Bri2 BRICHOS domain delivers proteins to CNS neurons

Abstract

An isolated protein for use as a pharmaceutical, comprising: (i) a first protein portion selected from the group consisting of proteins comprising an amino acid sequence having at least 70% identity with residues 113 to 231 of human Bri2 (SEQ ID NO: 2); and proteins comprising an amino acid sequence having at least 70% identity with any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively); and (ii) a second protein or polypeptide portion, preferably of at least 50 amino acid residues, selected from the group consisting of protein pharmaceuticals, polypeptide pharmaceuticals, protein tags, fluorescent proteins, antibodies, enzymes, and / or neurotrophic factors, wherein the isolated protein does not comprise an amino acid sequence having at least 70% identity with residues 1 to 89 of human Bri2 (SEQ ID NO: 3) and does not comprise an amino acid sequence having at least 70% identity with human ABri23 (SEQ ID NO: 4).

Description

[Technical Field]

[0001] This invention relates to the field of medicine. More specifically, this invention relates to the use of substances and agents to improve the treatment of Alzheimer's disease in mammals, including humans. Furthermore, this invention relates to substances and agents that transport proteins across the blood-brain barrier in mammals, including humans. This invention also relates to the delivery of proteins to CNS neurons and the treatment of neurological disorders. [Background technology]

[0002] Neurodegenerative disorders associated with protein misfolding and aggregation, such as Alzheimer's disease and familial British-Danish dementia, are on the rise. These diseases are characterized by protein deposition in the brain parenchyma and arteries and occur in both hereditary and sporadic forms. Although the clinical manifestations of these diseases differ, they share several common pathological features, such as neuronal loss, protein aggregates, and the presence of tau tangles. From a biochemical perspective, the proteins involved tend to form beta-sheet structures and aggregate into amyloid fibrils. Alzheimer's disease and familial British-Danish dementia share several similar neuropathological features, including amyloid plaques, neurofibrillar tangles, congophilic amyloid angiopathy, and neurodegeneration. Alzheimer's disease is one of the most common causes of dementia in humans. It is a chronic, fatal disease accompanied by neuronal degeneration in the brains of patients and characterized by the presence of amyloid plaques, consisting of extracellular deposits of amyloid beta peptide (Aβ peptide). Aβ aggregation leads to neuronal atrophy, resulting in a lack of acetylcholine and other signaling molecules. It is known that the 40-42 amino acid Aβ peptide is generated by processing of the amyloid precursor protein (APP, 695-770 amino acid residues), a type I membrane protein normally expressed in neurons of the central nervous system. However, the mechanism behind this processing is not fully understood. The released Aβ peptide contains part of the transmembrane domain of APP (29-40 / 42 of the Aβ residues) and contains a disconcerted helix, i.e., a helix composed of amino acids with a high propensity to form β-strands. When Aβ is removed from its stable membrane environment, it tends to misfold and aggregate.

[0003] Bri2 (SEQ ID NO: 1, also known as integral membrane protein 2B, ITM2B) is a 266-residue type II membrane protein (Figure 1) that is ubiquitously expressed, but its function and folding are unknown. Bri2 undergoes proteolytic cleavage at three sites: cleavage of the C-terminal region by furin to generate a 23-residue peptide (ABri23); processing of the extracellular domain by ADAM10 releases the BRICHOS domain from the membrane-bound N-terminus; and intramembrane cleavage by SPPL2a / 2b to release the intracellular domain. Familial British-Danish dementia is caused by a genetic mutation that results in the loss of a stop codon, resulting in two different 11-residue extensions (ABri) at the C-terminus. After cleavage by furin, a 34-residue peptide (ADan) is generated instead of the normally released ABri23. The long peptide tends to aggregate into amyloid fibrils and deposit in brain tissue or blood vessels, causing 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 proteins interact at some stage of misfolding and aggregation. Using transfected cell lines, Bri2 has been found to interact with APP and regulate APP processing by increasing the amount of β-secretase-generated fragments. Generation of a Bri2 / Aβ40 fusion protein indicates that Bri proteins may affect the aggregation properties of Aβ, and experiments using transgenic mouse models suggest that Bri23 interacts with Aβ42 and prevents its aggregation (Kim et al. J. Neurosci. 28: 6030-6036 (2008); WO 2009 / 009396). It has also been suggested that a protein containing the first 102 amino acid residues of Bri2 can reduce or prevent Aβ production (WO 2006 / 138355).

[0005] The BRICHOS domain is a natural chaperone with anti-amyloid properties. There are 10 human proproteins, one of which (proSP-C) is associated with amyloid lung disease, and another (Bri2 / ITM2b) is associated with amyloid-associated dementia and familial British-Danish dementia, as previously described. Recombinant (rh) human BRICHOS domains of proSP-C and Bri2 delay the formation of Aβ40 and Aβ42 fibrils and reduce the neurotoxicity associated with Aβ42 fibril formation in vitro and in vivo (WO 2011 / 62655).

[0006] The blood-brain barrier (BBB) ​​maintains the delicate homeostasis required for normal neuronal function. It also acts as a barrier against brain-targeting substances and drugs, and large molecules cannot spontaneously cross the BBB. Nevertheless, the BBB provides an efficient route for the transport of compounds, such as drugs, medicinal agents, and biopharmaceuticals (e.g., proteins), into the central nervous system (CNS). Only lipophilic small molecules have been shown to be able to passively cross the BBB. Recent studies have shown that isolated recombinant Bri2 BRICHOS protein tagged with an immunodetectable AU1 tag (Bri2 BRICHOS-AU1) was detected in the brain parenchyma after peripheral (intravenous) administration in wild-type mice, whereas recombinant proSP-C BRICHOS protein was translocated exclusively to the cerebrospinal fluid (CSF). This suggests that there is an unknown mechanism for transporting 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] Various methods for delivering compositions across the BBB have been developed. The combination of focused ultrasound and intravenous lipid microbubbles (MBs) locally and reversibly opens the BBB, allowing the transport of macromolecules, including proteins, into the brain parenchyma surrounding the ultrasound target area (Mikitsh et al. Perspective Medicin Chem. 6: 11-24 (2014); Brasnjevic et al. Prog Neurobiol. 87: 212-51 (2009); Konofagou et al. Theranostics. 2: 1223-37 (2012); Sierra et al. J Cereb Blood Flow Metab. 37:1236-1250 (2017)). This method requires specialized ultrasound equipment. Furthermore, it has been thought that ultrasound treatment may cause vascular damage under certain conditions.

[0008] Biverstal et al., Scientific Reports, 10:21765 (2020) discloses the functionalization of amyloid fibrils using a fusion protein containing the Bri2 BRICHOS domain, which is reported to be useful for synthesizing amyloids modified with various protein functional groups.

[0009] Current treatments for Alzheimer's disease are primarily directed at treating symptoms and include choline replacement therapy (e.g., inhibition of acetylcholinesterase), small molecule inhibitors that interact with soluble Aβ oligomers, and so-called β-sheet breakers that prevent the elongation of already formed β-sheet structures. Furthermore, it is interesting to note that ultrasound treatment can be combined with other therapies to reduce the side effects associated with it, potentially reducing the dosage of therapeutic drugs. Summary of the Invention

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

[0011] It is also an object of the present invention to provide a powerful and simple method and means for efficiently delivering proteins, such as therapeutic agents, to the CNS via the blood-brain barrier, and in particular to achieve delivery of proteins, such as therapeutic agents, to CNS neurons.

[0012] One of the aims of the present invention is to provide simple and effective methods and means for the efficient delivery of Bri2 BRICHOS and its variants 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 to prevent fibrillation-prone proteins from aggregating into amyloid fibrils.

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

[0015] The objective of the present invention is to influence the distribution of isolated recombinant Bri2 BRICHOS and its variants so as to increase the therapeutically effective amount of isolated recombinant Bri2 BRICHOS that 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 subsequent prognosis, and / or imaging of Alzheimer's disease and other neurological disorders in mammals, including humans.

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

[0018] The present invention is generally based on the discovery that the isolated recombinant protein Bri2 BRICHOS and its variants, when administered in combination with lipid microbubbles and / or nanodroplets, are efficiently delivered across the blood-brain barrier without ultrasound treatment of mammalian tissue. Surprisingly, co-administration with lipid microbubbles and / or nanodroplets improves uptake of Bri2 BRICHOS and its variants without ultrasound treatment, compared to both (a) administration without lipid microbubbles and / or nanodroplets and (b) administration with ultrasound treatment. This method does not involve treatment of mammalian tissue with light, sound, or ultrasound waves, which cavitates the microbubbles and / or nanodroplets in the tissue.

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

[0020] One aspect of the present invention is based on the discovery that isolated recombinant proteins, including Bri2 BRICHOS and its variants, including Bri2 BRICHOS R221E, can be efficiently delivered to CNS neurons across the blood-brain barrier. This method does not require the administration of lipid microbubbles and / or nanodroplets. This method does not require treatment of mammalian tissue with light waves, sonic waves, or ultrasound. Bri2 BRICHOS R221E is described in WO 2021 / 140140, the entire contents of which are incorporated herein by reference.

[0021] To achieve these and other objectives which will become apparent from the following description and claims, in a first aspect the invention provides an isolated recombinant protein comprising Bri2 BRICHOS and variants thereof for use in the treatment of Alzheimer's disease. In a second aspect there is provided a method for treating Alzheimer's disease, comprising administering an isolated recombinant protein comprising Bri2 BRICHOS and variants thereof and lipid microbubbles and / or nanodroplets without ultrasound treatment.

[0022] In a third aspect, there is provided a combination of a protein having a first protein portion that is Bri2 BRICHOS and variants thereof and a second protein or polypeptide portion with lipid microbubbles and / or nanodroplets, and in a fourth aspect, this combination is useful in a method of transporting a protein across the blood-brain barrier in a mammal without ultrasound treatment.

[0023] A third aspect of the protein, comprising a first protein portion that is Bri2 BRICHOS and variants thereof and a second protein or polypeptide portion, is useful for increasing transport of the second protein or polypeptide portion across the blood-brain barrier. The first (Bri2 BRICHOS) portion aids transport, which is achieved without the use of lipid microbubbles and / or nanodroplets. One particularly useful variant of the first protein portion is Bri2 BRICHOS R221E, as BRICHOS R221E has been shown herein to improve transport compared to wild-type Bri2 BRICHOS. Lipid microbubbles and / or nanodroplets can further improve transport. Even if lipid microbubbles and / or nanodroplets are included, the method does not require treatment of mammalian tissue with light, sound, or ultrasound waves to cavitate the microbubbles and / or nanodroplets in the tissue.

[0024] In another aspect, there is provided a treatment for Parkinson's disease comprising administering a protein comprising Bri2 BRICHOS and variants thereof. Proteins are further provided having a first protein portion that is Bri2 BRICHOS and variants thereof, and a second protein or polypeptide portion, the second protein or polypeptide portion being itself effective in treating Parkinson's disease. This combination is also useful for treating Parkinson's disease. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram of Bri2 (SEQ ID NO: 1) and the processing site. [Figure 2] FIG. 2 shows an alignment of mammalian Bri2 BRICHOS amino acid sequences (SEQ ID NOs: 5 to 10). [Figure 3] FIG. 3 shows the experimental design in the examples. [Figure 4] FIG. 4 shows that Bri2 BRICHOS, but not proSP-C BRICHOS, is detected in the non-FUS-targeted brain hemisphere after intravenous injection with lipid microbubbles and / or nanodroplets. [Figure 5] FIG. 5 shows that Bri2 BRICHOS is detected in both hemispheres after intravenous injection with lipid microbubbles and / or nanodroplets. [Figure 6] FIG. 6 shows intracellular immunostaining of Bri2 BRICHOS-AU1 in the cortex and hippocampus after intravenous injection with lipid microbubbles and / or nanodroplets. [Figure 7] FIG. 7 shows Western blots of Bri2 BRICHOS-AU1 in both hemispheres after systemic injection with lipid microbubbles and / or nanodroplets. [Figure 8] FIG. 8 shows that rhBri2 BRICHOS-mCherry is found in the striatum after systemic injection in combination with lipid microbubbles and / or nanodroplets. [Figure 9]FIG. 9 shows the detection of Bri2 BRICHOS in the brains of AppNL-GF and AppNL-F mice after repeated injections of Bri2 BRICHOS R221E. [Figure 10] FIG. 10 shows the permeability of various proteins and macromolecules through hCMEC / D3 monolayers. [Figure 11] FIG. 11 shows staining with anti-NT antibody of the brain cortex of mice injected with NT-Bri2 BRICHOS (A) or PBS (B). [Figure 12] FIG. 12 shows that rhBri2 BRICHOS mediates uptake into mouse primary neurons. [Figure 13] Figure 13 shows TEM images of post-fibrillation α-syn fibrils in the absence (a) or presence of 100% (b) of 70 μM molar equivalent of BRICHOS. (c) Diameter of α-syn fibrils incubated with or without BRICHOS. [Figure 14] FIG. 14 shows that BRICHOS monomers prevent the neurotoxic effects of sonicated α-syn fibrils.

[0026] [Table A-1]

[0027] [Table A-2] DETAILED DESCRIPTION OF THE INVENTION

[0028] Bri2 (SEQ ID NO: 1), also known as integral membrane protein 2B (ITM2B), contains an evolutionarily conserved BRICHOS domain at residues 137-231. BRICHOS domains are found in over 10 different families of functionally unrelated proteins expressed in various tissues. The name BRICHOS comes from the identification of the domain in Bri, chondromodulin-1, which is associated with chondrosarcoma, and pulmonary surfactant protein C precursor (proSP-C), which is involved in respiratory disease.

[0029] 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 and ABri / ADan peptides.

[0030] The blood-brain barrier (BBB) ​​maintains the delicate homeostasis required for normal neuronal function. The BBB also acts as a barrier to brain-targeting substances and drugs, and large molecules cannot spontaneously cross the BBB. Nevertheless, the BBB provides an efficient route for the transport of pharmaceutical compositions, such as proteins, into the central nervous system (CNS). Only small lipophilic molecules have been shown to be able to passively cross the BBB.

[0031] The present invention is generally based on the discovery that isolated Bri2 BRICHOS protein, either by itself or conjugated to a second protein or polypeptide moiety, can be efficiently delivered into the brain in combination with lipid microbubbles and / or nanodroplets, and is therefore useful in the treatment of Alzheimer's disease.

