] After intravenous injection of microbubbles and rh Bri2 BRICHOS-AU1 at a dose of 10 mg / kg, Rh Bri2 BRICHOS-AU1 was detected in the cortex, hippocampus, and choroid plexus of the lateral ventricles by immunohistochemistry using an antibody against the AU1 tag. Figure 5 A- Figure 5 G). No difference in staining intensity was observed between the two hemispheres ( Figure 5 A- Figure 5 G).

[0242] Figure 6 Figure 1 shows that intracellular immunostaining of rh Bri2 BRICHOS-AU1 was observed in the cortex and hippocampus following intravenous injection with lipid microbubbles. Images show the cortex (A, B) and hippocampus (C, D) of the right hemisphere of mice treated with microbubbles + 10 mg / kg rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11). Sections were stained for rh Bri2 BRICHOS-AU1 using an anti-AU1 antibody and counterstained with hematoxylin. Arrows in panels (B) and (D) indicate intracellular staining of rh Bri2 BRICHOS-AU1. Scale bars for panels A and C are 100 μm; and scale bars for panels B and D are 50 μm.

[0243] Thus, intracellular staining was observed in cells in the cortex and in the hippocampus ( Figure 6 A- Figure 6 D) Control brains from mice injected with rhproSP-C BRICHOS or PBS were stained with anti-AU1 antibody in the same manner but showed no immunoreactivity ( Figure 5 H- Figure 5 M).

[0244] Furthermore, Western blot analysis of homogenates from both hemispheres of mice administered microvesicles plus 10 mg / kg rh Bri2 BRICHOS-AU1 using both anti-AU1 and anti-Bri2 antibodies revealed bands migrating as rh Bri2 BRICHOS-AU1 and slightly slower, whereas the corresponding bands were absent in control samples. Figure 7 Western blot detection of rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11) in both brain hemispheres following systemic injection with lipid microbubbles is shown. Western blot analysis was performed on brain homogenates collected from the left (L) and right (R) hemispheres of one mouse two hours after intravenous injection of microbubbles + rh Bri2 BRICHOS-AU1, as well as from untreated control mice (negative control). Rh Bri2 BRICHOS-AU1 was detected using anti-AU1 antibodies (A) and anti-Bri2 antibodies (B), as indicated by the arrows on the right side of the gel. The lane labeled Rec protein contains 5 ng of rhBri2 BRICHOS-AU1.

[0245] Consistent with the immunohistochemistry and Western blot results, analysis of homogenates from both hemispheres of mice treated with microbubbles plus 10 mg / kg rh Bri2 BRICHOS-AU1 by sandwich ELISA 2 hours after injection showed rhBri2 BRICHOS-AU1 concentrations of 390 nM in the left hemisphere and 250 nM in the right hemisphere, which together corresponded to 1% of the total administered amount. Notably, a higher amount of isolated recombinant Bri2 BRICHOS-AU1 reached the brain parenchyma when lipid microbubbles were administered without sonication compared to when no microbubbles were administered. Two hours after intravenous injection, approximately 1% of the total isolated recombinant Bri2 BRICHOS-AU1 administered at a 10 mg / kg dose with lipid microbubbles was detected in the brain, while 0.1% to 1% (average 0.5%) was detected after administration of isolated recombinant Bri2 BRICHOS-AU1 at a 20 mg / kg dose without microbubbles. This was unexpected, as microbubbles alone (without sonication) have not been described to increase protein passage across the BBB or affect neuronal uptake.

[0246] The inventors also found that rh Bri2 BRICHOS-mCherry penetrated into the brain parenchyma of wild-type mice when lipid microbubbles were injected, but not when lipid microbubbles were not injected. The BBB permeability of rh Bri2 BRICHOS-mCherry in wild-type mice injected with or without microbubbles was assessed by macroscopic examination of the brain and observation of brain sections under a fluorescence microscope.

[0247] Figure 8 Figure 1 shows the finding of Rh Bri2 BRICHOS-mCherry (SEQ ID NO: 12) in the striatum following systemic injection in combination with lipid microbubbles. (AB) Photographs of mouse brains following injection of rh Bri2 BRICHOS-mCherry alone (A) or in combination with microbubbles (B). (CH) Fluorescence of striatal regions collected 2 hours after injection of (C, F) 20 mg / kg rh Bri2 BRICHOS-mCherry alone and (DE, GH) microbubbles and 20 mg / kg rh Bri2 BRICHOS-mCherry. Scale bars in panels C-E are 200 μm, and in panels F-H are 100 μm. The areas marked with white rectangles in panels C, D, and E are magnified in panels F, G, and H, respectively.

[0248] Brains of fully perfused mice injected with either rh Bri2 BRICHOS-mCherry alone (one mouse) or microbubbles and rh Bri2 BRICHOS-mCherry (two mice) were collected 2 hours after injection, showing clear coloration in the outer layers of the cortex and cerebellum only in mice with rh Bri2 BRICHOS-mCherry plus microbubbles ( Figure 8 White in ( Figure 8 A, Figure 8 B). In addition, fluorescence was detected in the striatum region of paraffin-embedded sections of two mice injected with microbubbles and rh Bri2 BRICHOS-mCherry ( Figure 8 White in ( Figure 8 D. Figure 8 E. Figure 8 G. Figure 8 H), but not in mice injected with rh Bri2BRICHOS-mCherry only ( Figure 8 C. Figure 8 F). Data strongly suggest that intravenously injected rh Bri2 BRICHOS-mCherry can penetrate into the brain parenchyma when microbubbles are injected into the circulation.

[0249] Further experiments with Bri2 BRICHOS

[0250] method

[0251] Cell culture

[0252] hCMEC / D3 cells (MilliporeSigma, Burlington, MA) were cultured on a plate coated with 5-10 μg / cm2 The cells were cultured in T75 tissue culture flasks (TC-coated, DeltaLab, Spain) on rat tail collagen type I (Millipore Sigma, Burlington, MA) supplemented with 1 ng / ml recombinant human basic fibroblast growth factor (FGF-2, Gibco, Invitrogen, Waltham, MA) and penicillin-streptomycin (100 IU / ml, 100 μg / ml, Gibco, Thermo Fisher Scientific, Waltham, MA) in EndoGRO-MV complete medium (Millipore Sigma, Burlington, MA). The human brain microvascular endothelial cell line hCMEC / D3 was used as an in vitro model of the human blood-brain barrier (BBB) ​​that produces an accurate in vivo phenotype that is reproducible and easy to maintain. The hCMEC / D3 cell line retains expression of most transporters and receptors intrinsic to the BBB and is therefore effectively used to study the dynamics and mechanisms of transport across the BBB. The hCMEC / D3 cell line has been previously described for transport of Aβ40 peptide, transthyretin, and several drug candidates across the BBB.

[0253] Human neuroblastoma (SH-SY5Y) cell line (ATCC, Manassas, Virginia) was grown in Dulbecco's modified Eagle's medium (DMEM, PAN-Biotech, Germany) supplemented with 4.5 g / L glucose, glutamine (2 mM) and 3.7 g / L NaHCO in T75 tissue culture flasks coated with TC (PAN-Biotech, Germany) (supplemented with 10% fetal bovine serum (FBS, Sera Plus, PAN-Biotech, Germany) and penicillin-streptomycin (100 IU / ml, 100 μg / ml). The human neuroblastoma cell line SH-SY5Y is a three-fold subclone cell line derived from the SK-N-SH neuroblastoma cell line. It is widely used as an in vitro neuronal model for studying neurodegenerative diseases.

[0254] Immortalized human astrocyte cell line (Innoprot, Spain) was coated with 2 μg / cm 2Cells were grown in astrocyte medium (Innoprot, Spain) containing 2% FBS, 1% astrocyte growth supplement, and 1% penicillin-streptomycin (100 IU / ml, 100 μg / ml) in T75 tissue culture flasks coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO). Human embryonic kidney 293 (HEK-293) cell line (ATCC, Manassas, VA) was grown in Eagle's Minimum Essential Medium (EMEM, ATCC, Manassas, VA) supplemented with 10% FBS and penicillin-streptomycin (100 IU / ml, 100 μg / ml) in T75 tissue culture flasks coated with TC. Cultures were maintained in a humidified atmosphere (5% CO2 / 95% air) at 37°C, and the culture medium was changed every other day. For hCMEC / D3, human astrocytes, and SH-SY5Y cell lines, cells were trypsinized (0.025% trypsin, 0.01% EDTA, Gibraltar, Thermo Fisher Scientific, Waltham, MA) at approximately 80% confluence and passaged up to passage 35.

[0255] Human embryonic kidney 293 (HEK-293) cell line (ATCC, Manassas, VA) was grown in Eagle's Minimum Essential Medium (EMEM, ATCC, Manassas, VA) supplemented with 10% FBS and penicillin-streptomycin (100 IU / ml, 100 μg / ml) containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g / L NaHCO in T75 tissue culture flasks coated with TC.

[0256] Cultures were maintained in a humidified atmosphere (5% CO2 / 95% air) at 37°C, and the medium was changed every other day. For hCMEC / D3 and SH-SY5Y cell lines, cells were trypsinized (0.025% trypsin, 0.01% EDTA, Gibraltar, Thermo Fisher Scientific, Waltham, MA) at approximately 80% confluence and passaged up to passage 35.

[0257] hCMEC / D3 cell monolayer preparation

[0258] To prepare hCMEC / D3 cell monolayers, transwell polyethylene terephthalate (PET) membrane inserts (24-well, clear, 0.4 μm pore size, CellQART, SABEU GmbH & Co. KG, Germany) were coated with rat tail type I collagen (150 μg / ml in 1× Dulbecco's phosphate-buffered saline, pH 7.4, 1× DPBS, Gibraltar, Thermo Fisher Scientific, Waltham, MA) for 2 h at 37°C. Excess collagen was removed, and the inserts were washed twice with 1× DPBS. hCMEC / D3 cells were plated at 50,000 cells / cm 2 Cells were seeded at a density of 100 μg / mL onto freshly prepared collagen-coated inserts and maintained in a humidified atmosphere (5% CO 2 / 95% air) at 37° C. The culture medium was changed every other day, and a monolayer of cells was established on day 6.

[0259] Analysis of hCMEC / D3 monolayer integrity

[0260] The integrity of monolayer cells was analyzed by crystal violet staining. The expression of tight junction proteins occluded zonula-1 (ZO-1), junction adhesion molecule A (JAM-A) and sealing protein-5 was confirmed by Western blotting and immunostaining. For crystal violet staining, cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, St. Louis, Missouri) for 2 min at room temperature, and permeabilized with 100% methanol (Sigma-Aldrich, St. Louis, Missouri) for 20 min at room temperature, then stained with 0.01% crystal violet (Sigma-Aldrich, St. Louis, Missouri) in 2% ethanol, and analyzed using a fluorescence microscope (Zeiss Axioskop 40, Germany). Images were captured using a fluorescence microscope using green (FITC / 488) and red (TexasRed / 570) channels.

[0261] For the determination of tight junction proteins, hCMEC / D3 cells were plated at 50,000 cells / cm 2Cells were plated at a seeding density of 1 / 400 μg / well onto collagen-coated 12-well plates (TC-coated, Cellstar, Greiner Bio-One, Austria) and maintained at 37°C in a humidified atmosphere (5% CO2 / 95% air) with a culture medium change every other day. After 6 days, cells were removed from the plates by scraping in ice-cold 1xPBS, centrifuged at 14,000 × g for 10 min at 4°C, lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Thermo Scientific, Waltham, MA) and 1× complete protease inhibitor (Roche, Switzerland), and then sonicated for 10 s at 50% amplitude and incubated on ice for 20 min. Cell lysates were clarified by centrifugation at 14,000 × g for 15 min at 4°C, and the total amount of protein in the cell lysates was determined by bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. For Western blotting (see below), anti-clauin-5, anti-JAM-A, and anti-ZO-1 polyclonal rabbit primary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA) were used at a dilution of 1:1000.