[0032] Furthermore, isolated Bri2 BRICHOS proteins conjugated to a second (cargo) protein or polypeptide moiety can be efficiently delivered into the brain in the absence of lipid microbubbles and nanodroplets.

[0033] Lipid microbubbles and / or nanodroplets and isolated proteins may be administered in combination. As used herein, the term "combination" refers to the fact that the isolated protein and lipid microbubbles and / or nanodroplets may be administered individually, independently of each other, in any order. The term may also refer to the fact that the recombinant protein and lipid microbubbles and / or nanodroplets may be administered individually and simultaneously. Furthermore, the term may refer to the fact that the isolated protein and lipid microbubbles and / or nanodroplets may be administered in the same pharmaceutical composition.

[0034] The isolated protein of the present invention can be formulated into a pharmaceutical composition. Such compositions typically contain the isolated protein of the present invention and a suitable pharmaceutically acceptable carrier. As used herein, "suitable pharmaceutical carrier" includes solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The composition can also contain a supplementary active compound.

[0035] In conventional methods, ultrasound is the key local stimulus for inducing drug release in target tissues, and may be a pressure wave with a frequency of 20 kHz or higher. Like light and sound waves, ultrasound waves pass through media and are focused, reflected, and refracted.

[0036] Microbubbles and / or nanodroplets combined with ultrasound treatment are commonly used for medical diagnosis and noninvasive delivery of pharmaceutical compositions and genes to various tissues. Focused ultrasound combined with intravenously administered microbubbles and / or nanodroplets has been shown in multiple in vivo models to efficiently and minimally invasively deliver small and large molecules across the BBB to specifically targeted brain regions. This technique involves administering lipid-based microbubbles and / or nanodroplets along with the pharmaceutical composition to be delivered. Ultrasound induces cavitation of the microbubbles and / or nanodroplets within brain capillaries. At such low pressures, the microbubbles and / or nanodroplets exhibit stable cavitation, which induces increased BBB opening. This temporarily loosens tight junctions between endothelial cells, resulting in temporary and localized BBB opening. This results in the delivery of blood-borne macromolecules to the brain parenchyma (Galan-Acosta et al. Mol Cell Neurosci, 103498 (2020)).

[0037] This paper describes a simple method for efficiently delivering compositions to neurons in the CNS through tissues such as the blood-brain barrier. This method combines the administration of isolated proteins with the administration of lipid microbubbles and / or nanodroplets. Importantly, this method does not involve any ultrasound treatment steps, which has the advantage of not requiring an ultrasound device. Furthermore, it avoids side effects associated with ultrasound treatment, such as vascular damage. Surprisingly, coadministration with lipid microbubbles and / or nanodroplets improves the uptake of Bri2 BRICHOS and its variants, even without ultrasound treatment, compared to both (a) administration without lipid microbubbles and / or nanodroplets and (b) administration with ultrasound treatment.

[0038] The method does not include treating mammalian tissue with light, sound or ultrasound waves to cavitate microbubbles and / or nanodroplets in the tissue.

[0039] To these and other objects which will become apparent from the following description, in a first aspect the present invention comprises: an isolated recombinant protein selected from the group consisting of a protein consisting of an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) and an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: The isolated recombinant protein is not contained within the microbubbles and / or nanodroplets, and the method does not include an ultrasonic treatment step on the mammalian tissue;

[0040] In this specification, we experimentally demonstrate that lipid microbubbles and / or nanodroplets alone, i.e., without ultrasound treatment, can uniformly transport Bri2 BRICHOS across the BBB, and that administering Bri2 BRICHOS in combination with lipid microbubbles and / or nanodroplets delivers Bri2 BRICHOS to the brain parenchyma and efficiently takes up into neurons in the cortex and hippocampus.

[0041] Surprisingly, we found that Bri2 BRICHOS protein was delivered to the brains of wild-type mice when administered in combination with lipid microbubbles and / or nanodroplets. This is particularly surprising in light of the various methods previously developed for delivering drugs and other compositions across the BBB using focused ultrasound in combination with intravenous lipid microbubbles and / or nanodroplets, which enable the transport of macromolecules, including proteins, into the brain parenchyma surrounding the ultrasound target area.

[0042] This invention is based on the surprising discovery disclosed herein that microbubbles and / or nanodroplets alone, without being combined with ultrasound treatment of mammalian tissue, are capable of mediating increased transport of isolated recombinant proteins across the BBB. Microbubbles and / or nanodroplets increase BBB penetration and neuronal uptake of Bri2 BRICHOS.

[0043] This aspect of the invention has the advantage that a temporary, localized opening of the BBB is achieved without the use of additional steps, such as ultrasound treatment, or the additional equipment associated with such steps. Thus, a convenient method for efficiently delivering compositions to neurons of the CNS through tissues such as the blood-brain barrier is provided. Another advantage is that, as shown herein, the uptake of the isolated recombinant protein into the brain parenchyma is significantly increased when administered in combination with lipid microbubbles and / or nanodroplets, as compared to administration of the isolated recombinant protein alone, as described in WO 2011 / 162655.

[0044] In this specification and claims, the term "percent similarity" is calculated as described under "percent 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 natural amino acid Gly is not similar to any other amino acid.

[0045] Throughout this description, alternative embodiments will substitute a corresponding percentage of similarity for a specific percentage of identity. Other alternative embodiments will combine a specific percentage of identity with another higher percentage of similarity selected from a group of preferred percentages of identity for each sequence. For example, the sequence of an isolated recombinant protein may be 70% similar to another protein sequence, 70% identical to another sequence, or 70% identical and even 90% similar to another sequence.

[0046] For the avoidance of doubt, an amino acid sequence having at least a certain percentage identity to either residues 113-231 of human Bri2 or the BRICHOS domain of Bri2 consists of 70 or more amino acid residues, such as 80 or more, for example 90 or more amino acid residues. A preferred size is 70-100 amino acid residues, for example 80-100 amino acid residues, for example 90-100 amino acid residues.

[0047] It is noted that the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOS: 5, 6, 7, 8, 9, and 10, respectively) are highly conserved (see alignment in Figure 2). Without wishing to be bound by any particular theory, it is believed that the BRICHOS domains have the desired activity against Aβ and ABri / ADan peptides. Preferably, the isolated recombinant protein of the present invention is selected from the group consisting of proteins comprising an amino acid sequence having at least 80%, preferably at least 90%, e.g., at least 95%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOS: 5, 6, 7, 8, 9, and 10, respectively). In a preferred embodiment, the isolated recombinant protein of the present invention comprises all amino acid residues conserved in the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), i.e., all amino acid residues of SEQ ID NO: 5 except for residues 42, 76, and 82 (corresponding to residues 178, 212, and 218 in full-length Bri2 (SEQ ID NO: 1)). In a specific embodiment, the isolated recombinant protein of the present invention is selected from the group consisting of proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), i.e., comprises one of these BRICHOS domains, preferably the human BRICHOS domain (SEQ ID NO: 5).

[0048] In a preferred embodiment, the amino acid residue corresponding to position 221 of SEQ ID NO:1 is selected from Glu and Asp. In a specific embodiment, the amino acid residue corresponding to position 221 of SEQ ID NO:1 is Glu. rhBri2BRICHOS was mutated so that monomers are more stable than larger oligomers, and rhBri2BRICHOS R221E (SEQ ID NO:13) selectively reduces Aβ42 oligomer formation and alleviates Aβ42-induced neurotoxicity in hippocampal slice preparations. rhBri2BRICHOS R221E crosses the BBB in mice and tends to have a higher permeability than the wild-type protein. This result is consistent with previous observations that wild-type rhBri2BRICHOS monomers cross the BBB more efficiently than large oligomers.

[0049] Contrary to conventional teachings, the isolated recombinant proteins of the present invention do not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO:3). In one aspect, the isolated recombinant proteins of the present invention do not comprise an amino acid sequence having at least 50% identity to residues 1-89 of human Bri2 (SEQ ID NO:3). This means that the isolated recombinant proteins of the present invention have a core amino acid sequence that shows high similarity or identity to residues 113-231 of human Bri2 (SEQ ID NO:2) and / or the mammalian BRICHOS domain of Bri2 (SEQ ID NOs:5-10), and, optionally, one or more other amino acid sequences that may not show high similarity or identity to residues 1-89 of human Bri2 (SEQ ID NO:3).

[0050] For the avoidance of doubt, amino acid sequences shorter than 10 amino acid residues are not considered relevant in the context of being excluded from the isolated recombinant protein of the present invention. Accordingly, the isolated recombinant protein of the present invention does not include an amino acid sequence consisting of 10 or more amino acid residues that has at least a certain percentage identity to residues 1-89 of human Bri2 (SEQ ID NO: 3).

[0051] Furthermore, isolated recombinant proteins of the invention do not comprise residues 244-266 of human Bri2, i.e., an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO:4). In one aspect, isolated recombinant proteins of the invention do not comprise an amino acid sequence having at least 50% identity to residues of human ABri23 (SEQ ID NO:4). As noted above, this means that isolated recombinant proteins of the invention have a core amino acid sequence that shows high similarity or identity to residues 113-231 of human Bri2 (SEQ ID NO:2) and / or the mammalian BRICHOS domain of Bri2 (SEQ ID NOs:5-10), as well as, optionally, one or more other amino acid sequences that may not show high similarity or identity to residues 1-89 of human Bri2 (SEQ ID NO:3).

[0052] For the avoidance of doubt, amino acid sequences shorter than 10 amino acid residues are not considered relevant in the context of being excluded from the isolated recombinant protein of the present invention. Accordingly, the isolated recombinant protein of the present invention does not include amino acid sequences consisting of 10 or more amino acid residues that have at least a certain percentage identity to human ABri23 (SEQ ID NO: 4).

[0053] In a preferred embodiment, the isolated recombinant protein used in the present invention is selected from the group consisting of a protein comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70% identity to the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0054] In a preferred embodiment, an isolated recombinant protein selected from the group consisting of a protein consisting of an amino acid sequence having at least 70% identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1 to 89 of human Bri2 (SEQ ID NO: 3) and an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; administering said isolated recombinant protein to said mammal; wherein the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets.

[0055] In certain embodiments, a combination of isolated recombinant protein and lipid microbubbles and / or nanodroplets, administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; A combination is provided wherein the method does not include a step of subjecting the mammalian tissue to ultrasound treatment.

[0056] In certain embodiments, a combination of isolated recombinant protein and lipid microbubbles and / or nanodroplets, administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: Combinations are provided wherein said isolated recombinant protein is not contained within said microbubbles and / or nanodroplets.

[0057] Pharmaceutical compositions comprising the isolated recombinant proteins may be useful as medicines, particularly for the treatment of diseases such as Alzheimer's disease in mammals, including humans.

[0058] Pharmaceutical compositions are formulated to be compatible with the route of administration, examples of which include parenteral (e.g., intravenous, intraarterial), intraperitoneal, intramuscular, intradermal, and intranasal.

[0059] Administration of the microbubbles and / or nanodroplets and the isolated recombinant protein may be by infusion.

[0060] Sterile injectable solutions can be prepared by mixing the required amount of the isolated recombinant protein of the present invention in an appropriate solvent with one or more of the ingredients listed above, followed by sterile filtration, if necessary. Generally, dispersions can be prepared by mixing the isolated recombinant protein of the present invention with a sterile vehicle containing the required other ingredients and a dispersion medium. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the isolated recombinant protein of the present invention and any other desired ingredients from a previously sterile-filtered solution.

[0061] Generally, sterile injection solutions of lipid microbubbles and / or nanodroplets may be prepared, for example, as described in Feshitan, JA et al., J. Colloid Interface Sci. 329 (2), 316-324 (2009). Briefly, microbubbles and / or nanodroplets can be formed using the gas perfluorobutane (PFB). PFB can act as a gas core that is introduced to activate the lipid microbubbles and / or nanodroplets and then isolated. The microbubbles and / or nanodroplets can be coated with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), polyoxyethylene-40 stearate (PEG40S), DSPC, and PEG40S. The resulting dried lipid film can be hydrated with filtered PBS and mixed into the final lipid suspension.

[0062] The lipid mixture can be sonicated to disperse lipid aggregates into small unilamellar liposomes. PFB gas can be introduced by flowing it over the surface of the lipid suspension. The air-liquid interface of the suspension can then be subjected to high-power sonication to generate microbubbles and / or nanodroplets. After separation, the microbubbles and / or nanodroplets of the present invention can be mixed with a sterile medium; for example, the microbubble and / or nanodroplet suspension can be collected in a 30 mL syringe, and washing and size fractionation can be achieved by centrifugation to collect all microbubbles and / or nanodroplets in the suspension in a cake that contacts the syringe plunger. The remaining suspension (precipitate), which may contain lipids or vesicles that did not form microbubbles, can be reused for subsequent microbubble and / or nanodroplet production. The entire cake obtained can be resuspended in PBS to improve overall yield.

[0063] In one embodiment, the isolated recombinant protein and the lipid microbubbles and / or nanodroplets are administered intravenously.

[0064] When administering the isolated proteins of the present invention to animals (e.g., humans) to treat Alzheimer's disease, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose initially, and then increase the dose until an appropriate response is obtained. Furthermore, it will be understood that the specific dose for a particular animal subject will depend on a variety of factors, including the activity of the particular isolated recombinant protein used, the age, weight, general condition, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any associated medications, and the degree of expression or activity to be modulated.

[0065] The data from cell culture assays and animal studies may be used in formulating a range of dosage for humans.

[0066] The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For compounds 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 cell death is observed. IC determined in cell culture 50 Doses may be formulated in animal models to achieve a plasma concentration range that includes (the concentration of the test compound that achieves 50% of the maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0067] As defined herein, a therapeutically effective amount (effective dose) of the isolated recombinant protein of the present invention is about 1 to 50 mg / kg body weight. In one embodiment, a therapeutically effective amount of at least 1 mg / kg, e.g., at least 5 mg / kg, more preferably at least 10 mg / kg, of the isolated recombinant protein is administered.

[0068] In another embodiment, a therapeutically effective amount of the isolated recombinant protein is administered that is less than 50 mg / kg, for example, less than 30 mg / kg, more preferably less than 20 mg / kg.