[0262] Preparation of hCMEC / D3 and human astrocyte co-culture

[0263] For the direct co-culture model, first coat the PET membrane on the top side of the cell culture insert with rat tail type I collagen (see hCMEC / D3 monolayer preparation). Flip the insert and incubate at 37°C with poly-L-lysine (2 μg / cm 2 The basolateral side of the cell culture inserts was coated overnight in ddH2O. Excess poly-L-lysine was removed and the membrane was washed twice with ddH2O. Human astrocytes were plated at 20,000 cells / cm 2 hCMEC / D3 cells were seeded at a density of 50,000 cells / cm on the basolateral side of the cell culture inserts and placed in an inverted orientation at 37°C for 4 h to allow attachment. The inserts were then placed in a normal orientation and grown in astrocyte culture medium for 48 h. 2 hCMEC / D3 cells were grown in EndoGro complete medium in the apical compartment, and human astrocytes were grown in astrocyte medium in the basolateral compartment.

[0264] For the indirect co-culture model, 24-well plates were coated with poly-L-lysine similar to the cell culture inserts. Human astrocytes were cultured at 20,000 cells / cm 2Cell culture inserts were seeded at a density of 100 μg / mL and incubated for 48 hours. The top side of the cell culture inserts was coated with rat tail type I collagen and seeded with hCMEC / D3 cells in a manner similar to the direct co-culture model. The hCMEC / D3-coated inserts were incubated overnight at 37°C in EndoGro complete medium and then inserted into 24-well plates containing human astrocytes in astrocyte culture medium.

[0265] The culture medium was changed every other day, and FITC-dextran permeability and TEER values ​​were measured on days 3–9 after seeding, similar to the hCMEC / D3 monolayer cell model.

[0266] Analysis of hCMEC / D3 cell monolayer permeability

[0267] On day 6 of culture, experiments were performed by adding different BRICHOS protein constructs or relevant controls to the apical side of hCMEC / D3 monolayers. The BRICHOS constructs used were rh proSP-CBRICHOS wt, rh proSP-C BRICHOST187R, rh Bri2 BRICHOS wt monomers and oligomers, rh Bri2 BRICHOS R221E monomers, S-tag-Bri2 BRICHOS monomers and oligomers, mCherry-Bri2 BRICHOS monomers, NT-Bri2 BRICHOS monomers and oligomers, and unresolved mixtures of different S-tag-Bri3 BRICHOS assembly states. Controls used were mCherry, NT*-tag, rhAβ42, and apolipoprotein AI (purified from >98% pure human plasma; Chemicon, Sigma-Aldrich, St. Louis, MO). The concentrations used were 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, or 0.05 μM; the incubation time was 2 h or 24 h. The monolayers were incubated at 37°C in a humidified atmosphere (5% CO2 / 95% air). After incubation, the culture medium on the apical and basal sides of the monolayers was removed and analyzed by Western blot and band intensity was measured by ImageJ. For Western blotting (see below), anti-Bri2 BRICHOS primary antibody (raised in goat and purified in-house) was diluted 1:1000, anti-proSP-C BRICHOS primary antibody (raised in goat and purified in-house) was diluted 1:2000, anti-S-tag primary antibody (S-tag protein HRP conjugate Novagen, Merck, Germany) was diluted 1:5000, anti-NT primary antibody (raised in rabbit and purified in-house) was diluted 1:5000, anti-RFP primary antibody (raised in mouse, Thermo Fisher Scientific, Waltham, USA) was diluted 1:1000, and anti-human apolipoprotein AI primary antibody (raised in goat, Calbiochem, San Diego, CA) was diluted 1:1000. For mCherry and mCherry-Bri2 BRICHOS monomers, the λ ex 570nm and λ em The fluorescence intensity of the apical and basolateral culture media was recorded at 610 nm, and the cells were then fixed and analyzed by fluorescence microscopy to analyze the uptake of mCherry or mCherry-Bri2 BRICHOS into the cells.

[0268] The permeability of the monolayers was also analyzed for fluorescein isothiocyanate dextran (FITC-dextran, Thermo Fisher Scientific, Waltham, MA) as a negative control and for human recombinant insulin (Sigma-Aldrich, St. Louis, MO) as a positive control. To determine the permeability of FITC-dextran, insulin, and propidium iodide, 1 μM solutions in cell culture medium were applied to the apical side of the monolayers and incubated at 37°C in a humidified atmosphere (5% CO2 / 95% air) for 24 h, after which the culture medium was removed from both the apical and basolateral sides. ex 492nm and λ em FITC fluorescence was measured at 518 nm. Insulin permeability was determined by Western blotting with a 1:1000 dilution of anti-insulin monoclonal primary antibody (Sigma, St. Louis, MO).

[0269] Cell viability

[0270] Cell viability was assessed by the tetrazolium-based thiazolyl blue tetrazolium bromide (MTT) assay. For the MTT assay, 50,000 cells / cm 2 Cells were seeded into clear 96-well plates (TC, Cellstar, Greiner Biotech, Austria) and grown to confluence, and then treated with rh BRICHOS domains (8 replicates) for 24 h in a humidified atmosphere (5% CO2 / 95% air) at 37°C. 2 The cells were incubated in a 0.5 mg / ml MTT (Sigma-Aldrich, St. Louis, MO) solution in FBS-free cell culture medium for 4 h. The MTT solution was removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) and incubated for 15 min with shaking. The absorbance at 570 nm was measured using a FLUOstar OPTIMA microplate reader (BMG Lab Tech, Germany). Untreated cells were used as a positive control, and culture medium without cells was used as a negative control. 2 μM staurosporine from Streptomyces sp. (SP, Sigma-Aldrich, St. Louis, MO) was also used as a negative control.

[0271] Cellular uptake

[0272] For cellular uptake analysis, hCMEC / D3 cells were seeded onto collagen-coated 12-well plates, and undifferentiated SH-SY5Y cells were seeded onto collagen-untreated 12-well plates at a seeding density of 75,000 cells / cm 2, and maintained at 37°C in a humidified atmosphere (5% CO2 / 95% air), with medium replacement every other day. Once confluent, hCMEC / D3 cells were treated with 1 μM rh Bri2 BRICHOS monomers, oligomers, or proSP-C monomers for 2 and 24 hours. SH-SY5Y cells were treated with rh Bri2 BRICHOS monomers at concentrations of 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, and 0.05 μM for 24 hours; rh Bri2 BRICHOS monomers at concentrations of 1 μM and 0.25 μM for 2 and 24 hours; rh Bri2 BRICHOS oligomers at a concentration of 1 μM for 24 hours; or rh proSP-C at 0.25 μM and 1 μM for 2 and 24 hours. Untreated cells served as controls. Following treatment, cells were lysed as described for HEK-293 cells. For Western blotting (see below), anti-Bri2 BRICHOS primary antibody (raised in goat and purified in-house) was used at a dilution of 1:1000, and anti-proSP-C BRICHOS primary antibody (raised in goat and purified in-house) was used at a dilution of 1:2000.

[0273] Transcytosis inhibition

[0274] Monolayer cell and cellular uptake experiments were performed essentially as described above, but after the monolayers were formed, they were serum starved for 4 h in EndoGro medium without FBS. The monolayers were then incubated with 30 μM Dyngo-4a (Selleck Chemicals, Boston, MA) in FBS-free medium for 20 min. Subsequently, monolayer permeability assays were performed with 1 μM rh Bri2 BRICHOS R221E monomer in EndoGro medium without FBS together with 30 μM Dyngo-4a in a humidified atmosphere (5% CO2 / 95% air) at 37°C for 24 h. Control experiments without Dyngo-4a were performed similarly in serum-free medium. The apical and basolateral sides were analyzed by Western blotting to determine the net average transport across the monolayers. For cellular uptake, cells were serum starved for 4 h in EndoGro medium without FBS and incubated with 30 μM Dyngo-4a for 20 min. Uptake of 1 μM rh Bri2 BRICHOS R221E monomer in FBS-free EndoGro medium was analyzed after 4 and 24 hours in the presence of 30 μM Dyngo-4a. A control experiment without Dyngo-4a was performed similarly in serum-free medium. Cell culture media and lysates were analyzed by Western blotting.

[0275] Western blotting

[0276] Samples were separated by Tris-glycine SDS-PAGE using 12% separating gels under reducing conditions (including 5% β-mercaptoethanol (Sigma, St. Louis, MO) and 5 mM DTT in sample buffer) and heated at 95°C for 5 min. Spectra Multicolor Broad Range Protein Molecular Weight Standards (Thermo Fisher Scientific, Waltham, MA, USA) were used. For monolayer cell permeability assessments, 4 μl of protein sample in cell culture medium was loaded into the wells. For cellular uptake assessments, 7 μg of cell lysate was loaded.

[0277] Electrophoresis was performed at 120 V, and electrotransfer was achieved using a transfer buffer formulation (25 mM Tris-HCl pH 8.3, 192 mM glycine, 0.05% SDS, 20% methanol) at 15 V for 30 min using a semi-dry transfer cell (Bio-Rad, Hercules, CA) to a polyvinylidene fluoride (PVDF) membrane (0.2 μm, Amersham Hybond, GE Healthcare, Chicago, IL).

[0278] The membrane was soaked in blocking buffer [5% skim milk powder (AppliChem, Germany) diluted in 1× PBS (Sigma, St. Louis, MO) containing 0.1% Tween-20 (1× PBS-T)] for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. The membrane was washed three times with 0.5% skim milk powder in 1× TPBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG from Abcam, UK, diluted 1:3000; rabbit anti-goat IgG from Invitrogen, Waltham, MA, diluted 1:10,000; rabbit anti-mouse IgG from Invitrogen, Waltham, MA, diluted 1:10,000; goat anti-human IgG Fc from Invitrogen, Waltham, MA, diluted 1:1000) at room temperature for 1 h. The membrane was washed again as above and washed twice with 1× PBS. Blots were developed using a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher Scientific, Waltham, MA, USA). For cell lysates, measurements were normalized with β-actin (0.01 μg / ml) (R&D Systems, Minneapolis, MI) after gentle stripping (0.2 M glycine, 0.1% SDS, 1% Tween-20 pH 2.2). Intensity was assessed using ImageJ software.

[0279] For insulin and NT*, additional experimental procedures were added. The blot membrane was soaked in blocking buffer [1% skim milk powder and 0.1% BSA in TBS-T (TBS [50mM Tris-HCl (pH 7.4), 150mM NaCl] containing 0.1% Tween 20)] for 5 min and washed with PBS-T for 3 min. The membrane was incubated in 0.2% glutaraldehyde in PBS-T for 15 min, washed 3 times with PBS-T, then immersed in citrate repair buffer (10mM citric acid (pH 6.0), 1mM EDTA, 0.05% Tween 20) and microwaved at 600W for 10 min after boiling. After cooling to room temperature, the membrane was soaked in quenching buffer (200mM glycine in PBS-T) for 10 min. After these additional steps, blot was performed as described above.

[0280] Analysis of NT-BRICHOS fusion protein crossing the BBB in wild-type mice

[0281] A gene fragment encoding the rh NT*-Bri2 BRICHOS fusion protein was cloned and expressed, where NT* is a solubility tag (as described in WO 2017 / 081239 A1) followed by human Bri2 residues 113-231. The protein was expressed in Shuffle T7 competent E. coli cells grown at 30°C in Lysogeny Broth (LB) medium supplemented with 15 μg / mL kanamycin. When OD 600nm When the pH reached about 0.9, the temperature was lowered to 20 ° C and overnight protein expression was induced by adding 0.5 mM isopropyl bD-1-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation (3,000 × g, 4 ° C) and the cell pellet was resuspended in 20 mM Tris-HCl pH 8.0, followed by 5 min of ultrasonic treatment (on 2 s, off 2 s, 65% power, on ice). The lysate was centrifuged (24,000 × g, 4 ° C) for 30 min, and the supernatant containing the target protein was purified using an immobilized metal affinity chromatography (IMAC) column (Ni SepharoseTM 6Fast Flow; General Healthcare Group, UK), which was equilibrated with 20 mM Tris-HCl pH 8.0. The fusion protein was eluted with 300 mM imidazole in 20 mM Tris-HCl pH 8.0 and dialyzed against 20 mM Tris-HCl pH 8.0 (regenerated cellulose RC, 6-8 kDa membrane; Spectroscopy Laboratories) overnight in the cold room.

[0282] All animal handling and experimental procedures were performed in accordance with local ethical guidelines in the animal facility of the Karolinska Institutet Huddinge campus and were approved by the Animal Ethics Committee of South Stockholm (dnr S 6-15) and the Linköping Animal Ethics Committee (ID 855). Three-month-old C57BL / 6NTac (Taconic, Denmark) mice and 11-month-old C57BL / 6J mice (Janvier Laboratories, France) were kept in controlled humidity and temperature, under a 12-hour light-dark cycle, and group-housed (seven per cage) (with ad libitum access to food and water). Three mice received a single intravenous injection of NT*-Bri2 BRICHOS fusion protein (10 mg / kg) or an equal volume of PBS into the lateral tail vein using a 0.3 mL syringe with a 30-gauge needle. Before injection, mice were placed in individual cages under a heating lamp for 5 minutes to dilate the tail vein. Two hours after injection, 3-month-old mice were anesthetized with isoflurane and perfused intracardially with 40 mL of saline (0.9% NaCl). 11-month-old recipient mice received a single intravenous injection of NT*-Bri2BRICHOS fusion protein (10 mg / kg) or an equal volume of PBS, anesthetized and perfused 2 hours after injection. The brains were quickly removed and snap-frozen in dry ice and stored at -80°C until analysis.

[0283] Immunohistochemical staining for the NT*-Bri2 BRICHOS fusion protein was performed on 5-μm-thick coronal sections of paraffin-embedded mouse brain tissue. Sections were deparaffinized in xylene and rehydrated in a graded alcohol series ranging from 99% to 70%. Brain sections were pretreated for antigen retrieval in DIVA decapsulation 1× solution (Bonjian Medical) at 110°C for 30 min in an autoclave (Bonjian Medical). Slides were cooled at room temperature (RT) for 30 min and then washed with Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and incubated with peroxidase blocking solution (Danko) for 5 min. After washing with TBS-T, brain sections were additionally blocked with background inhibitor (Danko) for 10 min. Slides were then incubated overnight at 4°C with a solution containing primary anti-NT antiserum from rabbit diluted in DAKO (Agilent) antibody diluent. After washing with TBS-T, sections were incubated with a secondary antibody cocktail containing a goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) for 30 min at room temperature (RT). HRP immunoreactivity was detected by permanent green (Biosite) solution. Sections were counterstained with hematoxylin (Mayer), dehydrated, cleared in xylene, and mounted with DEPEX mounting medium (Merck). Images were acquired using a Nikon Eclipse E800 light microscope connected to a high-resolution camera using a 20× objective and a 40× objective.

[0284] result

[0285] Bri2 BRICHOS accumulates in the CNS after intravenous administration

[0286] Aβ precursor protein (App) knock-in mouse model (Saito T. et al. (2014) Nat Neurosci. 17, 661-663) NL-G-F Mice (expressing human Aβ with the Arctic mutation) and App NL-F Mice (expressing wild-type human Aβ) were treated with repeated intravenous injections of rh Bri2 BRICHOS R221E (SEQ ID NO: 13). After 10-12 weeks of treatment, the amount of Bri2 BRICHOS in the CNS was analyzed.

[0287] Figure 9 Shown is the effect of repeated injections of Bri2 BRICHOS R221E on the App NL-G-F and App NL-F Bri2 BRICHOS in mouse brain. App in rh Bri2 BRICHOS R221E or PBS treated NL-G-F and AppNL-F Representative sections (A, D) of mice were double-stained with anti-Bri2 BRICHOS antibody (red / pink) and 82E1 anti-Aβ antibody (green) and counterstained with hematoxylin (Mayer) (blue). NL-G-F (B, C) and App NL-F (E, F) Average intensity of Bri2 BRICHOS in the hippocampus (B, E) and cortex (C, F) of mice. Data are shown as mean ± SEM (n = 3-4 mice / group, 4 histological sections analyzed per mouse). Unpaired parametric two-tailed t-test was used to calculate p values. Scale bars represent 400 μm (upper four sub-graphs in A and D) and 100 μm (lower two sub-graphs in A and D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0288] Compared with PBS-treated control mice, rh Bri2 BRICHOS R221E-treated App knock-in mice showed more abundant overall Bri2 BRICHOS staining in brain tissues, including neurons and around Aβ plaques ( Figure 9 , Part A and Part D). From the App NL-G-F and App NL-F Average intensity analysis of Bri2BRICHOS between PBS and rh Bri2BRICHOS R221E treated samples in mice showed a significant increase in overall Bri2BRICHOS staining ( Figure 9 (Panels B, C, E, and F). Notably, mice were sacrificed 2–4 weeks after the last dose of rh Bri2 BRICHOS R221E. These results strongly support that intravenously injected rh Bri2 BRICHOS R221E crosses the BBB and resides in the CNS parenchyma and cells for a considerable period of time in the App knock-in AD mouse model.

[0289] Cell viability

[0290] After 24 hours of incubation, the effects of the rh BRICHOS domain on hCMEC / D3 cells and undifferentiated SH-SY5Y cells were assessed by tetrazolium-MTT cell viability assay. Rh Bri2 BRICHOS monomers showed no toxicity towards the human brain microvascular endothelial cell line hCMEC / D3 and the human neuroblastoma cell line SH-SY5Y, but mitochondrial activity was slightly reduced in the presence of high concentrations of rh proSP-C BRICHOS and rh Bri2 BRICHOS oligomers or crude solutions.

[0291] The effects of rh Bri2 BRICHOS (113-231) wt monomers and oligomers on the human cell lines hCMEC / D3 and SH-SY5Y were concentration-dependent, with increasing concentrations decreasing cell viability. For rh Bri2 BRICHOS monomers in the concentration range of 1.0-0.1 μM, SH-SY5Y cell viability increased from 94.0% to 99%, and hCMEC / D3 viability increased from 100.4% to 106.6%. With lower concentrations of rh Bri2 BRICHOS monomers, hCMEC / D3 cell metabolic activity increased slightly. At a 1 μM concentration, rh Bri2 BRICHOS oligomers exhibited 83.5% viability on SH-SY5Y cells and 86.7% viability on hCMEC / D3 cells. rh proSP-C BRICHOS showed 88.9% viability against SH-SY5Y cells at 0.5 μM and 90.3% viability against hCMEC / D3 cells at 1 μM. High concentrations of rh Bri2BRICHOS oligomers (like Aβ peptides) may initiate mild stress in the endoplasmic reticulum (ER) and release Ca 2+ In the case of rhBri2 BRICHOS monomers, the integrity of the cell membrane remained similar to that of the positive control. Given the low physiological concentrations of BRICHOS domains in human plasma, it can be assumed that BRICHOS domains are not detrimental to the integrity of the BBB under normal conditions.

[0292] Cellular uptake

[0293] The cellular uptake of the rhBRICHOS domains was assessed by Western blotting of the human cell lines SH-SY5Y and hCMEC / D3 after 2 h and 24 h of incubation and determined by band intensity.

[0294] Incubation of the human neuroblastoma cell line SH-SY5Y with rh Bri2 BRICHOS monomers at concentrations ranging from 0.05 μM to 1 μM for 24 hours showed linear uptake. Bri2 BRICHOS oligomers were detected in cell lysates to a lesser extent than Bri2 BRICHOS, likely indicating that the oligomers adhere to the cell surface. ProSP-C BRICHOS was similarly taken up by SH-SY5Y cells, but to a lesser extent. The endogenous BRICHOS domain could not be detected in untreated cells, indicating that the detectable BRICHOS domain was taken up from the cell culture medium.

[0295] The cellular uptake of different rh BRICHOS domain constructs and concentrations was also investigated in human brain microvascular endothelial cells (hCMEC / D3) after 2 and 24 hours. Similar to SH-SY5Y cells, Bri2 BRICHOS monomers and proSP-C BRICHOS were taken up by hCMEC / D3 cells, but Bri2 BRICHOS oligomers did not appear to be taken up. Endogenous BRICHOS domains were not detected in untreated cells, indicating that detectable BRICHOS domains were taken up from the cell culture medium.

[0296] rh Bri2 BRICHOS wt monomers also efficiently passed through the astrocyte / hCMEC / D3 coculture model. The passage rate of direct astrocyte / hCMEC / D3 cocultures (7.0%) was slightly lower than that of indirect astrocyte / hCMEC / D3 cocultures (8.6%) and hCMEC / D3 monocultures (10.5%). This may be partially related to the higher TEER (transepithelial electrical resistance) values ​​of direct astrocyte / hCMEC / D3 cocultures compared to indirect astrocyte / hCMEC / D3 cocultures and hCMEC / D3 monocultures. Interestingly, astrocytes internalized rh Bri2 BRICHOS, suggesting that the lower passage of rh Bri2 BRICHOS into the basolateral compartment in the presence of astrocytes, particularly when grown close to hCMEC / D3 cells in the direct model, is partly due to astrocytic uptake.

[0297] Transcytosis inhibition

[0298] Addition of Dyngo-4a, a dynamin GTPase inhibitor that blocks endocytic vesicle formation (McCluskey et al., 2013), reduced rh Bri2 BRICHOS passage through hCMEC / D3 monolayers by more than fourfold. We observed that the presence of Dyngo-4a also resulted in a higher degree of rh Bri2 BRICHOS uptake or association with hCMEC / D3 cells and an increase in the amount of "residual" rh Bri2 BRICHOS (i.e., the amount that could not be detected in either the apical or basolateral compartments) after addition to hCMEC / D3 monolayers. Notably, in the presence of Dyngo-4a, both cellular uptake and residual rh Bri2 BRICHOS were approximately 5% after 24 hours, supporting the view that both measurements represent cell-associated rh Bri2 BRICHOS. These results suggest that functional endocytosis of rhBri2 BRICHOS is required for transport across hCMEC / D3 monolayers, and that blocking dynamin GTPase activity prevents rhBri2 BRICHOS from being internalized and instead accumulates on the cell surface. Nonspecific effects of dynamin GTPase inhibitors have been described, and further studies are needed to delineate the molecular details of rhBri2 BRICHOS trafficking across hCMEC / D3 monolayers.

[0299] To investigate the cellular uptake of rh Bri2 and proSP-C BRICHOS, we used hCMEC / D3 cells, human astrocytes, and undifferentiated and differentiated human neuroblastoma-derived SH-SY5Y cells, which are widely used as in vitro neuronal models. All BRICHOS variants investigated were taken up by cells, albeit to varying degrees. A correlation was observed between passage through hCMEC / D3 monolayers and cellular uptake, with rh Bri2 BRICHOS monomers exhibiting the highest values ​​for both parameters. rh Bri2 BRICHOS oligomers, which showed no detectable passage through hCMEC / D3 monolayers, were taken up to a lower extent in undifferentiated and differentiated SH-SY5Y cells but not in hCMEC / D3 cells. Apparent cellular uptake of rh Bri2 BRICHOS was concentration-dependent, showing saturation of approximately 2%, and residual values ​​(i.e., amounts not detectable in either the apical or basolateral compartments) determined from experiments with hCMEC / D3 monolayers were also approximately 1%-2%. Monolayer passage and cellular uptake studies, as well as the effects observed after dynamin inhibition, strongly support that BRICHOS is transported across endothelial monolayers by transcytosis rather than nonspecific leakage. Uptake observed in SH-SY5Y cells and human astrocytes suggests that neurons and astrocytes can also internalize BRICHOS.