[0069] The isolated recombinant protein can be administered to a subject long-term, for example, for the duration of the subject's life. Doses of 1 mg / kg to 50 mg / kg body weight are typically appropriate. Administration of the isolated recombinant protein in combination with the lipid microbubbles and / or nanodroplets of the present invention allows for the administration of significantly lower doses of the isolated recombinant protein than those described in WO 2011 / 162655. Administration of lipid microbubbles and / or nanodroplets locally and reversibly opens the BBB, allowing for efficient transport and increased uptake of the isolated recombinant protein into the brain parenchyma. This aspect of the invention is advantageous because it minimizes the risk of side effects associated with the administration of therapeutic compositions in both prophylactic and therapeutic treatments for subjects suffering from or at risk for (or susceptible to) Alzheimer's disease.

[0070] In some cases, the lipid microbubbles and / or nanodroplets and isolated recombinant protein can be administered once a week for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5, or 6 weeks. The compounds can also be administered over a longer period. Those skilled in the art will appreciate that factors including, but not limited to, the severity of the disease or disorder, previous treatment, the subject's general condition and / or age, and other diseases present can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of lipid microbubbles and / or nanodroplets and isolated recombinant protein can be a single treatment or, preferably, a series of treatments.

[0071] In a further aspect, the isolated protein is selected from a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identity to the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0072] In a preferred embodiment, the isolated 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).

[0073] In a related aspect, the invention provides an isolated protein selected from the group of proteins comprising an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

[0074] In another embodiment, the isolated protein is selected from proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

[0075] In particular embodiments, the isolated protein is selected from any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively).

[0076] In some embodiments, the isolated protein consists of 200 amino acid residues or less, such as 150 or less, or even 100. In some embodiments, the isolated protein consists of 90 or more amino acid residues, such as 100 or more amino acid residues.

[0077] The preferred size of the isolated protein is 80 to 200 amino acid residues, for example, 90 to 150 amino acid residues, for example, 90 to 100 amino acid residues.

[0078] Microbubbles and / or nanodroplets are tiny, gas-filled spheres. They may consist of gas surrounded by a lipid, lipopolymer, or polymer shell. They may also be similar in size to red blood cells, ranging from 0.5 to 10 μm.

[0079] Gas-filled microbubbles and / or nanodroplets vibrate when exposed to acoustic energy. Microbubbles and / or nanodroplets with a hydrophilic outer layer that interacts with the bloodstream and a hydrophobic inner layer that accommodates gas molecules are thermodynamically most stable. Air, sulfur hexafluoride, and perfluorocarbon gases may serve as components of the microbubble interior. Gases with large molecular weights and low blood solubility are attractive candidates for the gas core of microbubbles to increase stability and persistence in the bloodstream. Microbubbles and / or nanodroplets may also be used for drug delivery, potentially serving not only as a drug vehicle but also as a means to cross normally impermeable barriers, particularly the blood-brain barrier.

[0080] In a further aspect of the invention, the microbubbles and / or nanodroplets are lipid coated.

[0081] As disclosed herein, the microbubbles and / or nanodroplets may contain a gas core of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)2000), and perfluorobutane. The microbubbles and / or nanodroplets may contain, for example, sulfur hexafluoride, polyethylene glycol (PEG, macrogol), distearylphosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol sodium, and palmitic acid.

[0082] In a preferred embodiment of the invention, the individual microbubbles and / or nanodroplets of the invention have a diameter of 1-8 μm, such as 2-6 μm, for example 4-5 μm.

[0083] Those skilled in the art will understand that the aspects discussed above with respect to the first aspect of this disclosure equally apply in relation to the second, third and further aspects disclosed herein. This is particularly true of aspects relating to the combination of isolated recombinant proteins and lipid microbubbles and / or nanodroplets, the efficient and uniform transfer of the isolated recombinant proteins across the BBB into the brain parenchyma and their efficient uptake by cortical and hippocampal neurons, as well as aspects relating to methods and routes of administration. For the sake of brevity, these will only be mentioned briefly in the specification without repeating the subject matter.

[0084] In a second aspect of the invention, there is provided a method of treating Alzheimer's disease in a mammal, including a human, in need thereof, comprising: administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; administering to the mammal an isolated recombinant protein selected from the group consisting of a protein consisting of an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), provided that the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) or an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); A method is provided, comprising: wherein the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets, and the method does not include an ultrasonic treatment step on the mammalian tissue.

[0085] In an embodiment of the second aspect, there is provided a method of treating Alzheimer's disease in a mammal, including a human, in need thereof, comprising: administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; administering to the mammal an isolated recombinant protein selected from the group consisting of a protein consisting of an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), provided that the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) or an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); A method is provided, comprising: wherein said isolated recombinant protein is not contained within said microbubbles and / or nanodroplets.

[0086] In a further aspect of the invention, there is provided an isolated recombinant protein consisting of residues 113 to 231 of human Bri2 (SEQ ID NO: 2).

[0087] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk for (or susceptible to) Alzheimer's disease. As used herein, the term "treatment" is defined as the application or administration of an isolated recombinant protein and lipid microbubbles and / or nanodroplets of the present invention to a patient suffering from, exhibiting symptoms of, or predisposed to Alzheimer's disease, or to a tissue or cell line isolated from such a patient, with the intent to cure, heal, alleviate, mitigate, alter, treat, ameliorate, improve, or affect the disease, symptoms of the disease, or predisposition to the disease. In certain embodiments, the treatment is selected from the group consisting of preventative treatment, palliative treatment, and definitive treatment.

[0088] In one aspect, the invention provides a method for preventing (i.e., reducing the risk of developing or reducing the rate at which symptoms associated with the disease or condition appear) a disease or condition associated with fibril formation caused by Aβ peptides and / or ABri / ADan peptides by administering to a subject lipid microbubbles and / or nanodroplets with an isolated recombinant protein of the invention that reduces polypeptide aggregation. Subjects at risk for Alzheimer's disease can be identified, for example, by any one or combination of suitable diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur before the onset of symptoms characteristic of the disease, such that the disease is prevented or its progression is delayed.

[0089] The isolated recombinant proteins and lipid microbubbles and / or nanodroplets of the present invention can be administered to patients at therapeutically effective doses to prevent, treat, or ameliorate disorders, including fibril formation, associated with Alzheimer's disease. A therapeutically effective dose refers to that amount of compound sufficient to ameliorate the symptoms of the disorder. The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as previously described.

[0090] According to another aspect of the invention, Bri2 BRICHOS proteins and variants thereof are useful in the delivery of proteins or polypeptides (e.g., therapeutic agents, antibodies, protein tags), for example, by offering distinct advantages in improving drug therapeutic potential, drug targeting, in vivo diagnosis and prognosis, and in vivo imaging. Thus, a protein may comprise at least one other protein or polypeptide moiety, which may be a biological polymer, oligomer, or oligopeptide, such as a peptide or polypeptide.

[0091] In a third aspect of the present invention, (i) a first protein portion selected from the group consisting of a protein comprising an amino acid sequence having at least 70% identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively); and (ii) a second protein or polypeptide portion, preferably of at least 50 amino acid residues; 1. An isolated protein comprising: does not contain an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3); and An isolated protein is provided that does not include an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4).

[0092] Proteins comprising a first protein moiety are unique for in vivo therapeutic use by providing tissue-specific targeting and / or drug release when used in combination with microbubbles and / or nanodroplets for use in treatment and therapy, in vivo diagnosis and prognosis, and in vivo imaging.

[0093] The Bri2 BRICHOS domain provides the ability to transport a second protein or polypeptide moiety across the blood-brain barrier in mammals without ultrasound treatment. Isolated proteins comprising the Bri2 BRICHOS domain and a second protein or polypeptide moiety, such as a protein drug, polypeptide drug, protein tag, fluorescent protein, antibody, enzyme, and / or neurotrophic factor, are advantageous because they facilitate and enhance the treatment of Alzheimer's disease and other neurological disorders.

[0094] In one embodiment, the first protein portion of the isolated protein is selected from the group of proteins comprising an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

[0095] In another embodiment, the first protein moiety is selected from the group of proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

[0096] In one embodiment of the invention, the first protein portion of the isolated protein is selected from the group of proteins comprising an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

[0097] In a further aspect, the first protein portion 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).

[0098] In one embodiment, the amino acid residue in the first protein portion corresponding to position 221 of SEQ ID NO: 1 is selected from Glu and Asp, preferably Glu. rhBri2BRICHOS was mutated so that monomers are more stable than larger oligomers, and rhBri2BRICHOS R221E (SEQ ID NOs: 13-14) selectively reduces Aβ42 oligomer formation and alleviates Aβ42-induced neurotoxicity in hippocampal slice preparations. rhBri2BRICHOS R221E crosses the BBB in mice and tends to have a higher permeability than the wild-type protein. This result is consistent with previous observations that wild-type rhBri2BRICHOS monomers cross the BBB more efficiently than large oligomers. The improved permeability of the R221D / E variant is particularly advantageous when efficient delivery to the brain is desired without the simultaneous administration of lipid microbubbles and nanodroplets.

[0099] In one embodiment, the number of amino acid residues in the first protein portion is 200 or less, such as 150 or less. In one embodiment, the number of amino acid residues in the first protein portion is 90 or more.

[0100] In general, delivery of large therapeutic molecules to the brain to treat central nervous system (CNS) diseases is a major challenge in drug development. The BBB acts to restrict the movement of substances from the blood into the CNS. Therefore, only approximately 0.1% of antibodies in the blood cross an intact BBB, severely limiting the usefulness of antibody therapeutics for CNS diseases. Furthermore, the BBB excludes all large neurotherapeutic molecules and over 98% of small molecule drugs from the brain.

[0101] In one embodiment, the isolated protein does not have a cleavage site between the first protein portion and Bri2-BRICHOS sequence and the second protein or polypeptide portion, hi another embodiment, the isolated protein has a cleavage site (e.g., a cleavage site in Bri2 that, in the native protein, is naturally cleaved by a proprotein convertase to release the Abri peptide) between the first protein portion and Bri2-BRICHOS sequence and the second protein or polypeptide portion.

[0102] An object of the present invention is to provide an isolated protein, wherein the amino acid residues of the second protein or polypeptide portion of the isolated protein are 50 to 2000, for example, 50 to 1000, for example, 50 to 500, for example, 50 to 100. In another object of the present invention, the size of the second protein or polypeptide portion of the isolated protein is 5 to 200 kDa, for example, 5 to 100 kDa, for example, 5 to 50 kDa, for example, 5 to 10 kDa.

[0103] Surprisingly, the inventors have found that an isolated protein comprising Bri2 BRICHOS and a second (cargo) protein or polypeptide moiety is able to cross the BBB despite its large size.

[0104] In some aspects of the invention, a first protein moiety of the isolated protein is linked directly or indirectly to the amino or carboxy terminus of a second protein or polypeptide moiety.

[0105] In another aspect of the invention, the second protein or polypeptide portion of the isolated protein constitutes the amino and / or carboxy terminus of the isolated protein.

[0106] As mentioned above, the BBB often impedes the delivery of large therapeutic molecules (e.g., protein drugs, polypeptide drugs, protein tags, fluorescent proteins, antibodies, enzymes and / or neurotrophic factors) to the brain.

[0107] Protein and polypeptide drugs can be used to replace proteins that are abnormal or missing in certain diseases. Protein and polypeptide drugs can reduce the impact of treating diseases by increasing the supply of beneficial proteins in the body. Examples of common and widely used protein drugs are insulin, interferon alpha, and interleukin 2.

[0108] A protein tag is a peptide sequence grafted onto a protein, for example a peptide sequence genetically grafted onto a recombinant protein. Examples of protein tags include solubilization tags, epitope tags, and fluorescent tags.

[0109] Solubilization tags are used to aid in proper folding of proteins and prevent precipitation, especially in recombinant proteins expressed in chaperone-deficient species such as E. coli.

[0110] Epitope tags are short peptide sequences selected to ensure the production of high affinity antibodies in a variety of species.

[0111] Fluorescent tags and proteins are used to provide visual readouts of proteins. They may be used to tag components within cells, tissues, or organs, allowing for experiments using fluorescence spectroscopy, fluorescence microscopy, and other imaging techniques. GFP and its variants are the most commonly used fluorescent tags. mCherry, on the other hand, is a member of the mFruits family of red fluorescent protein monomers (mRFPs), and belongs to a group of fluorescent protein chromophores used as a means to visualize genes and analyze their function in experiments.

[0112] The second protein moiety can increase the stability (e.g., extend half-life) of the first protein moiety and the isolated protein, which may itself be useful as a therapeutic agent, thereby reducing the frequency of treatments. One possibility is that the second protein moiety is an Fc moiety, i.e., the crystallizable region of an antibody tail (Fc region) that interacts with cell surface receptors called Fc receptors and proteins of the complement system.

[0113] In another aspect, the second protein or polypeptide portion of the isolated protein is selected from the group consisting of a protein pharmaceutical, a polypeptide pharmaceutical, a protein tag, a fluorescent protein, an antibody, an enzyme and / or a neurotrophic factor.

[0114] Antibodies, also known as immunoglobulins (Ig), are large, Y-shaped proteins used by the immune system to identify and neutralize foreign substances, such as pathogenic bacteria and viruses. Antibodies are widely used in therapeutics due to their binding mechanism, treating diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and many cancers. Monoclonal antibodies being studied and / or used to treat Alzheimer's disease include aducanumab, gantenerumab, 3D6 (bapineuzumab), and m266 (solanezumab).

[0115] In a preferred embodiment, the second protein or polypeptide portion of the isolated protein is an antibody, e.g., a monoclonal antibody. Preferred monoclonal antibodies are selected from (i) aducanumab, gantenerumab, 3D6 (bapineuzumab), m266 (solanezumab), donanemab, and lecanemab, or (ii) aducanumab, gantenerumab, 3D6 (bapineuzumab), and m266 (solanezumab). A preferred monoclonal antibody is donanemab. Another preferred monoclonal antibody is lecanemab.

[0116] Neurotrophic factors (NTFs) are a class 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 early growth and development of neurons in the central and peripheral nervous systems and have the ability to regrow damaged neurons in vitro and in animal models. Some neurotrophic factors are also released by target tissues to induce axonal growth during development. In experiments, neurotrophic factors are typically used in combination with other techniques, and neurotrophic factors can be immobilized on scaffold structures. In neurodrug delivery systems, they are loosely immobilized for selective release in specific amounts at specific times.

[0117] In one 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.