[0300] To further rule out the possibility that BRICHOS passage through hCMEC / D3 monolayers or uptake in hCMEC / D3 and SH-SY5Y cells was related to toxic effects of the studied proteins, we determined cell integrity using a propidium iodide (PI) assay and mitochondrial function using a tetrazolium-MTT cell assay. The results showed that rh Bri2 BRICHOS monomer had no effect on cell integrity or mitochondrial function in either hCMEC / D3 or SH-SY5Y cells, while rh Bri2 BRICHOS oligomers and proSP-C BRICHOS had minimal effects on these cell viability measures. This means that the BRICHOS variant that exhibited the highest passage rate in hCMEC / D3 monolayers is clearly non-toxic, thus ruling out the possibility that the observed passage was due to cytotoxic effects. Furthermore, in the presence of rh Bri2 BRICHOS, TEER values ​​and FITC-dextran permeability measurements of monolayers from day 3 to 9 were unchanged, indicating that the protein does not negatively impact tight junctions or overall monolayer integrity.

[0301] rh Bri2 BRICHOS fused to other proteins were efficiently transported across cells in a human BBB model in vitro and In wild-type mice,

[0302] We used cultured human brain microvascular endothelial hCMEC / D3 cell monolayers as an in vitro model of the human BBB (Weksler, B. et al. (2013) Fluids Barriers CNS. [Central Nervous System Fluids and Barriers] 10, 16; and Markoutsa, E. et al. (2011) Eur J Pharm Biopharm. [European Journal of Pharmacy and Biopharmaceutics] 77, 265-274) to investigate whether rh Bri2 BRICHOS could be used as a transport vehicle to facilitate CNS uptake of other proteins. In particular, we tested the passage of two unrelated proteins fused to rh Bri2 BRICHOS: mCherry (a ~30 kDa fluorescent protein derived from sea anemones) and NT (a ~15 kDa protein domain derived from spider silk).

[0303] Six days after seeding, when tight junction monolayers are formed and highly intact, BRICHOS domain permeability was investigated on the hCMEC / D3 cell line. Different concentrations and incubation times of rh Bri2 BRICHOS and proSP-C constructs were evaluated and determined by band intensity.

[0304] Permeability studies of recombinant human Bri2 BRICHOS (113-231) wild-type proteins with different quaternary structures demonstrated that monomers permeated hCMEC / D3 cell monolayers, but oligomeric species did not. The permeability of rh Bri2 BRICHOS monomers was time- and concentration-dependent. Interestingly, the permeability of Bri2 BRICHOS R221E monomers was higher than that of Bri2 BRICHOS wt monomers. Furthermore, rh proSP-C BRICHOS wt was determined to have a lower permeability across hCMEC / D3 cell monolayers than the proSP-C BRICHOS 187R mutant.

[0305] Table 1. Permeability of different Bri2 and proSP-C BRICHOS domains after 2 h, 24 h, or 48 h of incubation. 1 μM BRICHOS solution was used. Results are expressed as the mean of three replicates.

[0306]

[0307] Table 2. Rh Bri2 BRICHOS wild-type monomeric hCMEC / D3 permeability at different concentrations and incubation times. Results are presented as the mean of three replicates.

[0308]

[0309]

[0310] The permeability of Bri2 and Bri3 BRICHOS solubility tag conjugates appeared similar to that of the unconjugated Bri2 BRICHOS construct, with monomeric species passing through hCMEC / D3 monolayers, but oligomers or crude solutions did not.

[0311] The NT-Bri2 BRICHOS fusion protein and the mCherry-Bri2 BRICHOS fusion protein showed the same trend as the unconjugated Bri2 BRICHOS (113-231) wt and S-tag fusion proteins, where only the monomer showed permeability. On the other hand, neither NT-tag alone nor mCherry alone showed any permeability, demonstrating that the Bri2 BRICHOS domain is capable of transporting conjugated molecules through hCMEC / D3 cell monolayers.

[0312] The data clearly demonstrate that endothelial cell uptake and permeability of BRICHOS domains is dependent on quaternary structure, with monomeric species permeating the hCMEC / D3 monolayer, but oligomers not. The hCMEC / D3 cell line has previously been shown to exhibit size selectivity, with permeability restricted in the apical to basolateral direction, while barrier formation is more efficient for larger molecules. Compared to their wild-type counterparts, Bri2BRICHOS R221E monomers and proSP-C BRICHOS T187R monomers exhibited enhanced permeability due to the formation of stable monomers.

[0313] Table 3. Summary of osmolarity of 1 μM solutions after 24 h incubation.

[0314] The results are expressed as the mean (SD) of three or four replicates.

[0315] ** indicates 0.5 μM solution. *** indicates aggregation in solution.

[0316]

[0317]

[0318] Figure 10 Table 3 shows the permeability of hCMEC / D3 monolayers with 1 μM solutions of various proteins and macromolecules (expressed as a percentage of the total amount added to the apical side).

[0319] Both rh wild-type and R221E Bri2 BRICHOS monomers showed significant passage from the apical to the basolateral side of the monolayer, i.e., they were transported across the cell, whereas rh wild-type Bri2 BRICHOS oligomers were not transported across the cell ( Figure 10 Neither mCherry nor NT alone could significantly cross the cell monolayer, but the fusion construct with the rh Bri2 BRICHOS domain was significantly transported across the cells ( Figure 10 ).

[0320] Whether the abundance of Bri2 BRICHOS-mCherry can be further enhanced by the addition of microbubbles in the in vitro human BBB model remains to be determined, as does the extent to which the in vitro human BBB model predicts all features of the in vivo mouse BBB.

[0321] To test whether rh Bri2 BRICHOS function as a transport vehicle across the BBB in vivo, we repeated the same type of experiments as previously used to test BBB crossing of recombinant Bri2 and proSP-C BRICHOS in wild-type mice. Figure 11Figure 3 shows staining of the cerebral cortex of mice injected with NT-Bri2 BRICHOS (A) or PBS (B) with anti-NT antibody (blue). This shows that two hours after intravenous injection, recombinant NT-Bri2 BRICHOS can be detected in the brain parenchyma and neuronal cells, while mice injected with PBS showed only background staining ( Figure 11 ).

[0322] These findings strongly suggest that the ability of rh Bri2 BRICHOS to cross the BBB could be used to transport other proteins across the BBB and that, after passage through the CNS, the fusion protein diffused within the parenchyma and was taken up by cells. Proteins do not typically cross the BBB passively; for example, up to 0.1% of peripherally administered antibodies cross the BBB (18), suggesting that an active mechanism mediates BBB translocation of rh Bri2 BRICHOS.

[0323] NT*-Bri2 BRICHOS monomers also efficiently cross the astrocyte / hCMEC / D3 co-culture model. Like rhBri2 BRICHOS wt monomers, the passage of the fusion protein through co-culture models, particularly in the direct astrocyte / hCMEC / D3 co-culture model, was lower than that observed in hCMEC / D3 monocultures. Fluorescence images of hCMEC / D3 monolayers after addition of rh Bri2BRICHOS-mCherry revealed abundant red fluorescent puncta, whereas no such puncta were observed after addition of mCherry alone. Like rh Bri2 BRICHOS alone, oligomeric forms of the fusion protein, S-tag-Bri2BRICHOS or NT*-Bri2 BRICHOS, did not cross the hCMEC / D3 monolayers.

[0324] rh Bri2 BRICHOS fused to target proteins are internalized and trafficked to lysosomes in primary neurons.

[0325] Figure 11 The data in Figure A demonstrate that the fusion protein rhNT-Bri2 BRICHOS is internalized into CNS cells and present in intracellular vesicles. We investigated intracellular localization in more detail using cultured primary mouse neurons and the recombinant fusion protein mCherry-Bri2 BRICHOS. Recombinant mCherry-Bri2 BRICHOS was added to the culture medium at low nanomolar concentrations, and neurons were fixed 24–48 hours later and visualized under a fluorescence microscope.

[0326] Figure 12Figure 2 shows Rh Bri2 BRICHOS-mediated uptake into mouse primary neurons. Following addition to the culture medium, a fusion protein consisting of Bri2 BRICHOS linked to mCherry (denoted as mCh-Bri2, results in the upper row) was detected in abundant intracellular vesicles. mCherry alone (results in the lower row) was not taken up.

[0327] Thus, mCherry-Bri2 BRICHOS is internalized and can be detected in intracellular vesicles, whereas the control protein mCherry alone is not taken up to any detectable level. Figure 10 The results of transcytosis experiments were highly consistent with those in the previous study.

[0328] Finally, Rh Bri2 BRICHOS connected to the cargo protein is endocytosed and transported to the lysosome. Primary mouse neurons treated with the fusion protein mCherry-Bri2 BRICHOS showed intracellular uptake and lysosomal presence, as shown by colocalization with the lysosome-specific dye SiR-Lyso (data not shown). Therefore, neurons treated with mCherry-Bri2 BRICHOS were marked with the lysosome-specific dye SiR-lysosome and showed clear colocalization.

[0329] in conclusion

[0330] This study demonstrates that rh Bri2 BRICHOS is efficiently transported across hCMEC / D3 cell monolayers (an in vitro model of the human BBB), apparently via a dynamin-dependent endocytosis mechanism. The mechanism of action of BRICHOS in vivo appears to be similar to that observed in vitro and ex vivo, suggesting that the selective reduction of toxic oligomers by Bri2 BRICHOS is an important component of its therapeutic efficacy. Our results demonstrate that rh Bri2 BRICHOS can enter neurons and astrocytes following intravenous injection in mice, suggesting that Bri2 BRICHOS can also mediate cellular effects. Bri2 BRICHOS can carry cargo proteins across the human BBB model used here, and this property also applies to the BBB in vivo, as rh NT*-Bri2 BRICHOS can be found in early endosomes of neurons and astrocytes in key brain regions following intravenous injection in mice. This raises the exciting possibility that the ability of BRICHOS to reduce Alzheimer's disease pathology can be combined with additional activities mediated by fusion partners, potentially creating multimodal biopharmaceuticals that are also effective across the BBB. One candidate group for fusion with Bri2 BRICHOS is anti-Aβ monoclonal antibodies, which have shown therapeutic efficacy in clinical trials for Alzheimer's disease (van Dyck et al., N Engl J Med 388(1):9-21, 2023), but are likely to benefit from increased BBB passage and combining this with the anti-Aβ toxicity effects mediated by Bri2 BRICHOS. Notably, BRICHOS is more effective in reducing the number of toxic Aβ oligomers in vitro than clinically tested anti-Aβ monoclonal antibodies (Linse et al., Nat Struct Mol Biol, 27:1125-1133, 2020). Bri2 BRICHOS combines a relatively small size, scalable recombinant production, lack of detectable toxicity to cultured cells or after repeated intravenous injections, and a high ability to carry cargo proteins across the BBB. The preparation and administration protocols for rh Bri2 BRICHOS fusions are also quite simple compared to the preparation of transport vesicles by encapsulation strategies. This makes rh Bri2 BRICHOS an interesting vehicle for delivering a variety of macromolecules across the BBB and into cells in the CNS.

[0331] Experiments on inhibiting the formation of α-synuclein oligomers

[0332] Materials and methods

[0333] Preparation of Bri2 BRICHOS monomer

[0334] SHuffle T7 (K12 strain) E. coli cells were transformed with a plasmid vector carrying the human Bri2 BRICHOS R221E mutant sequence (113–231 of full-length human Bri2) fused to His6x-NT at the N-terminus and separated by a thrombin cleavage site. The cells were incubated at 30°C in a 15 μg / ml -1 The LB culture medium of kanamycin is incubated until OD=0.8 is reached. Then, cells are induced with 0.5mM IPTG and grown overnight at 20°C. At 4°C, cells are harvested by centrifugation at 5000x g, and the precipitation is resuspended in 20mM Tris HCl (pH 8.0). Two precipitations are ultrasonically processed for 5min (2s on, 2s off, 65% maximum power), and the resulting lysates are centrifuged for 30min at 4°C with 20000x g. The supernatant collected is then loaded onto a Ni-NTA column (GE Healthcare Bio-Science AB, Sweden). Subsequently, the column is washed with 20mM Tris HCl, 15mM imidazoles (pH 8.0) of 30 column volumes (CV), then eluted with 20mMTris HCl, 300mM imidazoles (pH 8.0). The fractions were combined and dialyzed overnight at 4°C using 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.4) added with thrombin (1: 1000 enzyme and substrate, w / w). The sample was then loaded onto a Ni-NTA gravity column to remove His-NT material and uncut protein of interest. The flow-through was concentrated using a 10 kDa cut-off concentrator (Vivaspin 20, 10 kDa MWCO Cytiva) and directly loaded onto a Hiload superdex 75PG size exclusion column (Cytiva, USA) to collect the peak of interest corresponding to the monomer.

[0335] Preparation of α-syn monomers

[0336] BL21 (DE3) Escherichia coli is transformed with the bacterial expression plasmid pET21a-α-synuclein (Addgene) containing the human α-syn coding sequence. Cells are grown at 37°C in LB medium containing 100ug.ml-1 ampicillin until OD=0.8 is reached. The culture is inoculated with 0.5mM IPTG and incubated overnight at 20°C. The cells are centrifuged at 5000x g for 20min at 4°C, and the bacterial pellet is resuspended in 20mM Tris HCl (pH 8.0) by gentle vortexing. The cell pellet from 1L is heated and cracked at 65% maximum power for 5min using a sonicator (G. Heinemann Ultrasonics and Laboratory Technology Co., Ltd. (G. Heinemann Ultraschall-un Labortechnik)), with a pulse setting of 2 seconds on and 2 seconds off. The sample is then boiled for 10 or 5min. The sample is then centrifuged at 4°C for 20min at 24000x g. The supernatant was loaded onto a HiTrap QFF anion exchange chromatography column. The protein was gradient eluted over 10 CV using 20 mM Tris HCl, 1 M NaCl (pH 8). The eluted peak was then loaded onto a reverse phase chromatography column using 99.9% H2O, 0.1% TFA as running buffer. Gradient elution was performed over 20 CV using 70% ACN, 0.1% TFA buffer. The fractions corresponding to the peak of interest were combined and the optical density at 280 nm was measured. The sample was lyophilized overnight using a vacuum apparatus (LabConco CentriVap concentrator). The lyophilized samples not directly used were stored at -20°C. For further purification, an aliquot of approximately 8 mg of lyophilized protein was dissolved in 550 μl of 7 M Gdn-HCl (pH 8.0) and then loaded onto a Superose 6 Increase column in 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.4) as a running buffer to obtain a homogeneous, pure α-syn monomer sample that was further stored at −20°C.

[0337] ThT aggregation assay

[0338] For kinetic experiments, each sample contained 30 or 70 μM α-syn monomers, in the presence of 20 μM ThT and varying concentrations of Bri2 BRICHOS monomers in 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.4) buffer or with the addition of 154 mM NaCl salt. Two glass beads with a diameter of 1 mm were used in each well. Fluorescence was recorded using a 448-10 nm excitation filter and a 482-10 nm emission filter (FLUOStar Omega from BMG Labtech, Offenburg, Germany), while the plate was oscillated at 300 rpm between cycles, with 9 min turned on and 1 min turned off. Each cycle was set to a duration of 10 min and the temperature was set to 37 ° C. In order to prepare seeds, only 70 μM α-syn monomers were used to obtain α-syn fibrils from previous ThT kinetic runs. The fibrils were then sonicated using a probe sonicator (SONICS Vibra Cell™) for 3 min, 2 s on, 2 s off. For all experiments, aggregation trajectories were normalized and averaged using 6 replicates per sample.

[0339] Analysis of α-syn aggregation kinetics

[0340] Aggregation trajectories of 70 and 30 μM α-Syn monomers with different concentrations of BRICHOS at molar ratios between 0 and 100% were normalized, averaged across 6 replicates, and truncated at the plateau of the aggregation reaction. The aggregation kinetics were fitted by applying the unseeded nucleation model dominated by secondary nucleation in Amylofit, which includes primary nucleation, secondary nucleation, and elongation as the combined rate k + k n and k + k². Individual fits were first performed to obtain the dependence of these parameters on BRICHOS concentration. To assess the contribution of the single nucleation rate, we then applied a secondary nucleation dominance model with the initial aggregate number and concentration set to zero. The two nucleation rate constants were set as global fit parameters, meaning they were constrained to the same value at all BRICHOS concentrations, while the one nucleation constant was set as an individual fit parameter.

[0341] High-seeded aggregation kinetics were analyzed using GraphPad. Elongation rates were extracted by fitting a straight line to the first 2 h of the aggregation curve.

[0342] Preparation of fibrils incubated with Bri2 BRICHOS

[0343] α-syn monomers (70 μM) were incubated in a similar manner to the ThT kinetics experiments, but without ThT. After approximately 100 h of the experiment, the fibrils were spun at 16,200 x g for 30 min at 4°C and washed twice with fresh buffer (20 mM sodium phosphate buffer 0.2 mM EDTA pH 7.4).

[0344] Transmission electron microscopy

[0345] A diluted solution of 5 μl α-syn fibrils (about 15 μM) was applied to a 200-mesh formvar-coated nickel grid and incubated for 10 min before removing excess solution with blotting paper. The cells were then washed twice with 10 μl MQ water and stained with 1% uranyl formate for 5 min. Excess stain was removed with blotting paper and then air-dried. Transmission electron microscopy (FEI Tecnai 12 Spirit BioTWIN, operated at 100 kV) was performed using a 2k × 2k Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany) to analyze fibril morphology. 15-20 images were randomly recorded for each sample, and fibril diameter was measured using Image J software.

[0346] Native PAGE

[0347] Equimolar concentrations of BRICHOS were added to α-syn fibril samples (8 μM). Samples were prepared under non-denaturing conditions and subsequently run on a 7.5% native gel. BRICHOS was visualized using Coomassie blue staining.

[0348] Oligomer capture

[0349] Similar to the ThT kinetics experiment, α-syn monomers (70 μM) were incubated with and without equimolar concentrations of BRICHOS. Samples were collected at different time points, quickly frozen in liquid nitrogen, and stored at -80°C until further use. Samples were prepared under native conditions and run on native PAGE 10% and stained with Coomassie blue.

[0350] surface plasmon resonance

[0351] Binding assays were performed using a Biacore 3000 instrument at 25° C. These experiments were performed using different ChiPs: α-syn fibrils, salted α-syn fibrils and previously immobilized α-syn monomers.

[0352] The system was pre-filled with the following running buffer: 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.4). Different concentrations of Bri2 BRICHOS (0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) were injected into channels 2 and 4 at a flow rate of 20 μl / min for 5 minutes. Channels 1 and 3 were left empty and used as references to control for possible nonspecific binding of α-syn. The BRICHOS protein (if bound) was washed off with 20 mM NaOH, and the buffer was equilibrated on the chip for 15 minutes before being injected into the sample again. Each sample was run in duplicate.

[0353] NMR experiments

[0354] The 15N-labeled α-syn or 15N-labeled BRICHOS were analyzed at 281 K on a Bruker 700 MHz spectrometer in 20 mM NaP, 0.2 mM EDTA (pH 7.4) supplemented with BRICHOS or α-syn sonicated fibrils, respectively. 1 H- 15 N HSQC titration. All NMR data were processed using Bruker Topspin and analyzed using Poky.

[0355] ITC

[0356] 216 μM BRICHOS monomer was injected 18 times with a 2 μl syringe into a sample cell containing 20 μM α-syn monomer or buffer (20 mM NaP, 0.2 mM EDTA, pH 7.4) as a control (iTC200 α-syn Microcal, GE Healthcare). The experiment was performed at room temperature with a stirring speed of 1000 rpm.

[0357] FIDA

[0358] Recombinant wild-type human α-syn was expressed in Escherichia coli using the pET11-D construct and purified as previously described (Lorenzen et al., J. Am. Chem. Soc., 136:3859, 2014; Paslawski et al., Methods Mol. Biol., 1345:133, 2016). Oligomeric and monomeric α-syn were prepared by incubating 8 mg / ml of pure α-syn dissolved in PBS with shaking at 900 rpm on a heated digital shaking dry bath (Thermo Scientific) at 37 ° C for 3 h, followed by size exclusion on a Superose 6 prep grade XK 26 / 100 column (Universal Health Medical Group). Monomeric and oligomeric fractions were collected and stored at 4 ° C. Prior to analysis, oligomeric fractions were combined and concentrated using a 100 kDa spin filter (Amicon Ultra). α-syn fibrils were prepared by dissolving lyophilized α-syn in PBS and placing the filtered solution in a 96-well plate with 3 mm glass beads at a concentration of 1.1 mg / mL while shaking at 600 rpm at 37°C for 3 days. The fibrils were isolated by centrifugation at 13.000 rpm for 10 min, redissolved in PBS to a concentration of 1 mg / ml, and broken by sonication using a Q500 sonicator (Qsonica, Connecticut, USA) equipped with a 2 mm Microtip probe, set to pulse 10 s at 20% intensity, pause 10 s, and sonicate for a total of 30 s. BRICHOS was prepared by mixing Alexa-488 NHS ester (Thermo Scientific) with 43.6 μM BRICHOS dissolved in 100 mM bicarbonate buffer (pH 8.3) and Alexa-488 dissolved in DMSO in a 1:2 molar ratio and then incubated at room temperature for 1 hour. The reaction was quenched by adding 100 mM Tris-HCl pH 7.4, and the labeled BRICHOS was separated from the unreacted label by a PD-10 column (Universal Health Medical Group) equilibrated with PBS. Protein concentration and labeling efficiency were estimated using DeNovix DS-11 (DeNovix). Flow-induced dispersion analysis was performed using LED-induced fluorescence detection (excitation wavelength of 480 nm) on a FIDA One instrument (FIDA Biosystems). A standard capillary (inner diameter: 75 μm, outer diameter: 375 μm, total length: 100 cm, length of detection window: 84 cm) (Fida Biosystems) was rinsed with 1 M NaOH and equilibrated with PBS at 25 ° C. The experiment was performed by perfusing the capillary with running buffer at a pressure of 3500 mbar.The analyte of α-syn is carried out to the analyte of α-syn monomer, oligomer or protofibril and 50nM indicator (BRICHOS-Alexa488) and is injected into capillary successively, and continues 20 seconds at every turn, and wherein analyte injection pressure is 3500 millibars, and indicator injection pressure is 50 millibars.In order to make indicator and analyte move towards detector, the final injection of analyte of 180 seconds is carried out under the pressure of 400 millibars.The Taylor diagram of gained is analyzed using Fida software V2.3 (FIDA Biosystems company), and this software provides hydrodynamic radius (R for each binding reaction). h The resulting isothermal binding curve was fitted to the following equation:

[0359]

[0360] where K D is the dissociation constant, [A] is the analyte concentration, R I and R IA are the R of the indicator and the complex, respectively. h .

[0361] Electrophysiological studies

[0362] For electrophysiological experiments, all chemical compounds used in the extracellular solution were obtained from Sigma-Aldrich Sweden (Stockholm, Sweden). Kainic acid (KA) was obtained from Tocris Bioscience (Bristol, UK). α-syn fibrils were prepared by incubating α-syn monomers at 37°C at 900 rpm in PBS, 0.1% sodium azide (pH 7.4) for 7 days. They were then sonicated using a probe sonicator for 3 min, 2 seconds on and 2 seconds off.