[0118] Enzymes are proteins that act as biological catalysts and facilitate chemical reactions. Enzymes are necessary for many chemical interconversions that support life and accelerate all biochemical processes in the body. These characteristics distinguish enzymes from other drugs. Because of these characteristics, enzymes are widely used medically, either alone or in combination with other therapies, for the safe treatment of a variety of diseases. Examples of therapeutic enzymes used to treat various disorders and for various therapies include α-L-iduronidase, iduronate sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase, and / or lysosomal acid lipase. Other examples of therapeutic enzymes that can constitute the second protein include enzymes known to be genetically mutated and therefore functionally deficient in lysosomal storage diseases (LSDs).

[0119] In another aspect, the second protein or polypeptide portion of the isolated protein is selected from the group consisting of α-L-iduronidase, iduronate sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase, and / or lysosomal acid lipase.

[0120] A fusion protein is a protein created by joining genes encoding two or more separate proteins. When the fusion gene is translated, it results in a single protein that possesses the functional properties of each of the original proteins. Recombinant fusion proteins are artificially prepared using recombinant DNA technology for use in biological experiments and therapeutics.

[0121] In some aspects of the invention, the isolated protein is a recombinant fusion protein.

[0122] Chemical conjugation is the process of chemically joining two or more protein molecules, such as chemically linking a first protein moiety to a second protein or polypeptide moiety. Chemical conjugation allows for the attachment of moieties such as proteins, polypeptides, protein drugs, tags, and fluorescent molecules to another protein molecule for delivery and targeting to cells and tissues, providing target-specific therapy and treatment, and / or improving in vivo diagnosis and prognosis, as well as aiding in molecular imaging and detection.

[0123] In another aspect of the invention, the first protein moiety of the isolated protein is chemically conjugated to said second protein or polypeptide moiety.

[0124] A further aspect of the invention provides a combination of the disclosed isolated protein with a plurality of lipid microbubbles and / or nanodroplets, wherein the isolated protein is not contained within the microbubbles and / or nanodroplets.

[0125] In one aspect, a kit is provided that includes a disclosed isolated protein and a plurality of lipid microbubbles and / or nanodroplets, wherein the isolated protein is not contained within the microbubbles and / or nanodroplets.

[0126] Brain-penetrating molecules, recombinant proteins, and / or biologics generally will not be successful unless the drugs cross the BBB, and therefore BBB drug delivery will be a limiting factor in the future development of new therapeutics for the brain.

[0127] The inventors have designed a convenient method for efficiently delivering compositions to neurons of the CNS through tissues such as the BBB.

[0128] To these and other ends, in a further aspect, the present invention provides a method for transporting an isolated protein disclosed herein across the blood-brain barrier in a mammal, including a human, in need thereof, comprising: administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; and administering the isolated protein to the mammal; The present invention provides a method comprising:

[0129] In certain embodiments, the method does not include the step of ultrasonically treating mammalian tissue. administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; administering the isolated protein to the mammal; Including, providing an isolated protein disclosed herein for use in therapy, comprising transporting said isolated protein across the blood-brain barrier in said mammal; The isolated protein is not contained within the microbubbles and / or the nanodroplets.

[0130] In some embodiments, the method does not include the step of administering ultrasound to mammalian tissue.

[0131] The method does not include treating mammalian tissue with light, sound or ultrasound waves to cavitate microbubbles and / or nanodroplets in the tissue.

[0132] In another aspect, a method for transporting the isolated proteins disclosed herein across the blood-brain barrier in a mammal, including a human in need thereof, comprises: administering the isolated protein to the mammal; It comprises or consists of.

[0133] In certain embodiments, administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; and administering the isolated protein to the mammal; Consists of: An isolated protein as disclosed herein is provided for use in therapy, comprising transporting said protein across the blood-brain barrier in said mammal, The isolated protein is not contained within the microbubbles and / or the nanodroplets.

[0134] Surprisingly, we found that rhBri2 BRICHOS fused to short peptide tags or globular proteins was detected in the brain parenchyma 2 hours after administration with lipid microbubbles and / or nanodroplets. Immunohistochemistry and Western blot analysis of the brain parenchyma clearly demonstrated similar distribution of the delivered protein in both hemispheres, with strong staining in the cortex, hippocampus, and choroid plexus after administration of rhBri2 BRICHOS-AU1 with microbubbles and / or nanodroplets. Intracellular translocation of rhBri2 BRICHOS-AU1 delivered with microbubbles and / or nanodroplets was observed, consistent with the results of FUS-mediated delivery of rhBri2 BRICHOS-AU1 (Galan-Acosta et al., Mol Cell Neurosci. 2020:103498). Bri2 is expressed not only in the CNS but also in peripheral tissues, suggesting the existence of a transport system that allows crosstalk between these sites.

[0135] Drug transport into the cerebrospinal fluid (CSF) cannot be estimated from BBB crossing. Therefore, it is noteworthy that another BRICHOS protein, rh proSP-C BRICHOS, was detected in the CSF after intravenous injection in mice but not in brain homogenates. This indicates that crossing 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)). BBB permeability varies depending on the brain region, e.g., it is greater in areas close to the choroid plexus and in the periventricular region than in the rest of the brain.

[0136] Furthermore, quantitative analysis of the total rhBri2 BRICHOS domains that reached the brain parenchyma by sandwich ELISA demonstrated significant rhBri2 BRICHOS-AU1 presence in both hemispheres. In this analysis, 1% of the infused rhBri2 BRICHOS domains administered with lipid microbubbles and / or nanodroplets was detected in the brain 2 hours after infusion, a 2- to 10-fold increase over the passage observed when rhBri2 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 and FUS (Galan-Acosta et al., Mol Cell Neurosci. 2020:103498). Although the extent of microbubble- and / or nanodroplet-mediated increased BBB crossing was not quantified for the rh Bri2 BRICHOS-mCherry fusion protein, the results indicate an effect, as it was detected in the brain only when the fusion protein was administered with microbubbles and / or nanodroplets.

[0137] In summary, the current data demonstrate that microbubbles and / or nanodroplets significantly increase BBB crossing of rhBri2BRICHOS, even when fused to a large globular protein (30 kDa). While incorporating or associating drugs with lipid nanoparticles can increase bioavailability, in this invention, the rhBri2BRICHOS protein and microbubbles and / or nanodroplets are administered independently, making it unlikely that rhBri2BRICHOS crosses the BBB with the microbubbles and / or nanodroplets. Without wishing to be bound by theory, the inventors speculate that the increased crossing of the BBB of rhBri2BRICHOS or rhBri2BRICHOS-mCherry in the presence of microbubbles and / or nanodroplets is due to an increased half-life of rhBri2BRICHOS in the blood. Given that functional mCherry has been shown to be transportable to the brain of wild-type mice, other means of increasing the half-life of rhBri2BRICHOS in the blood are likely to increase its crossing of the BBB; for example, fusion of rhBri2BRICHOS to other proteins, including antibodies, enzymes, or neurotrophic factors, may increase its translocation into the brain parenchyma.

[0138] This finding indicates that in the presence of microbubbles and / or nanodroplets, rhBri2BRICHOS crosses the BBB 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). Similarly, BBB crossing of rhBri2BRICHOS was also significantly more efficient than affibodies designed to improve CNS uptake, which were detected in only small amounts in the cerebrospinal fluid after injection (Meister et al. Int J Mol Sci. 2020;21(8)).

[0139] As described above, proteins, including the isolated recombinant Bri2 BRICHOS protein and its variants (including Bri2 BRICHOS R221E), can be efficiently delivered to CNS neurons across the blood-brain barrier. This method does not require the administration of lipid microbubbles and / or nanodroplets. This method does not require the treatment of mammalian tissue with light waves, sound waves, or ultrasound. Thus, the isolated Bri2 BRICHOS protein conjugated to a second protein or polypeptide moiety can be efficiently delivered to the brain even in the absence of lipid microbubbles or nanodroplets.

[0140] We demonstrate that cargo proteins fused to rhBri2BRICHOS cross a human BBB model prepared with brain microvascular endothelial cells and astrocytes as efficiently as proteins known to be transported across the BBB. In contrast, 4 kDa dextran or the fusion partner alone do not, and rhBri2BRICHOS does not cause nonspecific leakage or cytotoxicity. Intravenously administered Bri2BRICHOS fusion proteins cross the mouse BBB and localize to early endosomes in cerebral and cerebellar neurons. Our results demonstrate that rhBri2BRICHOS fusions can provide diverse biopharmaceuticals for CNS diseases.

[0141] As described herein, an isolated protein comprising a first protein portion that is Bri2 BRICHOS or a variant thereof and at least one other protein or polypeptide portion may be prepared by: - administering said isolated protein to a mammal, including a human, in need thereof; The present invention is useful in a method of transporting an isolated protein across the blood-brain barrier in said mammal, comprising or consisting of:

[0142] Additionally, an isolated protein comprising a first protein portion that is Bri2 BRICHOS or a variant thereof and at least one other protein or polypeptide portion may be - administering said isolated protein to a mammal, including a human, in need thereof; The present invention is useful in a method for treating a disease in a mammal, comprising or consisting of:

[0143] Therefore, the isolated protein is useful as a pharmaceutical. - administering said isolated protein to a mammal, including a human, in need thereof; The present invention is useful in a method for treating a disease in a mammal, comprising or consisting of:

[0144] In these methods, an isolated protein comprising a first protein moiety that is Bri2 BRICHOS or a variant thereof and at least one other protein or polypeptide moiety can be efficiently delivered to the brain, even without the use of lipid microbubbles and nanodroplets.

[0145] The isolated proteins of the present invention can be administered by gene therapy, such as by introducing them into cells of the nervous system, preferably the brain, using expression vectors, plasmids, or viruses, and the isolated proteins are believed to be expressed in these cells of the central nervous system. This is useful for treating Alzheimer's disease. It may also be useful for treating other neurological disorders.

[0146] The isolated Bri2 BRICHOS protein itself can also be efficiently delivered to the brain and is useful for treating Parkinson's disease.

[0147] Aggregation of α-synuclein (α-syn) protein is associated with several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy (MSA), which are characterized by the abnormal accumulation of misfolded α-syn in intracellular inclusions called Lewy bodies within neurons. While the specific molecular mechanisms underlying this disease pathogenesis remain unclear, there is strong evidence that aggregation of α-syn into amyloid fibrils plays a key role, as known mutations in the gene encoding α-syn cause early onset or significant progression of PD. Although significant efforts have been made to inhibit amyloid formation in PD, no effective treatment has been developed to date. In particular, the generation of intermediate α-syn aggregate oligomers has been suggested to be responsible for α-syn-associated neurotoxic effects, creating a need for methods to inhibit toxic aggregation pathways and species.

[0148] Without wishing to be limited to a particular mechanism, we show here that BRICHOS effectively retards α-synuclein self-assembly by binding to the surface of fibrils, thereby specifically inhibiting secondary nucleation and the resulting oligomer formation, as well as fibril end extension. Furthermore, our findings provide evidence that BRICHOS directly binds to oligomers and reduces α-synuclein-associated toxicity.

[0149] We show 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, a similar inhibitory mechanism has been reported for BRICHOS against Aβ42 aggregation (Chen et al., Nat Commun, 8(1):2081, 2017; Chen et al., Communications biology, 3(1):1-12, 2020). Previous studies have shown that BRICHOS binds to the surface of fibrils and along Aβ42 fibrils (Biverstal et al., Sci Rep, 10(1):21765, 2020). One hypothesis is that BRICHOS binds to secondary nucleation spots along the surface of fibrils, competing with α-syn monomers as shown for Aβ42.

[0150] We demonstrate for the first time the interaction between BRICHOS and α-syn oligomers. BRICHOS not only prevents secondary nucleation by binding to α-syn oligomers and fibrils, but also binds to its products, which often correlate with toxicity in in vitro experiments. This binding ability may be important for reducing oligomer-induced toxicity.

[0151] Although oligomers are the most toxic molecules, sonicated α-syn fibrils also exhibit toxicity in various in vitro and in vivo models. Here, we show that BRICHOS prevents the neurotoxicity associated with sonicated α-syn fibrils in mouse hippocampal slices. This can be explained by the fact that BRICHOS can bind to α-syn fibrils and inhibit the formation of oligomers through a secondary nucleation process with endogenous α-syn. BRICHOS may also interfere with oligomers released from the ends of pre-existing α-syn fibrils that are toxic in neurons.

[0152] Relevant in vitro models suggest that BRICHOS may be a potential therapeutic candidate for α-synucleinopathies, including Parkinson's disease.

[0153] Bri2 BRICHOS protein can also be conjugated to a second protein or polypeptide that is effective in treating Parkinson's disease, such as glucocerebrosidase, progranulin, prosaposin, cathepsin D, and antibodies. Without wishing to be bound by any particular theory, it is believed that the combined effect of Bri2 BRICHOS and the second protein / polypeptide is highly useful in treating Parkinson's disease. The greater effectiveness of this drug may also allow for less frequent administration.

[0154] therefore, - administering the isolated protein as defined herein to a mammal, including a human, in need thereof; and a method for treating an alpha-synucleinopathy, including Parkinson's disease, in said mammal, comprising or consisting of:

[0155] moreover, - administering the isolated protein as defined herein to a mammal, including a human, in need thereof; A method of slowing the accumulation of alpha-synuclein in a mammal is provided, comprising or consisting of:

[0156] The present invention is further illustrated by the following non-limiting examples. [Example]

[0157] The examples demonstrate that the use of microbubbles can enhance the delivery of Bri2 BRICHOS and its fusion proteins across the BBB, facilitating the treatment of AD and other neurological disorders.

[0158] Recombinant protein expression and purification An AU1 tag or mCherry protein was ligated to the C-terminus of the rh Bri2 BRICHOS domain, corresponding to residues 113–231 of the full-length Bri2 protein. The BRICHOS-containing proteins, Bri2 BRICHOS-AU1 (SEQ ID NO: 11) and Bri2 BRICHOS-mCherry (SEQ ID NO: 12), were expressed in E. coli and purified. Prior to injection into mice, rh Bri2 BRICHOS-AU1 was dialyzed against filtered, autoclaved phosphate-buffered saline (PBS), pH 7.4 (6–8 kDa membrane, Spectrum Lab). Endotoxin was removed 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.), stored at −20°C, and thawed several minutes before injection.

[0159] As a control, the proSP-C BRICHOS domain (proSP-C residues 59-197) was prepared as described in Galan-Acosta et al., Mol Cell Neurosci. 2020:103498.

[0160] animal Female and male C57BL / 6J mice, 4-6 months old and weighing 24-30 g, were used. All animals were provided with free access to food and water and housed in cages of five mice each under a 12-h light-dark cycle. All experiments were approved and conducted in accordance with the ethical committees of Södra Stockholms Djurforsoksetiska Namnd (dnr 03049), Linkopings Etiska Namnd (ID855), or under the guidelines of the Columbia University Institutional Animal Care and Use Committee.

[0161] Experimental design Figure 3 shows the experimental design of an example. (A) Mice were injected twice into the tail vein with microbubbles and 1, 10, or 20 mg / kg of rhBri2BRICHOS-AU1 (SEQ ID NO: 11), or PBS as a control. (B) Mice were administered 20 mg / kg of (i) rhBri2BRICHOS-mCherry (SEQ ID NO: 12) and microbubbles, or (ii) rhBri2BRICHOS-mCherry alone.

[0162] Two commercially available microbubbles, Definity® (Lantheus Medical Imaging, MA, USA) or SonoVue® (Bracco, Milan, Italy), were administered at a dose of 5 μl / g body weight. In the first part of the experiment (Figure 3A), a total of 14 mice were divided into four groups and intravenously injected with microbubbles + PBS (n = 4), microbubbles + rhBri2BRICHOS-AU1 (SEQ ID NO: 11) at 1 mg / kg body weight (n = 4), 10 mg / kg body weight (n = 6), or 20 mg / kg body weight (n = 4). In the second part of the experiment (Figure 3B), a total of three mice were intravenously injected with 20 mg / kg of rhBri2BRICHOS-mCherry (SEQ ID NO: 12) (n = 1) or microbubbles + 20 mg / kg of rhBri2BRICHOS-mCherry (n = 2). Two mice receiving 10 mg / kg rh Bri2 BRICHOS-AU1 were infused with Definity® microbubbles, and the remaining mice received SonoVue® microbubbles.

[0163] All animals received two doses of rhBri2 BRICHOS protein into the lateral tail vein using a 29-gauge needle. Prior to administration, the microbubble vial was mechanically agitated for 20 seconds to activate the microbubble solution, 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 (2% oxygen) and placed under a heat lamp to dilate the tail vein. Two hours after injection, mice were anesthetized and transcardially perfused with 120 ml of PBS. One mouse administered Definity® and 10 mg / kg rhBri2 BRICHOS-AU1 was perfused with PBS for 5 minutes, followed by 4% paraformaldehyde (PFA) for 7 minutes. Brains were removed from the skull and either flash-frozen on dry ice and stored at -80°C, or fixed in 4% PFA for 48 hours and embedded in paraffin.

[0164] antibody For immunohistochemistry (IHC), a polyclonal rabbit anti-AU1 primary antibody (Abcam Cat# ab3401) was used at a dilution of 1:200, and a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (GE Healthcare Cat# NA934) was used at a dilution of 1:2000. For Western blot, a polyclonal rabbit anti-AU1 (Abcam Cat# ab3401) was used at a dilution of 1:600, and a fluorescently labeled secondary anti-rabbit antibody (Li-Cor, Cat# 926-32213) was used at a dilution of 1:10,000. For sandwich ELISA, a goat anti-Bri2 BRICHOS antibody was used at a dilution of 1:250 for capture, and a polyclonal rabbit anti-AU1 (Abcam Cat# ab3401) antibody was used at a dilution of 1:2000 for detection.

[0165] Rabbit anti-proSP-C antibody was used as described in Galan-Acosta et al., Mol Cell Neurosci. 2020:103498.

[0166] Immunohistochemical analysis (IHC) Coronal sections of paraffin-embedded tissues, 5 μm thick, were placed on Superfrost Plus microscope slides (Thermo Scientific) and dried overnight at room temperature (RT) to remove residual water. Sections were deparaffinized by washing with xylene and rehydrated with decreasing concentrations of ethanol (99% to 70%). Sections were immersed in DIVA decloaker 1x solution (Biocare Medical, Concord, USA) in a decloaking chamber (Biocare Medical) and incubated at 110°C for 30 minutes under pressure or in a 95°C water bath for 30 minutes. Slides were cooled at RT for 20 minutes, then washed with PBS buffer containing 0.1% Tween® 20 (PBST) and incubated with peroxidase blocking solution (Dako) for 5 minutes. Sections were washed with Tris-buffered saline (TBS) and additionally blocked with Background Punisher (Biocare) for 10 minutes. Primary antibodies diluted in DAKO (Agilent) antibody diluent were incubated for 45 minutes at room temperature. Slides were then washed with TBS and incubated with Mach 2 Double stain 2 alkaline phosphatase (AP)-conjugated secondary anti-rabbit antibody for 30 minutes at room temperature. AP staining was detected with permanent red (Biosite). Sections were counterstained with hematoxylin (Mayer), dehydrated in ethanol (70% to 99%), cleared in xylene, and mounted with DEPEX mounting medium (Merck).

[0167] Microscopy Stained sections were visualized with a Nikon Eclipse E800M light microscope equipped with 10x and 20x Plan-Apochromate objectives. rhBri2BRICHOS-mCherry protein was detected in brain samples using a Nikon fluorescence microscope and recorded with a 20x objective.

[0168] Western blot Brain tissue from one mouse treated with rhBri2 BRICHOS-AU1 + microbubbles and one negative control mouse 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 × g) at 4 °C for 30 minutes, and the supernatant was collected and stored at -20 °C. Protein concentration was measured using the BCA method. The homogenate was diluted with homogenization buffer and 1 × SDS reducing buffer (containing 2-mercaptoethanol) to load a total of 100 μg of protein per sample and well. Samples were heated at 97°C for 10 minutes, separated on a 4-20% precast polyacrylamide gel (Bio-Rad), and blotted onto a nitrocellulose membrane (GE Healthcare). After blotting, the membrane was blocked with 5% nonfat dry milk in 0.1% Tween® / TBS for 1 hour at room temperature. After rinsing with 0.1% Tween® / TBS, primary antibodies diluted in 0.1% Tween® / TBS were added overnight at 4°C. The membrane was washed three times with 0.1% Tween® / TBS and then incubated with secondary antibodies prepared in 0.1% Tween® / TBS for 1 hour at room temperature. After rinsing unbound secondary antibodies with 0.1% Tween® / TBS, images were captured using a fluorescent imaging system (Li-Cor, Odyssey CLx).

[0169] Sandwich ELISA A 96-well plate (Nunc MicroWell™) was 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 plate was blocked with 1% BSA / PBS for 1 hour. Brain samples (250 μg / ml) and standards diluted in 0.05% Tween® / PBS were then incubated for 2 hours at room temperature. The plate was washed, and rabbit anti-AU1 primary antibody diluted in 0.05% Tween® / PBS was added overnight at 4°C. The plate was washed three times, and secondary anti-rabbit antibody diluted in 0.05% Tween® / PBS was added for 2 hours at room temperature. After washing, tetramethylbenzidine (TMB) (Thermo Fisher) solution was added and the plate was incubated for 30 minutes in the dark. The reaction was stopped by adding stop solution for TMB substrate (Thermo Fisher), and the absorbance was measured at 450 nm using brain homogenate from an untreated mouse as a blank. A standard curve was generated from 0.1 to 64 ng of rh Bri2 BRICHOS-AU1 protein.

[0170] Calculation of the concentration and total amount of rh Bri2 BRICHOS-AU1 in each brain hemisphere was assessed by assuming a brain density of 1.04 mg / ml, an average brain hemisphere weight of 225 mg, and an average total protein in the brain hemisphere homogenate of 25.6 mg.

[0171] result BBB penetration of rh Bri2 BRICHOS but not proSP-C BRICHOS in the non-ultrasound-treated hemisphere in the presence of lipid microbubbles The presence of rh Bri2 BRICHOS-AU1 (SEQ ID NO: 11) in the brain parenchyma was investigated. Figure 4 shows that rh Bri2 BRICHOS, but not rh proSP-C BRICHOS, is detected in the non-FUS-targeted hemisphere after intravenous injection with lipid microbubbles. (A-H) Immunohistochemistry of cortical and hippocampal sections from mice treated with rh proSP-C BRICHOS + microbubbles (A-B, E-F, I-J) or rh Bri2 BRICHOS-AU1 + microbubbles (C-D, G-H) in the contralateral non-FUS-targeted hemisphere. (I-J) Controls showing no staining for rh Bri2 BRICHOS-AU1 in contralateral or ipsilateral slides from mice treated with intravenous proSP-C BRICHOS + microbubbles and FUS. Tissues were stained with rabbit anti-proSP-C antibody (A-B, E-F) or rabbit anti-AU1 antibody (C-D, G-H), followed by AP-conjugated secondary antibodies, and developed with permanent red AP solution. All samples were counterstained with hematoxylin. Scale bars are 100 μm for A-H and 200 μm for I-J.

[0172] Two hours after intravenous administration of 10 mg / kg of rhBri2BRICHOS-AU1 in the presence of microbubbles to wild-type mice, rhBri2BRICHOS-AU1 was found in the cortex and hippocampus of the non-FUS-targeted contralateral hemisphere (Figure 4C-D, G-H). In FUS-treated wild-type mice, rh proSP-C BRICHOS was not observed in the non-ultrasound-treated hemisphere 2 hours after intravenous administration of 10 mg / kg of rh proSP-C BRICHOS and microbubbles (Figure 4A-B, E-F). Immunohistochemical staining for rhBri2BRICHOS-AU1 was negative in brain sections from mice administered 10 mg / kg of rh proSP-C BRICHOS and then treated with FUS + microbubbles 2 hours later, indicating that the anti-AU1 antibody immunoreactivity was not an artifact of the injection of recombinant protein (Figure 4I-J).

[0173] Microbubbles increase BBB permeability of rhBri2 BRICHOS in wild-type mice The effect of rhBri2BRICHOS microbubbles alone, i.e., without FUS application, on BBB permeability was further investigated by administering the rhBri2BRICHOS domain with an AU1 tag (rhBri2BRICHOS-AU1; SEQ ID NO: 11) or mCherry protein (rhBri2BRICHOS-mCherry; SEQ ID NO: 12) recombinantly linked to its C-terminus. SonoVue® microbubbles (5 μl / g body weight) and various doses of rhBri2BRICHOS-AU1 were injected into the lateral tail vein of individual adult wild-type mice, and 2 hours later, brains were perfused and collected for analysis. Control mice received SonoVue® microbubbles and PBS. The presence of rhBri2BRICHOS-AU1 in the brain parenchyma was examined by immunohistochemistry and Western blot, and the amount of rhBri2BRICHOS-AU1 was assessed by sandwich ELISA (Figure 3A).

[0174] To investigate the effect of microbubbles on BBB permeability of rhBri2BRICHOS fused to a folded globular protein, we selected rhBri2BRICHOS-mCherry (total molecular weight 44 kDa), which can be detected by the fluorescent properties of folded mCherry, which are unchanged upon fusion with rhBri2BRICHOS. In this case, two wild-type mice were intravenously injected with SonoVue® and 20 mg / kg of rhBri2BRICHOS-mCherry (SEQ ID NO: 12), while control mice were injected with rhBri2BRICHOS-mCherry alone (Figure 3B). Brains were collected 2 hours after injection and analyzed macroscopically and under a fluorescent microscope.

[0175] Surprisingly, microbubbles alone were found to increase BBB penetration of rhBri2 BRICHOS-AU1.

[0176] Figure 5 shows that Bri2 BRICHOS is detected in both hemispheres after intravenous injection with lipid microbubbles. (A-G) Images of the cortex, hippocampus, and lateral ventricles of a mouse treated with microbubbles plus 10 mg / kg rh Bri2 BRICHOS-AU1. (H-J) Images of a mouse injected with 10 mg / kg rh proSP-C BRICHOS and microbubbles. (K-M) Images of a control mouse that received no injection. All sections were incubated with anti-AU1 antibody followed by anti-rabbit antibody conjugated with AP, developed with permanent red AP solution, and counterstained with hematoxylin. Scale bars are 500 μm in panel A and 200 μm in panels B-M.

[0177] After intravenous injection of microbubbles and a dose of 10 mg / kg of rhBri2BRICHOS-AU1, rhBri2BRICHOS-AU1 was detected in the cortex, hippocampus, and choroid plexus in the lateral ventricles by immunohistochemistry using an antibody against the AU1 tag (Figure 5A-G). No difference in staining intensity was observed between the two hemispheres (Figure 5A-G).

[0178] Figure 6 shows that intracellular immunostaining of rhBri2BRICHOS-AU1 is observed in the cortex and hippocampus after intravenous injection with lipid microbubbles. Images show the cortex (A, B) and hippocampus (C, D) of the right hemisphere of a mouse treated with microbubbles plus 10 mg / kg of rhBri2BRICHOS-AU1 (SEQ ID NO: 11). Sections were stained for rhBri2BRICHOS-AU1 with anti-AU1 antibody and counterstained with hematoxylin. Arrows in (B) and (D) indicate intracellular staining of rhBri2BRICHOS-AU1. Scale bars are 100 μm in panels A and C; 50 μm in panels B and D.

[0179] Intracellular staining was observed in cells present in the cortex and hippocampus (Fig. 6A-D). Control brains from mice injected with rh proSP-C BRICHOS or PBS were stained in the same manner with anti-AU1 antibodies, but no immunoreactivity was observed (Fig. 5H-M).

[0180] Furthermore, analysis of homogenates from both hemispheres of mice administered microbubbles and 10 mg / kg of rhBri2BRICHOS-AU1 by Western blot with both anti-AU1 and anti-Bri2 antibodies revealed a slightly slower-migrating band for rhBri2BRICHOS-AU1, with no corresponding band present in control samples. Figure 7 shows Western blots of rhBri2BRICHOS-AU1 (SEQ ID NO: 11) in both hemispheres after systemic injection with lipid microbubbles. Western blots of the left (L) and right (R) hemispheres of one mouse collected 2 hours after intravenous injection of microbubbles + rhBri2BRICHOS-AU1, as well as brain homogenates from an untreated control mouse (negative control). As indicated by the arrows on the right side of the gel, rhBri2BRICHOS-AU1 is detected with anti-AU1 (A) and anti-Bri2 (B) antibodies. The Rec protein lane contains 5 ng of rh Bri2 BRICHOS-AU1.