[0363] We use wild-type (WT) mice (n=12) of postnatal 4-6 week, and test the influence of external α-syn protofibrils by isolated hippocampus γ oscillation incubation.For brain extraction, mice are deeply anesthetized with isoflurane.Brain is dissected out and placed in improved ice-cold artificial cerebrospinal fluid (ACSF), for dissecting, this cerebrospinal fluid contains (in mM): 80NaCl, 24NaHCO , 25 glucose, 1.25NaH PO , 1 ascorbic acid, 3Na-pyruvic acid, 2.5KCl, 4MgCl , 0.5CaCl , 75 sucrose, and with carbogen (carbogen) (95% O and 5%CO ) bubble.With Leica VT1200S vibrating microtome (Leica Microsystems (Leica Microsystems)) prepare the horizontal slice (350 μ m thickness) of the ventral hippocampus of two hemispheres. After cutting, slices were immediately transferred to a humidified interface holding chamber containing standard ACSF (in mM): 124 NaCl, 30 NaHCO₃, 10 glucose, 1.25 NaH₂PO₄, 3.5 KCl, 1.5 MgCl₂, 1.5 CaCl₂, and continuously aerated with carbogen. The chamber was maintained at 37°C during slicing and then cooled to room temperature (approximately 22°C) for at least 1 hour. Four conditions were selected to test the toxic effects of α-syn and BRICHOS R221E monomer on hippocampal gamma oscillations: sham (PBS, 0.1% sodium azide, pH 7.4), α-syn 500 nM, α-syn 1 μM, and α-syn 1 μM + Brichos 1 μM. Hippocampal slices in each condition were preincubated in a submerged incubation chamber containing ACSF for 30 minutes. During the incubation period, carbogen gas (5% CO2, 95% O2) was continuously purged into the slices and bubbled into the ACSF. After the incubation period, the slices were transferred to an interface recording chamber for extracellular recording.

[0364] Recordings were made in the CA3 region of the hippocampus using borosilicate glass microelectrodes filled with ACSF with a resistance of 3-6 MΩ. Local field potentials (LFPs) were recorded at 32°C in an interface chamber (perfusion rate of 4.5 mL per minute). Kainic acid (100 nM) triggered LFP gamma oscillations. The oscillations were allowed to stabilize for 20 min before any recording. Interface chamber LFP recordings were performed using a 4-channel amplifier / signal conditioner M102 amplifier (Electronics lab, University of Cologne, Germany). Signals were sampled at 10 kHz, conditioned using a Hum Bug 50 Hz noise canceller (Quest Scientific, North Vancouver, British Columbia, Canada), software low-pass filtered at 1 kHz, and digitized and stored using Digidata 1322A and Clampex 10.4 programs (Molecular Devices, California, USA). Power spectral density maps (from 60 s long LFP recordings) were calculated using an Axograph X (Kagi, Berkeley, CA, USA) in average Fourier segments of 8192 points. Gamma oscillation power was calculated by integrating the power spectral density between 20 and 80 Hz, and the results are expressed as the average obtained over a 1 min period.

[0365] result

[0366] BRICHOS effectively delays α-syn aggregation

[0367] We first monitored the aggregation kinetics of 70 μM α-syn using oscillatory conditions at physiological pH 7.4 in the presence of different concentrations of recombinant human (rh) Bri2 BRICHOS R221E monomers (hereinafter referred to as BRICHOS), with the molar equivalents of these monomeric α-syn ranging from 2% to 95%. We observed that after the addition of BRICHOS, the aggregation of α-syn was delayed in a concentration-dependent manner until 95% molar equivalents of BRICHOS were reached. This resulted in a delay in the aggregation half-life from 18.3 ± 1.8 h to 53.1 ± 3 h, obtained by sigmoid fitting of the individual aggregation trajectories. At higher BRICHOS concentrations, the inhibitory effect appeared to be saturated, and no further delay in aggregation was observed from 25% to 100% BRICHOS.

[0368] To test whether electrostatic interactions could mask the inhibitory effect, we performed the same experiment at physiological salt concentrations. We found that the presence of salt significantly accelerated the aggregation kinetics of α-syn, with an aggregation half-life of 9.6 ± 0.5 h, compared to 18.3 ± 1.8 h in the absence of salt. Remarkably, under these conditions, BRICHOS also effectively delayed the aggregation process, extending the aggregation half-life to 12.1 ± 5 h at 90% molar equivalents. Therefore, the inhibitory effect of BRICHOS is specific for α-syn and operates under near-physiological conditions.

[0369] We further aimed to confirm our results by performing the same experiment at lower α-syn concentrations (using 30 μM instead of 70 μM) and adding BRICHOS at the same molecular ratio. We indeed found that BRICHOS delayed α-syn aggregation at lower α-syn concentrations, both in the presence and absence of salt. These results suggest that the inhibitory effect is specific to BRICHOS interactions with strong electrostatic forces or other types of interactions, such as hydrophobic interactions with α-syn.

[0370] BRICHOS specifically inhibits secondary nucleation and fibril end elongation

[0371] Amyloid formation can generally be described by a nucleation-dependent polymerization mechanism. The formation of nuclei is called primary nucleation (k n ), through the elongation of the fibril ends (k + ) triggers the growth of additional fibrils. Other secondary processes may also occur, such as secondary nucleation (k2) or breakage (k - ). The α-syn aggregation trajectory can be fitted with these theoretical kinetic models to describe the contribution of specific microscopic nucleation events. Here, we applied a model consisting of three microscopic processes (primary nucleation, secondary nucleation, and fibril end elongation). In addition to monomer-dependent secondary nucleation, there may be monomer-independent secondary reactions associated with fibril breakage, and represented by the nucleation rate constant k - Typically, this process is associated with stirring during the fibril reaction.

[0372] Previous studies have shown that α-syn aggregation follows a secondary nucleation-dependent aggregation mechanism at physiological pH. By performing a detailed global fit analysis of the aggregation kinetics, we were able to analyze which nucleation rates are affected by BRICHOS. First, we applied a model that included only the monomer-dependent secondary nucleation reaction in addition to the primary nucleation and elongation reactions.

[0373] To account for the contribution of individual nucleation events, we set the two nucleation rate constants as global fit parameters, meaning that they were constrained to be the same value at all BRICHOS concentrations, and one nucleation constant as a separate fit parameter. The global fit that best describes the data is either k2 or k + is the fit of the free fitting parameter, which suggests that BRICHOS inhibits elongation and / or secondary nucleation. The same results were obtained in the presence of salt and at 30 μM α-syn in the presence of salt.

[0374] Second, we tested different nucleation models in which we neglected the monomer-dependent secondary nucleation k2 and used the breakage-dependent, monomer-independent secondary nucleation k - For this model, we also obtain k - or k + The free fitting parameters were found to be overall good fits, but the average residuals slightly favored the monomer-dependent secondary nucleation model.

[0375] To infer whether BRICHOS primarily affects elongation or secondary nucleation, or both, we performed high-seed kinetics. At high seed concentrations, a large number of free fibril ends are available, creating conditions where the onset of aggregation kinetics is dominated solely by fibril elongation events. Therefore, by analyzing the initial slope of the first 2 h of the kinetic trajectory, we can extract the elongation rate. We found that the initial slope depends on the BRICHOS concentration, indicating that BRICHOS prevents the elongation of α-syn fibrils. The combined fitting parameter k from the global fitting analysis was used as the + k2 is related to the initial slope from the high seed aggregation kinetics (and k + By comparing the dependence of k + The regulation in k cannot be explained only by + The large effect observed on k2 suggests that, in addition to k + In addition, k2 was also significantly reduced by the presence of BRICHOS. Therefore, these results indicate that BRICHOS inhibits secondary nucleation and fibril end elongation.

[0376] Although fracture-related processes may also exist, including k - The models for k2 exhibited several coupled fitting parameters, which increased the risk of overfitting. The slightly better fit for k2 and the mechanistic understanding of the inhibition of secondary nucleation on the fibril surface by binding to BRICHOS on α-syn fibrils provide a reductionist explanation for the mechanism by which BRICHOS prevents monomer-dependent secondary nucleation and fibril end elongation.

[0377] BRICHOS has reduced inhibitory effect on aggregation of C-terminally truncated α-syn variants

[0378] We then investigated the effects of BRICHOS on variants of α-syn with C-terminal truncations at positions 110 and 121 (referred to as α-syn110 and α-syn121, respectively). These variants have been shown to exhibit a tendency to accelerate aggregation (Farzadfard et al., Commun Biol, 5(1):123, 2022). Our results showed that BRICHOS had limited effects on α-syn121 and had a smaller inhibitory effect on α-syn110, whose data may not be fitted. These results suggest that the presence of the C-terminus is crucial for the optimal efficacy of BRICHOS in preventing α-syn aggregation. Given that secondary nucleation involves the interaction between the C-terminal segment of α-syn fibrils and the N-terminus of α-syn monomers (Kumari et al., Proc Natl Acad Sci USA, 118(10), 2021), the limited effect of BRICHOS on the aggregation kinetics of C-terminally truncated variants suggests that BRICHOS binds to the C-terminus of α-syn and competes with α-syn monomers for the secondary nucleation process.

[0379] BRICHOS does not affect the morphology of α-syn fibrils

[0380] To investigate whether BRICHOS regulates the morphology of α-syn fibrils, we collected electron microscopy images of α-syn fibrils obtained without and with 100% molar equivalent of BRICHOS during the aggregation reaction. We analyzed the diameters of 100 selected fibrils under both conditions and observed that the diameter of the first generation α-syn fibrils co-incubated with BRICHOS was approximately 10.32 ± 2.07 nm, while the diameter of the α-syn fibrils alone was approximately 11.52 ± 1.10 nm ( Figure 13 ), which is consistent with the published in vitro α-syn fibril 3D structure, which is approximately 10 nm and corresponds to two entangled protofilaments.

[0381] Figure 13 BRICHOS does not affect α-syn fibril morphology. TEM images of α-syn fibrils after fibrillation at 70 μM: (a) molar equivalents without BRICHOS, (b) molar equivalents with 100% BRICHOS. (c) Fibril diameters of α-syn fibrils incubated without or with BRICHOS.

[0382] In conclusion, BRICHOS did not affect the diameter of α-syn fibrils when incubated with α-syn monomers.

[0383] BRICHOS binds to α-syn fibrils but not to monomers

[0384] To evaluate the binding ability of BRICHOS to different α-syn species, we employed various biophysical techniques, including isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence intensity distribution analysis (FIDA). SPR experiments confirmed the binding of BRICHOS to α-syn fibrils.

[0385] To determine the amount of BRICHOS bound to α-syn fibrils, samples of co-incubated BRICHOS-α-syn fibrils were analyzed using native PAGE. These experiments showed that only 9 ± 4% of the BRICHOS molecules were bound to the fibrils when incubated at equimolar concentrations. This observation was confirmed by solution NMR experiments (2D 1 H- 15 N HSQC spectroscopy) was confirmed, in which sonicated α-syn fibrils were added to 15 N-labeled BRICHOS. Due to the dynamic nature of BRICHOS, only a portion of the resonance is expected to be visible in the spectrum. Furthermore, due to the large size of α-syn fibrils, bound BRICHOS is not expected to be visible in the solution NMR spectrum. We found that the addition of α-syn fibrils at equimolar concentrations resulted in a disappearance of approximately 10 ± 10% of the signal, indicating that only a small fraction of BRICHOS is bound. Furthermore, the addition of fibrils at half or double the molar concentration resulted in similar signal loss.

[0386] Fluorescent intracapillary FIDA experiments were performed using fluorescently labeled BRICHOS-Alexa488 to investigate its binding affinity to α-syn fibrils and monomers. Although BRICHOS-Alexa488 did not show any binding to α-syn monomers, higher molecular weight species were detected in the presence of fibrils, indicating binding between BRICHOS-Alexa488 and α-syn monomers. 15 These findings were confirmed by NMR HSQC experiments with N-labeled α-syn, which showed no observable binding between BRICHOS and α-syn monomers. This lack of interaction was further supported by ITC experiments, which demonstrated that BRICHOS did not bind to α-syn monomers and that the observed signal was solely due to the heat of dilution when the BRICHOS sample was injected into the fraction cell.

[0387] BRICHOS binds to α-syn oligomers

[0388] We further investigated the ability of BRICHOS to bind to α-syn oligomers. One challenge we faced stemmed from the complexity of generating α-synuclein oligomers, as various oligomeric forms can emerge under varying environmental conditions, displaying distinct characteristics and different structural arrangements. In our studies, α-synuclein oligomers were prepared by incubating α-synuclein monomers at 37°C for 5 hours, removing larger aggregates by centrifugation, and then separating the oligomeric species by size exclusion chromatography. These specific oligomers were found to be stable, exhibit a β-sheet structure, were kinetically trapped, and were able to permeabilize vesicles in vitro. Using FIDA experiments, we observed that BRICHOS-Alexa488 could bind to these α-syn oligomers. Using simulations using kinetic parameters derived from experimental data, we observed that the presence of BRICHOS reduced the rate of secondary nucleation, leading to the formation of oligomers.

[0389] BRICHOS prevents α-syn neurotoxicity

[0390] We then assessed how the effects of BRICHOS on α-syn aggregation translated into α-syn neurotoxicity. Electrophysiological measurements were performed on hippocampal slices from wild-type mice. Gamma oscillations are characteristic of neuronal activity and an important indicator of the neurotoxicity of the added compounds. Gamma oscillations (γ) were recorded after exposure to control buffer (PBS, 0.1% sodium azide, pH 7.4), sonicated α-syn fibrils alone, or sonicated α-syn fibrils combined with equimolar concentrations of BRICHOS. Figure 14 , ac).

[0391] Figure 14 BRICHOS monomers are shown to protect against the neurotoxic effects of sonicated α-syn fibrils.

[0392] (a) Schematic experimental setup for measuring toxicity in mouse hippocampal brain slices.

[0393] (b) Example traces of γ oscillations: under control conditions (grey), after incubation with 500 nM α-syn sonicated fibrils, after incubation with 1 μM sonicated α-syn fibrils, and after incubation with 1 μM sonicated α-syn fibrils and equimolar concentrations of BRICHOS.

[0394] (c) Example power spectra of gamma oscillations: under control conditions, after incubation with 500 nM α-syn sonicated fibrils, after incubation with 1 μM sonicated α-syn fibrils, and after incubation with 1 μM sonicated α-syn fibrils and equimolar concentrations of BRICHOS.

[0395] Ultrasonicated α-syn fibrils showed a concentration-dependent effect on the power of gamma oscillations. At a fibril concentration of 500 nM, there was no significant reduction in the power of gamma oscillations, whereas a concentration of 1 μM led to a substantial reduction in the power of gamma oscillations, indicating a significant neurotoxic effect. Interestingly, co-incubation of 1 μM ultrasonicated α-syn fibrils with an equimolar concentration of BRICHOS did not affect the power of gamma oscillations, indicating that the presence of BRICHOS effectively prevented the neurotoxic effects of α-syn fibrils.

[0396] Implementation Plan List

[0397] 1. An isolated recombinant protein for use in a method of treating Alzheimer's disease in a mammal in need thereof, the protein being selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4); the mammal comprising a human, the method comprising the steps of:

[0398] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0399] - administering said isolated recombinant protein to said mammal;

[0400] wherein the isolated recombinant protein is not contained within the microvesicles and / or the nanodroplets; and

[0401] The method does not include any step of sonicating any tissue of the mammal.

[0402] 2. An isolated recombinant protein for use in a method of treating Alzheimer's disease in a mammal in need thereof, selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4); the mammal including a human, the method consisting of the following steps:

[0403] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0404] - administering said isolated recombinant protein to said mammal;

[0405] The isolated recombinant protein is not contained in the microvesicles and / or the nanodroplets.

[0406] 3. A combination of an isolated recombinant protein and lipid microvesicles and / or nanodroplets according to any one of the preceding items, for use in a method for treating Alzheimer's disease in a mammal in need thereof, the mammal including a human, the method comprising the following steps;

[0407] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0408] - administering said isolated recombinant protein to said mammal;

[0409] wherein the isolated recombinant protein is not contained within the microvesicles and / or the nanodroplets; and

[0410] The method does not include any step of sonicating any tissue of the mammal.

[0411] 4. A combination of an isolated recombinant protein and lipid microvesicles and / or nanodroplets according to any one of the preceding items, for use in a method for treating Alzheimer's disease in a mammal in need thereof, including a human, the method comprising the following steps:

[0412] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0413] - administering said isolated recombinant protein to said mammal;

[0414] The isolated recombinant protein is not contained in the microvesicles and / or the nanodroplets.

[0415] 5. The isolated recombinant protein or combination for use according to any one of the preceding items, wherein the isolated recombinant protein and the microvesicles and / or the nanodroplets are administered intravenously.

[0416] 6. The isolated recombinant protein or combination for use according to any one of items 1 to 5, wherein the isolated recombinant protein is administered in a therapeutically effective amount of at least 1 mg / kg, such as at least 5 mg / kg, more preferably such as at least 10 mg / kg.

[0417] 7. The isolated recombinant protein or combination for use according to any one of items 1 to 5, wherein the isolated recombinant protein is administered in a therapeutically effective amount of less than 50 mg / kg, such as less than 30 mg / kg, more preferably less than 20 mg / kg.

[0418] 8. An isolated recombinant protein or combination for use according to any one of items 1 to 7, wherein the isolated recombinant protein is selected from the group consisting of: a protein comprising an amino acid sequence that is at least 70%, preferably at least 75%, identical to residues 113-231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 70%, preferably at least 75% identical to the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0419] 9. An isolated recombinant protein or combination for use according to item 8, wherein the isolated recombinant protein is selected from the group consisting of: a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to residues 113-231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 80%, preferably at least 85% identical to the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0420] 10. An isolated recombinant protein or combination for use according to item 9, wherein the isolated recombinant protein is selected from the group consisting of: a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, preferably at least 99% identical to residues 113-231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence that is at least 90% identical to the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0421] 11. The isolated recombinant protein or combination for use according to item 10, wherein the isolated recombinant protein is selected from the group consisting of residues 113-231 of human Bri2 (SEQ ID NO: 2); and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0422] 12. The isolated recombinant protein or combination for use according to any one of items 1 to 7, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0423] 13. The isolated recombinant protein or combination for use according to item 12, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0424] 14. The isolated recombinant protein or combination for use according to item 13, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0425] 15. The isolated recombinant protein or combination for use according to item 14, wherein the isolated recombinant protein is selected from the group consisting of any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0426] 16. The isolated recombinant protein or combination for use according to any one of items 1 to 15, wherein, in the isolated recombinant protein, the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is selected from the group consisting of Glu and Asp.

[0427] 17. The isolated recombinant protein or combination for use according to item 16, wherein, in the isolated recombinant protein, the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is Glu.

[0428] 18. The isolated recombinant protein or combination for use according to any one of items 1 to 17, wherein the isolated recombinant protein consists of less than or equal to 200 amino acid residues, such as less than or equal to 150 amino acid residues.

[0429] 19. The isolated recombinant protein or combination for use according to any one of items 1 to 18, wherein the isolated recombinant protein consists of more than or equal to 90 amino acid residues.

[0430] 20. The isolated recombinant protein or combination for use according to any one of the preceding items, wherein the microvesicles and / or the nanodroplets are coated with lipids.

[0431] 21. An isolated recombinant protein or combination for use according to any one of the preceding items, wherein the microbubbles and / or nanodroplets comprise a core of 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol) 2000) and perfluorobutane gas.

[0432] 22. An isolated recombinant protein or combination for use according to any one of items 1 to 21, wherein the microbubbles and / or nanodroplets comprise sulfur hexafluoride, polyethylene glycol (PEG, Macrogol), distearoylphosphatidylcholine (DSPC), sodium 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol and palmitic acid.

[0433] 23. The isolated recombinant protein or combination for use according to any one of the preceding items, wherein the microvesicles and / or the nanodroplets have a diameter in the range of 1-8 μm, such as 2-6 μm, such as 4-5 μm.

[0434] 24. The isolated recombinant protein or combination for use according to any one of items 1 to 23, wherein the treatment is selected from the group consisting of preventive treatment, palliative treatment and curative treatment.

[0435] 25. A method of treating Alzheimer's disease in a mammal in need thereof, including a human, comprising the steps of:

[0436] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0437] - administering to the mammal an isolated recombinant protein selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4);

[0438] wherein the isolated recombinant protein is not contained within the microvesicles and / or the nanodroplets; and

[0439] The method does not include any step of sonicating any tissue of the mammal.

[0440] 26. A method of treating Alzheimer's disease in a mammal in need thereof, including a human, comprising the steps of:

[0441] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0442] - administering to the mammal an isolated recombinant protein selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); provided that the protein does not comprise an amino acid sequence at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and the protein does not comprise an amino acid sequence at least 70% identical to human ABri23 (SEQ ID NO: 4);

[0443] The isolated recombinant protein is not contained in the microvesicles and / or the nanodroplets.

[0444] 27. The method of any one of items 25-26, wherein the isolated recombinant protein is selected from the group consisting of proteins comprising an amino acid sequence that is at least 70% identical to the BRICHOS domain of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cattle (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0445] 28. The method according to item 27, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0446] 29. The method according to item 28, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0447] 30. The method according to item 29, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0448] 31. The method according to any one of items 25-26, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 70% identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0449] 32. The method according to item 31, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0450] 33. The method according to item 31, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, and preferably at least 99% identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0451] 34. The method according to item 33, wherein the isolated recombinant protein is selected from the group consisting of: residues 113-231 from human Bri2 (SEQ ID NO: 2); and the BRICHOS domain from human Bri2 (SEQ ID NO: 5).

[0452] 35. The method according to any one of items 25-34, wherein the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is selected from the group consisting of Glu and Asp.

[0453] 36. The method according to item 35, wherein the amino acid residue corresponding to position 221 in SEQ ID NO: 1 is Glu.

[0454] 37. The method according to any one of items 25-34, wherein the lipid microvesicles and / or nanodroplets are defined according to any one of items 20-23.

[0455] 38. An isolated protein comprising

[0456] (i) a first protein portion selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); and

[0457] (ii) a second protein or polypeptide portion, preferably comprising at least 50 amino acid residues;

[0458] wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and

[0459] wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to human ABri23 (SEQ ID NO: 4).

[0460] 39. The isolated protein of item 38, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0461] 40. The isolated protein according to item 39, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0462] 41. The isolated protein of item 40, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0463] 42. The isolated protein of item 41, wherein the first protein portion is selected from the group consisting of a protein comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

[0464] 43. The isolated protein according to item 38, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence at least 70% identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0465] 44. The isolated protein according to item 43, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0466] 45. The isolated protein according to item 44, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0467] 46. ​​The isolated protein of item 45, wherein the first protein portion is selected from the group consisting of residues 113-231 from human Bri2 (SEQ ID NO: 2); and the BRICHOS domain from human Bri2 (SEQ ID NO: 5).

[0468] 47. The isolated protein according to any one of items 38-46, wherein the amino acid residue in the first protein portion corresponding to position 221 in SEQ ID NO: 1 is selected from the group consisting of Glu and Asp.

[0469] 48. The isolated protein according to item 47, wherein the amino acid residue in the first protein portion corresponding to position 221 in SEQ ID NO: 1 is Glu.

[0470] 49. The isolated protein according to any of items 38-48, wherein the first protein portion consists of less than or equal to 200 amino acid residues, such as less than or equal to 150 amino acid residues.

[0471] 50. The isolated protein according to any one of items 38-48, wherein the first protein portion consists of more than or equal to 90 amino acid residues.

[0472] 51. The isolated protein according to any one of items 38-50, wherein the second protein or polypeptide portion contains 50 to 2000 amino acid residues, such as 50 to 1000 amino acid residues, such as 50 to 500 amino acid residues, such as 50 to 100 amino acid residues.

[0473] 52. The isolated protein according to any one of items 38-51, wherein the size of the second protein or polypeptide portion is 5-200 kDa, such as 5-100 kDa, such as 5-50 kDa, such as 5-10 kDa.

[0474] 53. The isolated protein according to any one of items 38-52, wherein the first protein moiety is directly or indirectly linked to the amino terminus or carboxyl terminus of the second protein or polypeptide moiety.