[0181] Consistent with the immunohistochemistry and Western blot results, analysis of homogenates from both hemispheres of mice treated with microbubbles and 10 mg / kg rhBri2BRICHOS-AU1 by sandwich ELISA 2 h after injection showed that the concentration of rhBri2BRICHOS-AU1 in the left hemisphere was 390 nM and in the right hemisphere was 250 nM, both of which correspond to 1% of the total dose administered. Interestingly, more isolated recombinant Bri2BRICHOS-AU1 reached the brain parenchyma after administration with lipid microbubbles and without ultrasound treatment than after administration without microbubbles. Approximately 1% of isolated recombinant Bri2BRICHOS-AU1 administered at a dose of 10 mg / kg with lipid microbubbles was detected in the brain 2 h after intravenous injection, while 0.1–1% (average 0.5%) was detected after administration of isolated recombinant Bri2BRICHOS-AU1 at a dose of 20 mg / kg without microbubbles. This is surprising, as microbubbles alone (without ultrasound treatment) have not previously been shown to increase BBB crossing of proteins or affect neuronal uptake.

[0182] We further found that rhBri2BRICHOS-mCherry penetrated the brain parenchyma of wild-type mice after injection with, but not without, lipid microbubbles. The BBB permeability of rhBri2BRICHOS-mCherry in wild-type mice injected with, or without, microbubbles was assessed by macroscopic observation of the brains and by fluorescence microscopy of brain sections.

[0183] Figure 8 shows that rhBri2BRICHOS-mCherry (SEQ ID NO: 12) is found in the striatum after systemic injection in combination with lipid microbubbles. (A-B) Photographs of mouse brains after injection of rhBri2BRICHOS-mCherry alone (A) or in combination with microbubbles (B). (C-H) Fluorescence in striatal regions collected 2 hours after injection of 20 mg / kg rhBri2BRICHOS-mCherry alone (C, F) and microbubbles and 20 mg / kg rhBri2BRICHOS-mCherry (D-E, G-H). Scale bars are 200 μm in panels C-E and 100 μm in panels F-H. The areas marked with white rectangles in panels C, D, and E are enlarged in panels F, G, and H, respectively.

[0184] Brains collected 2 h after injection from thoroughly perfused mice injected with either rhBri2BRICHOS-mCherry alone (one mouse) or microbubbles and rhBri2BRICHOS-mCherry (two mice) revealed distinct color (white in Figure 8) in the outer layers of the cortex and cerebellum only in the rhBri2BRICHOS-mCherry and microbubble mice (Figure 8A, B). Fluorescence (white in Figure 8) was also detected in the striatum region of two mice injected with microbubbles and rhBri2BRICHOS-mCherry (Figure 8D, E, G, H), but not in mice injected with rhBri2BRICHOS-mCherry alone (Figure 8C, F). These data strongly suggest that intravenously administered rhBri2BRICHOS-mCherry penetrates the brain parenchyma when injected with microbubbles.

[0185] Further experiments with Bri2 BRICHOS administration method cell culture hCMEC / D3 cells (MilliporeSigma, Burlington, MA) at 5-10 μg / cm 2T75 tissue culture flasks (TC-Coat, DeltaLab, Spain) coated with rat tail type I collagen (MilliporeSigma, Burlington, MA) were cultured in EndoGRO-MV complete medium (MilliporeSigma, Burlington, MA) supplemented with 1 ng / ml basal recombinant human fibrillocyte growth factor (FGF-2, Gibco, Invitrogen, Waltham, MA) and penicillin-streptomycin (100 IU / ml, 100 μg / ml, Gibco, Thermo Fisher Scientific, Waltham, MA). The human brain microvascular endothelial cell line, hCMEC / D3, is used as a reproducible and easy-to-maintain in vitro model of the human blood-brain barrier (BBB), generating an accurate in vivo phenotype. The hCMEC / D3 cell line preserves the expression of most BBB-specific transporters and receptors, making it effective for investigating transport rates and mechanisms across the BBB. The hCMEC / D3 cell line, in which Aβ40 peptide, transthyretin and several drug candidates are transported across the BBB, has been previously described.

[0186] Human neuroblastoma (SH-SY5Y) cell line (ATCC, Manassas, VA) was grown in Dulbecco's modified Eagle's medium (DMEM, PAN-Biotech, Germany) with 4.5 g / L glucose, 2 mM glutamine, and 3.7 g / L NaHCO3, supplemented with 10% fetal bovine serum (FBS, Sera Plus, PAN-Biotech, Germany) and penicillin-streptomycin (100 IU / ml, 100 μg / ml) using TC-coated T75 tissue culture flasks. The human neuroblastoma cell line SH-SY5Y is a triple-subcloned cell line derived from the SK-N-SH neuroblastoma cell line. It is widely used in in vitro neuronal models for the investigation of neurodegenerative diseases.

[0187] An immortalized human astrocyte cell line (Innoprot, Spain) was administered at 2 μg / cm2 Cells were grown in astrocyte medium (Innoprot, Spain) containing 2% FBS, 1% astrocyte growth supplement, and 1% penicillin-streptomycin (100 IU / ml, 100 μg / ml) using T75 tissue culture flasks coated with 10% 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) containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g / L NaHCO3, supplemented with 10% FBS and penicillin-streptomycin (100 IU / ml, 100 μg / ml) using TC-coated T75 tissue culture flasks. Cultures were maintained in a humidified atmosphere (5% CO2 / 95% air) at 37°C, and medium was changed every other day. Upon reaching approximately 80% confluency, cells were trypsinized (0.025% trypsin, 0.01% EDTA, Gibco, Thermo Fisher Scientific, Waltham, MA), and hCMEC / D3, human astrocyte, and SH-SY5Y cell lines were passaged up to passage 35.

[0188] The human embryonic kidney 293 (HEK-293) cell line (ATCC, Manassas, VA) was grown in Eagle's minimum essential medium (EMEM, ATCC, Manassas, VA) containing 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g / L NaHCO3, supplemented with 10% FBS and penicillin-streptomycin (100 IU / ml, 100 μg / ml) using TC-coated T75 tissue culture flasks.

[0189] Cultures were maintained at 37°C in a humidified atmosphere (5% CO2 / 95% air), and medium was changed every other day. Upon reaching approximately 80% confluency, cells were trypsinized (0.025% trypsin, 0.01% EDTA, Gibco, Thermo Fisher Scientific, Waltham, MA) and passaged (35 passages for hCMEC / D3 and SH-SY5Y cell lines).

[0190] hCMEC / D3 monolayer preparation For the preparation of hCMEC / D3 cell monolayers, Transwell ethylene 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 (DPBS) pH 7.4, Gibco, 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 a density of 50,000 cells / cm. 2 Cells were seeded onto freshly prepared collagen-coated inserts at 27 °C and maintained in a humidified atmosphere (5% CO2 / 95% air) at 37 °C. Medium was changed every 2 days, and a cell monolayer was established by day 6.

[0191] Analysis of hCMEC / D3 monolayer integrity The integrity of the monolayer was analyzed by crystal violet staining. The expression of the tight junction proteins zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A), and claudin 5 was confirmed by Western blot and immunostaining. For crystal violet staining, cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, St. Louis, MO) for 2 minutes at room temperature and permeabilized with 100% methanol (Sigma-Aldrich, St. Louis, MO) for 20 minutes at room temperature. Cells were then stained with 0.01% crystal violet (Sigma-Aldrich, St. Louis, MO) in 2% ethanol and analyzed using a fluorescence microscope (Axioskop 40, Zeiss, Germany). Images were captured using the green (FITC / 488) and red (Texas Red / 570) channels.

[0192] For the determination of tight junction proteins, hCMEC / D3 cells were seeded at a density of 50,000 cells / cm. 2Cells were seeded onto collagen-coated 12-well plates (TC-Coat, Cellstar, Greiner Bio-One, Austria) at 100°C in a humidified atmosphere (5% CO2 / 95% air) and maintained at 37°C with medium changes every 2 days. After 6 days, cells were removed from the plates by scraping in ice-cold 1x PBS, centrifuged at 14,000 x g for 10 minutes at 4°C, lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Thermo Scientific, Waltham, MA) and 1x Complete protease inhibitor (Roche, Switzerland), sonicated at 50% amplitude for 10 seconds, and incubated on ice for 20 minutes. Cell lysates were cleared 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 the bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. For Western blots (see below), anti-claudin-5, anti-JAM-A, and anti-ZO-1 polyclonal rabbit primary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA) were used at a 1:1,000 dilution.

[0193] hCMEC / D3 and human astrocyte co-culture preparation For the direct co-culture model, the PET membrane on the apical side of the cell culture insert was first coated with rat tail type I collagen (see hCMEC / D3 monolayer preparation). The insert was inverted, and the basolateral side of the cell culture insert was coated with poly-L-lysine (2 μg / cm in ddHO). 2 ) overnight at 37°C. Excess poly-L-lysine was removed and the membrane was washed twice with ddH2O. Human astrocytes were cultured on the basolateral side of the cell culture inserts at a density of 20,000 cells / cm2. 2 hCMEC / D3 cells were seeded at a density of 50,000 cells / cm and allowed to adhere in an upside-down orientation for 4 hours at 37°C, after which the inserts were placed in the normal orientation and grown in astrocyte medium for 48 hours.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.

[0194] For the indirect co-culture model, 24-well plates were coated with poly-L-lysine, similar to the cell culture insert membrane. Human astrocytes were cultured at a density of 20,000 cells / cm. 2 The inserts were seeded with rat tail I cells and incubated for 48 hours. The cell culture inserts were coated on the apical side with rat tail I cells, and hCMEC / D3 cells were seeded as in the direct coculture model. The hCMEC / D3-coated inserts were separately incubated overnight at 37°C in EndoGro complete medium and then inserted into 24-well plates containing human astrocytes in astrocyte medium.

[0195] The medium was changed every 2 days, and FITC-dextran permeability and TEER values ​​were measured on days 3 to 9 after seeding, similar to the hCMEC / D3 monolayer model.

[0196] hCMEC / D3 monolayer permeability analysis Experiments were performed on day 6 of culture by adding various BRICHOS protein constructs or relevant controls to the apical side of hCMEC / D3 monolayers. BRICHOS constructs were rh proSP-C BRICHOS wt, rh proSP-C BRICHOS T187R, rh Bri2 BRICHOS wt monomers and oligomers, rh Bri2 BRICHOS R221E monomer, S-tag-Bri2 BRICHOS monomers and oligomers, mCherry-Bri2 BRICHOS monomer, NT-Bri2 BRICHOS monomers and oligomers, and an undefined mixture of various S-tag-Bri3 BRICHOS assembly states. Controls included mCherry, NT *The antibodies were TAG, rhAβ42, and apolipoprotein AI (purified from human plasma to >98% purity; Chemicon, Sigma-Aldrich, St. Louis, MO). The concentrations were 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, or 0.05 μM, and the incubation time was 2 or 24 h. The monolayers were incubated at 37°C in a humidified atmosphere (5% CO2 / 95% air). After incubation, the media from the apical and basolateral sides of the monolayers were removed and analyzed by Western blot, and band intensity was measured using ImageJ. For Western blotting (see below), we used anti-Bri2 BRICHOS primary antibody (produced in goat and purified in-house) at a dilution of 1:1000, anti-proSP-C BRICHOS primary antibody (produced in goat and purified in-house) at a dilution of 1:2000, anti-S-tag primary antibody (HRP-conjugated S-tag protein, Novagen, Merck, Germany) at a dilution of 1:5000, anti-NT primary antibody (produced in rabbit and purified in-house) at a dilution of 1:5000, anti-RFP primary antibody (produced in mouse, Thermo Fisher Scientific, Waltham, USA) at a dilution of 1:1000, and anti-human apolipoprotein AI primary antibody (produced in goat, Calbiochem, San Diego, CA). Fluorescence intensity in the apical and basolateral media for mCherry and mCherry-Bri2 BRICHOS monomers was measured using λ = 1. ex 570nm and λ em Cellular uptake of mCherry or mCherry-Bri2 BRICHOS was analyzed by recording at 610 nm, after which the cells were fixed and analyzed by fluorescence microscopy.

[0197] The permeability of the monolayers was also analyzed for fluorescein isothiocyanate dextran (FITC-dextran, Thermo Fisher Scientific, Waltham, MA) as a negative control and 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 medium was 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 apical and basolateral medium was removed. FITC fluorescence was measured as λ ex 492 nm and λ em Measurements were taken at 518 nm. Insulin permeability was determined by Western blot and a 1:1000 dilution of anti-insulin monoclonal primary antibody (Sigma, St. Louis, MO).

[0198] Cell viability Cell viability was assessed by the tetrazolium-based thiazolyl blue tetrazolium bromide (MTT) assay. For the MTT assay, 50,000 cells / cm were cultured. 2The cells were seeded into clear 96-well plates (TC, Cellstar, Greiner Bio-One, Austria) and grown to confluence. Afterward, the cells were treated with rh BRICHOS domains in eight replicates for 24 hours at 37°C in a humidified atmosphere (5% CO2 / 95% air). The treated cells were incubated with 0.5 mg / ml MTT solution (Sigma-Aldrich, St. Louis, MO) in FBS-free medium for 4 hours at 37°C in a humidified atmosphere (5% CO2 / 95% air). The MTT solution was removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) and incubated for 15 minutes with shaking. Absorbance was measured at 570 nm using a FLUOstar OPTIMA microplate reader (BMG Lab Tech, Germany). Untreated cells served as a positive control, and medium without cells served as a negative control. Staurosporine (SP, Sigma-Aldrich, St. Louis, MO) at 2 μM from Streptomyces sp. was also used as a negative control.

[0199] Cellular uptake For cell uptake analysis, a seeding density of 75,000 cells / cm 2hCMEC / D3 cells were seeded onto collagen-coated 12-well plates, and undifferentiated SH-SY5Y cells were seeded onto untreated 12-well plates. They were maintained at 37°C in a humidified atmosphere (5% CO2 / 95% air) with medium changes every 2 days. After reaching confluence, hCMEC / D3 cells were treated with 1 μM rhBri2 BRICHOS monomer, oligomer, or proSP-C monomer for 2 and 24 hours. SH-SY5Y cells were treated with rhBri2 BRICHOS monomer at concentrations of 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, or 0.05 μM for 24 hours; rhBri2 BRICHOS monomer at concentrations of 1 μM and 0.25 μM for 2 and 24 hours; rhBri2 BRICHOS oligomer at concentrations of 1 μM for 24 hours; or rhBri2 BRICHOS oligomer at concentrations of 0.25 μM and 1 μM for 2 and 24 hours. Untreated cells served as a control. After treatment, cells were lysed as for HEK-293 cells. For Western blots (see below), anti-Bri2 BRICHOS primary antibody (produced in goat and purified in-house) was used at a dilution of 1:1000, and anti-proSP-C BRICHOS primary antibody (produced in goat and purified in-house) was used at a dilution of 1:2000.

[0200] Transcytosis inhibition Monolayer and cellular uptake experiments were performed essentially as described above, except that monolayers were serum-starved for 4 hours in FBS-free EndoGro medium after formation. Monolayers were then incubated with 30 μM Dyngo-4a (Selleck Chemicals, Boston, MA) in FBS-free medium for 20 minutes. Monolayer permeability assays were then performed using 1 μM rhBri2 BRICHOS R221E monomer in FBS-free EndoGro medium supplemented with 30 μM Dyngo-4a for 24 hours at 37°C in a humidified atmosphere (5% CO2 / 95% air). Control experiments without Dyngo-4a were similarly performed in serum-free medium. The apical and basolateral sides were analyzed by Western blot to determine the net mean transport across the monolayer. For cellular uptake, cells were similarly serum-starved for 4 hours in FBS-free EndoGro medium and incubated with 30 μM Dyngo-4a for 20 minutes. Uptake of 1 μM rhBri2 BRICHOS R221E monomer in FBS-free EndoGro medium was analyzed after 4 and 24 hours in the presence of 30 μM Dyngo-4a. Control experiments without Dyngo-4a were similarly performed in serum-free medium. Culture media and lysates were analyzed by Western blot.

[0201] Western blot Samples were separated by Tris-glycine SDS-PAGE under reducing conditions using a 12% resolving gel containing 5% β-mercaptoethanol (Sigma, St. Louis, MO) and 5 mM DTT in sample buffer and heated at 95°C for 5 min. A Spectra Multicolor Broad Range protein ladder was used (Thermo Fisher Scientific, Waltham, USA). For monolayer permeability assessment, 4 μl of protein sample in medium was loaded into wells. For cellular uptake assessment, 7 μg of cell lysate was loaded.

[0202] Electrophoresis was performed at 120 V, and electrophoretic transfer to a polyvinylidene fluoride (PVDF) membrane (0.2 μm, Amersham Hybond, GE Healthcare, Chicago, IL) was achieved at 15 V for 30 min using a transfer buffer of 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 0.05% SDS, and 20% methanol (semi-dry transfer cell, Bio-Rad, Hercules, CA).

[0203] The membrane was immersed in blocking buffer (5% non-fat dry milk (AppliChem, Germany) diluted in 1x PBS containing 0.1% Tween® 20 (Sigma, St. Louis, MO) (1x PBS-T)) for 30 minutes at room temperature and then incubated with primary antibodies overnight at 4°C. The membrane was washed three times with 0.5% non-fat dry milk in 1x PBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG (abcam, UK) diluted 1:3000; rabbit anti-goat IgG (Invitrogen, Waltham, MA) diluted 1:10000; rabbit anti-mouse IgG (Invitrogen, Waltham, MA) diluted 1:10000; goat anti-human IgG Fc (Invitrogen, Waltham, MA) diluted 1:1000) at room temperature for 1 hour. The membrane was washed again as above and twice with 1x PBS. Blotting was performed using a chemiluminescence detection system (Pierce ECL plus, Thermo Fisher Scientific, Waltham, USA). After gentle stripping of cell lysates (0.2 M glycine, 0.1% SDS, 1% Tween® 20, pH 2.2), measurements were normalized to β-actin (0.01 μg / ml) (R&D Systems, Minneapolis, MI). Intensity was evaluated using ImageJ software.

[0204] Insulin and NT *Additional experiments were performed. The blotted membrane was immersed in blocking buffer [1% nonfat dry milk and 0.1% BSA in TBS-T (TBS [50 mM Tris-HCl (pH 7.4), 150 mM NaCl] containing 0.1% Tween® 20]] for 5 minutes and washed with PBS-T for 3 minutes. The membrane was incubated with 0.2% glutaraldehyde in PBS-T, washed three times with PBS-T, immersed in citrate activation buffer (10 mM citric acid pH 6.0, 1 mM EDTA, 0.05% Tween® 20), boiled, and microwaved at 600 W for 10 minutes. After cooling to room temperature, the membrane was immersed in quenching buffer (200 mM glycine in PBS-T) for 10 minutes. After these additional steps, blotting was performed as described above.

[0205] Analysis of BBB passage of NT-BRICHOS fusion protein in wild-type mice Soluble tag nt * (described in WO 2017 / 081239) followed by human Bri2 residues 113-231 * The gene fragment encoding the -Bri2 BRICHOS fusion protein was cloned and expressed. The protein was expressed using Shuffle T7 competent E. coli cells grown at 30°C in Lysogeny Broth (LB) medium supplemented with 15 μg / mL kanamycin. OD 600nmAfter reaching a pH of approximately 0.9, the temperature was lowered to 20°C, and overnight protein expression was induced by adding 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation (3000 × g, 4°C), and the cell pellet was resuspended in 20 mM Tris-HCl (pH 8.0) followed by 5 minutes of sonication (2 seconds on, 2 seconds off, 65% power, on ice). The lysate was centrifuged (24000 × g, 4°C) for 30 minutes, and the supernatant containing the target protein was purified on an immobilized metal affinity chromatography (IMAC) column (Ni Sepharose™ 6 Fast Flow; GE Healthcare, UK) 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 (regenerated cellulose RC, 6-8 kDa membrane; Spectrum Lab) against 20 mM Tris-HCl (pH 8.0) overnight in a cold room.

[0206] All animal handling and experiments were performed at the animal facility, Huddinge campus, Karolinska Institutet, in accordance with local ethical guidelines and approved by Södra Stockholm's Djurforsoksetiska Namnd (dnr S 6-15) and Linkoping's animal ethical board (ID 855). Three-month-old C57BL / 6NTac mice (Taconic, Denmark) and 11-month-old C57BL / 6J mice (Janvier labs, France) were housed seven mice per cage under a 12-h light / dark cycle, controlled humidity, and temperature with free access to food and water. A 0.3 mL syringe with a 30-gauge needle was used to administer 10 mg / kg NT. * Three mice were administered a single dose of either the Bri2-BRICHOS fusion protein or an equal volume of PBS into the lateral tail vein. Prior to injection, the mice were placed in a cage under a heat lamp for 5 minutes to allow the tail vein to dilate. Two hours after injection, 3-month-old mice were anesthetized with isoflurane and intracardially perfused with 40 mL of saline (0.9% NaCl). 10 mg / kg NT was administered. *Eleven-month-old mice received a single intravenous injection of either the -Bri2 BRICHOS fusion protein or an equal volume of PBS. Two hours after injection, they were anesthetized and perfused. Brains were rapidly removed, snap-frozen in dry ice, and stored at -80°C until analysis.

[0207] NT * Immunohistochemical staining of the -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 graded alcohols from 99% to 70%. Brain sections were pretreated for antigen retrieval in DIVA Decloaker 1x solution (Biocare Medical) at 110°C for 30 minutes inside a pressure cooker (Biocare Medical). Slides were cooled at room temperature (RT) for 30 minutes and then washed with Tris-buffered saline containing 0.05% Tween® 20 (TBS-T) and first incubated with peroxidase blocking solution (Dako) for 5 minutes. After washing with TBS-T, brain sections were further blocked with background punisher (Dako) for 10 minutes. The slides were then incubated overnight at 4°C with a solution containing primary anti-NT antiserum from rabbit diluted in DAKO antibody diluent (Agilent). After washing with TBS-T, the sections were incubated for 30 minutes at RT with a secondary cocktail containing horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody. HRP immunoreactivity was detected with permanent green solution (Biosite). 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 coupled to a high-resolution camera, using 20x and 40x objectives.

[0208] result Bri2 BRICHOS accumulation in the CNS after intravenous administration App NL-G-F Mice (expressing human Aβ with the Arctic mutation) and App NL-FMice (expressing wild-type human Aβ) were knocked in with the Aβ precursor protein (Aβ) (Saito T. et al. (2014) Nat Neurosci. 17, 661-663) and were given repeated intravenous injections of rhBri2BRICHOS R221E (SEQ ID NO: 13). After 10-12 weeks of treatment, the amount of Bri2BRICHOS in the CNS was analyzed.

[0209] Figure 9 shows the results of Bri2 BRICHOS R221E after repeated injections. NL-G-F and App NL-F Representative sections (A, D) show Bri2 BRICHOS in mouse brains treated with rh Bri2 BRICHOS R221E or PBS. NL-G-F and App NL-F In mice, double staining was performed with anti-Bri2 BRICHOS antibody (red / pink) and 82E1 anti-Aβ antibody (green), and counterstained with hematoxylin Mayer (blue). Histograms were obtained from App NL-G-F (B, C) and App NL-F (E, F) Average Bri2 BRICHOS intensity in the hippocampus (B, E) and cortex (C, F) of mice. Data are shown as mean ± SEM (3-4 mice / group, 4 tissue sections analyzed for each mouse). p values ​​were calculated using an unpaired parametric two-tailed t-test. Scale bars represent 400 µm (top four panels in A and D) and 100 µm (bottom two panels in A and D). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0210] rh Bri2 BRICHOS R221E-treated App knock-in mice showed more abundant overall Bri2 BRICHOS staining in brain tissue, including neuronal and peripheral Aβ plaques, compared with PBS-treated control mice (Figure 9, panels A and D). NL-G-F and App NL-FIn both mice, there was a significant increase in overall Bri2 BRICHOS staining, as analyzed by the average Bri2 BRICHOS intensity between PBS- and rhBri2 BRICHOS R221E-treated samples (Figure 9, panels B, C, E, and F). Mice were sacrificed 2 to 4 weeks after the last dose of rhBri2 BRICHOS R221E. These results strongly support the finding that intravenously infused rhBri2 BRICHOS R221E crosses the BBB and persists in the CNS parenchyma and cells of App knock-in AD model mice.

[0211] Cell viability The effects of rh BRICHOS domains on hCMEC / D3 and undifferentiated SH-SY5Y cells were assessed by tetrazolium-MTT cell viability assay after 24 hours of incubation. rh Bri2 BRICHOS monomers were not toxic to the human brain microvascular endothelial cell line hCMEC / D3 and the human neuroblastoma cell line SH-SY5Y, but slightly reduced cellular mitochondrial activity in the presence of high concentrations of rh proSP-C BRICHOS and rh Bri2 BRICHOS oligomers or crude solutions.

[0212] The effect of rhBri2 BRICHOS(113-231) wt monomer and oligomer on the human cell lines hCMEC / D3 and SH-SY5Y is concentration-dependent, with increasing concentrations resulting in decreased cell viability. At concentrations of 1.0–0.1 μM, rhBri2 BRICHOS monomer increases SH-SY5Y cell viability from 94.0% to 99% and hCMEC / D3 cell viability from 100.4% to 106.6%. Lower concentrations of rhBri2 BRICHOS monomer slightly increase hCMEC / D3 cell metabolic activity. At 1 μM, rhBri2 BRICHOS oligomer increases SH-SY5Y cell viability to 83.5% and hCMEC / D3 cell viability to 86.7%. At a concentration of 0.5 μM, rh proSP-C BRICHOS induced 88.9% viability in SH-SY5Y cells, and at a concentration of 1 μM, 90.3% viability in hCMEC / D3 cells. High concentrations of rh Bri2 BRICHOS oligomers, like Aβ peptides, caused mild stress on the endoplasmic reticulum (ER), resulting in Ca2+ upregulation. 2+ In the example of rhBri2 BRICHOS monomer, cell membrane integrity is similar to that of the positive control. Because the physiological concentration of BRICHOS domains in human plasma is low, BRICHOS domains are not thought to be detrimental to BBB integrity under normal conditions.

[0213] Cellular uptake Cellular uptake of rh BRICHOS domains was assessed by Western blot after 2 and 24 hours of incubation in the human cell lines SH-SY5Y and hCMEC / D3 and determined by band intensity.

[0214] Incubation of the human neuroblastoma cell line SH-SY5Y with rhBri2BRICHOS monomer at concentrations of 0.05 μM to 1 μM for 24 hours demonstrated linear uptake. Bri2BRICHOS oligomers were detected in cell lysates to a lesser extent than Bri2BRICHOS, possibly indicating that the oligomers are attached to the cell surface. Similarly, ProSP-C BRICHOS was taken up by SH-SY5Y cells, but to a lesser extent. No endogenous BRICHOS domain was detectable in untreated cells, indicating that the detectable BRICHOS domain was taken up from the culture medium.

[0215] The cellular uptake and concentration of rh BRICHOS domain constructs after 2 and 24 hours were also investigated in hCMEC / D3 human brain microvascular endothelial cells. Similar to SH-SY5Y cells, Bri2 BRICHOS monomers and proSP-C BRICHOS were taken up by hCMEC / D3 cells, whereas Bri2 BRICHOS oligomers appeared not to be. No endogenous BRICHOS domain was detectable in untreated cells, indicating that detectable BRICHOS domain was taken up from the culture medium.

[0216] rhBri2BRICHOS wt monomer also efficiently passed through the astrocyte / hCMEC / D3 coculture model. The passage rate through the astrocyte / hCMEC / D3 direct coculture (7.0%) was slightly lower than that of the astrocyte / hCMEC / D3 indirect coculture (8.6%) and hCMEC / D3 monoculture (10.5%). This may be partially related to the higher TEER (transepithelial electrical resistance) value for the astrocyte / hCMEC / D3 direct coculture compared with that of the astrocyte / hCMEC / D3 indirect coculture and hCMEC / D3 monoculture. It is intriguing that astrocytes endocytose rhBri2BRICHOS, especially when astrocytes grow adjacent to hCMEC / D3 cells in the direct coculture model. This suggests that the lower passage rate of rhBri2BRICHOS into the basolateral compartment in the presence of astrocytes may be due, in part, to astrocyte uptake.

[0217] Transcytosis inhibition Addition of Dyngo-4a, a dynamin GTPase inhibitor that blocks endocytic vesicle formation (McCluskey et al., 2013), reduced rhBri2BRICHOS penetration across hCMEC / D3 monolayers by more than fourfold. The presence of Dyngo-4a increased the extent to which rhBri2BRICHOS was internalized or associated with hCMEC / D3 cells, and we observed an increase in the "residual" amount of rhBri2BRICHOS after addition to hCMEC / D3 monolayers, i.e., the amount that could not be accounted for in the apical or basolateral compartments. Notably, cellular uptake and residual amounts of rhBri2BRICHOS were both approximately 5% after 24 h in the presence of Dyngo-4a, confirming that both of these measures represent cell-associated Bri2BRICHOS. These results suggest that functional endocytosis is required for rhBri2BRICHOS to cross the hCMEC / D3 monolayer, and that blocking dynamin GTPase activity prevents rhBri2BRICHOS bound to the hCMEC / D3 cell membrane from being endocytosed and accumulates on the cell surface. Although we have described the nonspecific effects of dynamin GTAase inhibitors, additional experiments are needed to clarify the molecular details of rhBri2BRICHOS transport across the hCMEC / D3 monolayer.