[0475] 54. The isolated protein according to any one of items 38-53, wherein the second protein or polypeptide portion constitutes the amino terminus and / or carboxyl terminus of the protein.

[0476] 55. The isolated protein according to any one of items 38-54, wherein the second protein or polypeptide portion is selected from the group consisting of a protein drug, a polypeptide drug, a protein tag, a fluorescent protein, an antibody, an enzyme and / or a neurotrophic factor.

[0477] 56. The isolated protein according to item 55, wherein the second protein or polypeptide portion is an antibody.

[0478] 57. The isolated protein of item 55, wherein the second protein or polypeptide portion is a neurotrophic factor selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), ephrins, epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and / or interleukins.

[0479] 58. The isolated protein of item 55, wherein the second protein or polypeptide portion is selected from the group consisting of α-L-iduronidase, iduronic acid sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase and / or lysosomal acid lipase.

[0480] 59. The isolated protein according to any one of items 38-58, wherein the isolated protein is a recombinant fusion protein.

[0481] 60. The isolated protein according to any one of items 38-58, wherein the first protein moiety is chemically linked to the second protein or polypeptide moiety.

[0482] 61. A combination of an isolated protein and a plurality of lipid microvesicles and / or nanodroplets according to any one of items 38-60, wherein the isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0483] 62. A kit comprising the isolated protein of any one of items 38-60, and a plurality of lipid microvesicles and / or nanodroplets, wherein the isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0484] 63. The combination or kit according to any one of items 61-62, wherein the lipid microvesicles and / or nanodroplets are defined according to any one of items 20-23.

[0485] 64. A method for transporting the isolated protein according to any one of items 38-60 across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the following steps:

[0486] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal;

[0487] - administering said isolated protein to said mammal.

[0488] 65. The method of claim 64, wherein the method does not comprise any step of sonicating any tissue of the mammal.

[0489] 66. The method according to any one of items 64-65, wherein the lipid microvesicles and / or nanodroplets are defined according to any one of items 20-23.

[0490] 67. The isolated protein according to any one of items 38 to 60, for use in a method of treatment involving transport of the isolated protein across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of:

[0491] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0492] - administering said isolated protein to said mammal;

[0493] The isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0494] 68. An isolated protein for use in the method according to item 67, wherein the method does not comprise any step of sonicating any tissue of the mammal.

[0495] 69. The isolated protein according to any one of items 38 to 60, for use in a method of treatment involving transport of the isolated protein across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of:

[0496] - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal

[0497] - administering said isolated protein to said mammal;

[0498] The isolated protein is not contained within the microvesicles and / or the nanodroplets.

[0499] 70. The isolated protein for use according to any one of items 67-69, wherein the lipid microvesicles and / or nanodroplets are defined according to any one of items 20-23.

[0500] 71. A method for transporting the isolated protein according to any one of items 38 to 60 across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of:

[0501] - administering said isolated protein to said mammal.

[0502] 72. A method for transporting the isolated protein according to any one of items 38 to 60 across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of:

[0503] - administering said isolated protein to said mammal.

[0504] 73. A method of treating a medical condition in a mammal, including a human, in need thereof, comprising the steps of:

[0505] - Administering to said mammal an isolated protein according to any one of items 55-60.

[0506] 74. A method of treating a medical condition in a mammal, including a human, in need thereof, comprising the steps of:

[0507] - Administering to said mammal an isolated protein according to any one of items 55-60.

[0508] 75. The isolated protein according to any one of items 55-60, for use as a medicament.

[0509] 76. The isolated protein according to any one of items 55 to 60, for use in a method of treating a medical condition in a mammal in need thereof, including a human, comprising the steps of:

[0510] - Administering to said mammal an isolated protein according to any one of items 55-60.

[0511] 77. The isolated protein according to any one of items 55 to 60, for use in a method of treating a medical condition in a mammal in need thereof, including a human, the method comprising the steps of:

[0512] - Administering to said mammal an isolated protein according to any one of items 55-60.

Claims

1. An isolated protein for use in a method of treating a medical condition in a mammal in need thereof, comprising (i) a first protein portion selected from the group consisting of: a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10); and (ii) a second protein or polypeptide portion, preferably comprising at least 50 amino acid residues, wherein the second protein or polypeptide portion is selected from the group consisting of a protein drug, a polypeptide drug, a protein tag, a fluorescent protein, an antibody, an enzyme and / or a neurotrophic factor; wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to residues 1-89 of Bri2 from human (SEQ ID NO: 3); and wherein the isolated protein does not comprise an amino acid sequence that is at least 70% identical to human ABri23 (SEQ ID NO: 4); The mammal includes a human, and the method comprises the following steps: - administering the isolated protein to said mammal; The administration step does not require lipid microbubbles or nanodroplets.

2. The isolated protein for use according to claim 1, which comprises the following steps: - administering the isolated protein to said mammal, wherein the administering step does not require lipid microbubbles or nanodroplets.

3. The isolated protein for use according to any one of claims 1-2, wherein the second protein or polypeptide moiety is an antibody.

4. The isolated protein for use according to claim 3, wherein the antibody is a monoclonal antibody.

5. The isolated protein for use according to claim 4, wherein the monoclonal antibody is selected from the group consisting of aducanumab, gantnerumab, 3D6 (bapinezumab), m266 (solanesinomab), donetumab and lencanirumab.

6. The isolated protein for use according to claim 5, wherein the monoclonal antibody is selected from the group consisting of aducanumab, gantnerumab, 3D6 (bapinezumab) and m266 (solanesinomab).

7. The isolated protein for use according to claim 5, wherein the antibody is donetumab.

8. The isolated protein for use according to claim 5, wherein the antibody is lencanezumab.

9. The isolated protein for use according to any one of claims 1 to 3, wherein the second protein or polypeptide portion is a neurotrophic factor selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), ephrins, epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and / or interleukins.

10. The isolated protein for use according to any one of claims 1 to 3, wherein the second protein or polypeptide portion is selected from the group consisting of α-L-iduronidase, iduronic acid sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase and / or lysosomal acid lipase.

11. The isolated protein for use according to any one of claims 1 to 3, wherein the second protein or polypeptide portion is effective for treating Parkinson's disease.

12. The isolated protein for use according to claim 11, wherein the second protein or polypeptide portion is selected from the group consisting of glucocerebrosidase, progranulin, prosaposin, cathepsin D and an antibody.

13. The isolated protein for use according to any one of claims 1 to 12, wherein the isolated protein is a recombinant fusion protein.

14. The isolated protein for use according to any one of claims 1 to 12, wherein the first protein moiety is chemically linked to the second protein or polypeptide moiety.

15. The isolated protein for use according to any one of claims 1 to 14, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

16. The isolated protein for use according to claim 15, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

17. The isolated protein for use according to claim 16, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), bovine (SEQ ID NO: 7), porcine (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

18. The isolated protein for use according to claim 17, wherein the first protein portion is selected from the group consisting of proteins comprising any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9) and rat (SEQ ID NO: 10).

19. The isolated protein for use according to any one of claims 1 to 14, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence at least 70% identical to any one of residues 113-231 of Bri2 from human (SEQ ID NO: 2) and the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

20. The isolated protein for use according to claim 19, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 80%, preferably at least 85%, identical to any one of residues 113-231 of Bri2 from human (SEQ ID NO: 2) and the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

21. The isolated protein for use according to claim 20, wherein the first protein portion is selected from the group consisting of a protein comprising an amino acid sequence that is at least 90%, preferably at least 95%, identical to any one of residues 113-231 of Bri2 from human (SEQ ID NO: 2) and the BRICHOS domain of Bri2 from human (SEQ ID NO: 5).

22. The isolated protein for use according to claim 21, wherein the first protein portion is selected from the group consisting of: residues 113-231 from human Bri2 (SEQ ID NO: 2); and the BRICHOS domain from human Bri2 (SEQ ID NO: 5).

23. The isolated protein for use according to any one of claims 1 to 22, wherein the amino acid residue in the first protein portion corresponding to position 221 in SEQ ID NO: 1 is selected from the group consisting of Glu and Asp.

24. The isolated protein for use according to claim 23, wherein the amino acid residue in the first protein portion corresponding to position 221 in SEQ ID NO: 1 is Glu.

25. The isolated protein for use according to any one of claims 1 to 24, wherein the first protein portion consists of less than or equal to 200 amino acid residues, such as less than or equal to 150 amino acid residues.

26. The isolated protein for use according to any one of claims 1 to 25, wherein the first protein portion consists of more than or equal to 90 amino acid residues.

27. An isolated protein for use according to any one of claims 1 to 26, wherein the second protein or polypeptide portion contains 50 to 2000 amino acid residues, such as 50 to 1000 amino acid residues, such as 50 to 500 amino acid residues, such as 50 to 100 amino acid residues.

28. An isolated protein for use according to any one of claims 1 to 27, wherein the size of the second protein or polypeptide portion is 5-200 kDa, such as 5-100 kDa, such as 5-50 kDa, such as 5-10 kDa.

29. The isolated protein for use according to any one of claims 1 to 28, wherein the first protein moiety is linked directly or indirectly to the amino terminus or carboxyl terminus of the second protein or polypeptide moiety.

30. The isolated protein for use according to any one of claims 1 to 29, wherein the second protein or polypeptide portion constitutes the amino terminus and / or carboxyl terminus of the protein.

31. The isolated protein of any one of claims 1-30, wherein the second protein or polypeptide portion is effective for treating Parkinson's disease.

32. The isolated protein of claim 31, wherein the second protein or polypeptide moiety is selected from the group consisting of glucocerebrosidase, progranulin, prosaposin, cathepsin D, and an antibody.

33. The isolated protein according to any one of claims 31-32, for use as a medicament.

34. The isolated protein of any one of claims 31-32 for use in a method of treating Parkinson's disease in a mammal, including a human, in need thereof.

35. An isolated protein for use in a method of treating Parkinson's disease in a mammal in need thereof, comprising a protein comprising an amino acid sequence at least 70% identical to residues 113-231 of Bri2 from human (SEQ ID NO: 2); and a protein comprising an amino acid sequence at least 70% identical to any one of the BRICHOS domains of Bri2 from human (SEQ ID NO: 5), chimpanzee (SEQ ID NO: 6), cow (SEQ ID NO: 7), pig (SEQ ID NO: 8), mouse (SEQ ID NO: 9), and rat (SEQ ID NO: 10), including human.

36. The isolated protein for use according to claim 35, wherein the isolated protein is further defined as the first protein portion of any one of claims 15 to 26.

37. The isolated protein of claim 4.

38. The isolated protein of claim 37, wherein the isolated protein is further defined as any one of claims 13-26 and 29-30.

39. The isolated protein of any one of claims 37-38, wherein the antibody is donetumab.

40. The isolated protein of any one of claims 37-38, wherein the antibody is lencanezumab.

41. A method for transporting the isolated protein according to any one of claims 1 to 30 across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of: - administering said isolated protein to said mammal; The administration step does not require lipid microbubbles or nanodroplets.

42. A method of treating a medical condition in a mammal, including a human, in need thereof, comprising the steps of: - administering to the mammal an isolated protein according to any one of claims 1 to 30; The administration step does not require lipid microbubbles or nanodroplets.

43. A method of treating Parkinson's disease in a mammal in need thereof, including a human, comprising the steps of: - Administering to said mammal an isolated protein according to any one of claims 31-32.

44. A method for delaying the accumulation of alpha-synuclein in a mammal in need thereof, including a human, comprising the steps of: - Administering to said mammal an isolated protein according to any one of claims 31-32.

45. A method for transporting the isolated protein according to any one of claims 31-32 and 37-40 across the blood-brain barrier in a mammal in need thereof, including a human, the method comprising the steps of: - administering said isolated protein to said mammal.

46. ​​A method of treating a medical condition in a mammal, including a human, in need thereof, comprising the steps of: - administering to said mammal an isolated protein according to any one of claims 31-32 and 37-40.

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