[0218] To investigate the cellular uptake of rhBri2 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 in vitro neuronal models. All BRICHOS variants tested were internalized by cells, albeit to varying degrees. There was a correlation between penetration of the hCMEC / D3 monolayer and cellular uptake, with rhBri2 BRICHOS monomers showing the highest values ​​for both parameters. rhBri2 BRICHOS oligomers, which were not detected passing through the hCMEC / D3 monolayer, were internalized to a low extent by undifferentiated and differentiated SH-SY5Y cells but not by hCMEC / D3 cells. The apparent cellular uptake of rhBri2 BRICHOS was concentration-dependent, saturating at around 2%, and the residual values ​​determined from hCMEC / D3 monolayer experiments (i.e., the amount not accounted for in the apical or basolateral compartments) were approximately 1–2%. The monolayer penetration and cellular uptake experiments, together with the effects observed after dynamin inhibition, strongly support that BRICHOS is transported across endothelial monolayers by transcellular transport and not by nonspecific leakage. The uptake observed in SH-SY5Y cells and human astrocytes suggests that neurons and astrocytes can also endocytose BRICHOS.

[0219] To further rule out the possibility that the penetration or uptake of BRICHOS through the hCMEC / D3 monolayer in hCMEC / D3 and SH-SY5Y cells was related to toxic effects of the proteins tested, we determined cell integrity using a propidium iodide (PI) assay and mitochondrial function using a tetrazolium-MTT cell assay. While rhBri2 BRICHOS monomer showed no effect on either cell integrity or mitochondrial function in either hCMEC / D3 or SH-SY5Y cells, rhBri2 BRICHOS oligomer and proSP-C BRICHOS had only a slight effect on the viability of these cells. This suggests that the BRICHOS variant with the highest penetration through the hCMEC / D3 monolayer is not clearly toxic, arguing against the possibility that the observed penetration is the result of a cytotoxic effect. Additionally, TEER values ​​and FITC-dextran permeability measurements of the monolayers on days 3–9 were unchanged in the presence of rh Bri2 BRICHOS, indicating that the protein does not negatively affect tight junctions or the integrity of the overall monolayer.

[0220] rhBri2 BRICHOS fused to other proteins is efficiently transcytosed across a human BBB model in vitro and is transported across the BBB in wild-type mice Using cultured human cerebral microvascular endothelial (hCMEC / D3) cell monolayers as an in vitro model of the human BBB (Weksler, B. et al. (2013) Fluids Barriers CNS. 10, 16 and Markoutsa, E. et al. (2011) Eur J Pharm Biopharm. 77, 265-274), we investigated whether rhBri2BRICHOS could be used as a transport vehicle to facilitate the uptake of other proteins into the CNS. Specifically, we tested the passage of rhBri2BRICHOS fused to mCherry, a fluorescent protein derived from sea anemones (approximately 30 kDa), and NT, a protein domain derived from spider silk (approximately 15 kDa).

[0221] The permeability of BRICHOS domains was investigated in the hCMEC / D3 cell line 6 days after seeding, when tight junction monolayers had formed with high integrity. Various constructs of rh Bri2 BRICHOS and proSP-C were evaluated at various concentrations and incubation times, as determined by band intensity.

[0222] Permeability studies of various quaternary structures of wild-type recombinant human Bri2 BRICHOS (113-231) show that monomers, but not oligomers, cross hCMEC / D3 monolayers. The permeability of rh Bri2 BRICHOS monomers is time- and concentration-dependent. Interestingly, the permeability of Bri2 BRICHOS R221E monomers is higher than that of Bri2 BRICHOS wt monomers. rh proSP-C BRICHOS wt is found to cross hCMEC / D3 monolayers less than the proSP-C BRICHOS T187R mutant.

[0223] [Table 1]

[0224] [Table 2]

[0225] The permeability of Bri2 and Bri3 BRICHOS soluble tag conjugates was similar to that of the unconjugated Bri2 BRICHOS construct, with monomers crossing the hCMEC / D3 monolayer but not oligomers or crude solutions.

[0226] The NT-Bri2 BRICHOS and mCherry-Bri2 BRICHOS fusion proteins show the same trend as the unconjugated Bri2 BRICHOS(113-231) wt and S-tag fusion proteins, where only the monomer is permeable, whereas the NT tag alone or mCherry alone is not permeable, demonstrating the ability of the Bri2 BRICHOS domain to transport conjugated molecules across hCMEC / D3 monolayers.

[0227] These data clearly demonstrate that endothelial cell uptake and permeability of BRICHOS domains depend on quaternary structure, with monomers crossing hCMEC / D3 monolayers, whereas oligomers do not. The hCMEC / D3 cell line has previously been shown to be size-sensitive, with limited permeability in the apical-to-basolateral direction, and barrier formation is more efficient for larger molecules. Bri2 BRICHOS R221E monomers and proSP-C BRICHOS T187R monomers exhibit higher permeability due to the formation of stable monomers compared to their wild-type counterparts.

[0228] [Table 3]

[0229] Figure 10 and Table 3 show the hCMEC / D3 monolayer permeability of 1 μM solutions of various proteins and macromolecules (percentage of total amount added to the apical side).

[0230] We demonstrate significant passage of rh wild-type and R221E Bri2 BRICHOS monomers from the apical to the basolateral side of the monolayer, i.e., they are transcytosed, whereas rh wild-type Bri2 BRICHOS oligomers are not (Figure 10). Neither mCherry nor NT alone crossed the monolayer substantially, but the fusion construct with the rh Bri2 BRICHOS domain was transcytosed in substantial amounts (Figure 10).

[0231] It remains to be determined whether the substantial passage of Bri2 BRICHOS-mCherry through an in vitro human BBB model can be further increased by adding microbubbles, and the extent to which the in vitro human BBB model predicts all characteristics of the mouse BBB in vivo remains to be determined.

[0232] To test whether rhBri2 BRICHOS acts to transport media across the BBB in vivo, we repeated the same type of experiment previously performed to test BBB penetration of recombinant Bri2 and proSP-C BRICHOS in wild-type mice. Figure 11 shows anti-NT antibody staining (blue) of the brain cortex of mice injected with NT-Bri2 BRICHOS (A) or PBS (B). This shows that recombinant NT-Bri2 BRICHOS was detectable in the brain parenchyma and neurons 2 hours after intravenous injection, whereas mice injected with PBS showed only background staining (Figure 11).

[0233] These findings strongly support the idea that the ability of rhBri2BRICHOS to cross the BBB can be exploited to transport other proteins across the BBB and that, after passage into the CNS, the fusion protein spreads in the parenchyma and is taken up by cells. Proteins generally do not passively cross the BBB; for example, only up to 0.1% of peripherally administered antibodies cross the BBB (18), indicating that an active mechanism mediates BBB passage of rhBri2BRICHOS.

[0234] NT *The rhBri2BRICHOS monomer also efficiently passed through an astrocyte / hCMEC / D3 coculture model. As with the rhBri2BRICHOS wt monomer, passage of the fusion protein through coculture models, particularly the astrocyte / hCMEC / D3 direct coculture model, was lower than that through hCMEC / D3 monoculture. Fluorescence images of hCMEC / D3 monolayers after addition of rhBri2BRICHOS-mCherry show abundant red fluorescent dots, whereas no such dots are observed after addition of mCherry alone. As with the rhBri2BRICHOS alone, the fusion proteins S-tag-Bri2BRICHOS or NT * -Bri2 BRICHOS oligomers do not cross hCMEC / D3 monolayers.

[0235] rhBri2 BRICHOS fused to target proteins is endocytosed and transported to lysosomes in primary neurons Figure 11A shows that the fusion protein rhNT-Bri2BRICHOS translocates into CNS cells and appears in vesicles. Subcellular localization was investigated in more detail using primary mouse neurons in culture and the recombinant fusion protein mCherry-Bri2BRICHOS. Recombinant mCherry-Bri2BRICHOS was added to the culture medium at low nanomolar concentrations, and after 24–48 hours, neurons were fixed and visualized by fluorescence microscopy.

[0236] Figure 12 shows that rhBri2BRICHOS mediates uptake into mouse primary neurons. After addition to the culture medium, a fusion protein consisting of Bri2BRICHOS bound to mCherry (mCh-Bri2) was detected in abundant intracellular vesicles (top panel). mCherry alone was not internalized (bottom panel).

[0237] Thus, mCherry-Bri2 BRICHOS is endocytosed and detected in intracellular vesicles, whereas the control protein, mCherry alone, is not internalized at detectable levels. These data are highly consistent with the results of the transcytosis experiments shown in Figure 10.

[0238] Finally, the cargo protein-bound rhBri2BRICHOS is endocytosed and transported to lysosomes. Primary mouse neurons treated with the fusion protein mCherry-Bri2BRICHOS exhibit cellular uptake and localization in lysosomes, as indicated by colocalization with the lysosome-specific dye SiR-Lyso (data not shown). Accordingly, labeling of neurons treated with mCherry-Bri2BRICHOS with the lysosome-specific dye SiR-Lysosome demonstrated clear colocalization.

[0239] conclusion This experiment demonstrates that rhBri2BRICHOS is efficiently transported across hCMEC / D3 monolayers, an in vitro model of the human BBB, apparently by a dynamin-dependent endocytic mechanism. The in vivo mechanism of action of BRICHOS is clearly similar to that found in in vitro and ex vivo applications, suggesting that selective reduction of toxic oligomers by Bri2BRICHOS is an important part of the efficacy of the treatment. The results presented here, showing that rhBri2BRICHOS can enter neurons and astrocytes after intravenous injection in mice, suggest that Bri2BRICHOS can mediate cellular effects as well. In mice, rhNT *Because Bri2 BRICHOS is found in early endosomes of neurons and astrocytes in key areas of the brain after intravenous injection, it can transport cargo proteins across the human BBB model used here, a property that also applies to the BBB in vivo. This opens up the exciting possibility that the ability of BRICHOS to reduce Alzheimer's pathology can be combined with additional activities mediated by fusion partners, thereby creating diverse biopharmaceuticals that also efficiently cross the BBB. One candidate for fusion with Bri2 BRICHOS is a monoclonal antibody against Aβ, which has shown therapeutic efficacy in clinical trials for Alzheimer's disease (van Dyck et al., N Engl J Med 388(1):9-21, 2023), which may benefit from combining increased BBB crossing with Bri2 BRICHOS-mediated effects on Aβ toxicity. Notably, BRICHOS is more effective in reducing the number of toxic Aβ oligomers in vitro compared with a monoclonal antibody against Aβ that has undergone clinical trials (Linse et al., Nat Struct Mol Biol, 27:1125-1133, 2020). Bri2BRICHOS is relatively small, scalable for recombinant production, shows no detectable toxicity in cells in culture or after repeated intravenous injections, and has a high ability to transport cargo across the BBB. The protocol for producing and administering rhBri2BRICHOS fusions is also rather simple compared with producing transport vesicles using encapsulation strategies. This makes rhBri2BRICHOS an interesting vehicle for delivering various macromolecules across the BBB to cells in the CNS.

[0240] Experiments on inhibition of α-synuclein oligomer formation Materials and Methods Preparation of Bri2 BRICHOS monomer SHuffle T7 (K12 strain) E. coli cells were transformed with a plasmid vector carrying the human Bri2 BRICHOS R221E mutant sequence (nucleotides 113-231 of full-length human Bri) fused to His6x-NT at the N-terminus separated by a thrombin cleavage site. Cells were incubated in LB medium with 15 μg / ml kanamycin at 30°C until an OD of 0.8 was reached. Cells were then induced with 0.5 mM IPTG and grown overnight at 20°C. Cells were harvested by centrifugation at 5,000 × g at 4°C, and the pellets were resuspended in 20 mM Tris-HCl (pH 8.0). The two pellets were sonicated for 5 minutes (2 seconds on, 2 seconds off, 65% of maximum power), and the resulting lysate was centrifuged at 20,000 × g for 30 minutes at 4°C. The collected supernatant was then loaded onto a Ni-NTA column (GE Healthcare Bio-Science AB, Sweden). The column was washed with 30 column volumes (CV) of 20 mM Tris-HCl, 15 mM imidazole, pH 8.0, followed by elution with 20 mM Tris-HCl, 300 mM imidazole, pH 8.0. Fractions were pooled, thrombin (1:1000 enzyme-to-substrate, w / w) was added, and the mixture was dialyzed overnight at 4°C against 20 mM sodium phosphate, 0.2 mM EDTA, pH 7.4. The sample was loaded onto a Ni-NTA gravity column to remove His-NT species and uncleaved target protein. The flow-through was concentrated with a cut-off concentrator (Vivaspin 20, 10 kDa MWCO Cytiva) and directly loaded onto a Hiload Superdex 75 PG size-exclusion column (Cytiva, USA) to collect the peak of interest corresponding to the monomer.

[0241] Preparation of α-syn monomers BL21(DE3) Escherichia coli was transformed with the bacterial expression plasmid pET21a-α-syn (Addgene), which contains the human α-synuclein coding sequence. Cells were grown in LB medium with 100 μg / ml ampicillin at 37°C to an OD of 0.8. The culture was inoculated with 0.5 mM IPTG and incubated overnight at 20°C. Cells were centrifuged at 5,000 × g for 20 minutes at 4°C, and the bacterial pellet was resuspended in 20 mM Tris-HCl (pH 8.0) by gentle vortexing. Cell pellets from 1 L were warmed and lysed using a G. Heinemann Ultraschall- un Labortechnik (G. Heinemann Ultraschall- un Labortechnik) for 5 minutes at 65% maximum power with a 2-second on, 2-second off pulse setting. Samples were then boiled for 10 minutes or 5 minutes. This was followed by 20 minutes of centrifugation at 24,000 × g for 20 minutes at 4°C. The supernatant was loaded onto a HiTrap QFF anion exchange chromatography column. Proteins were eluted with a 10 CV gradient of 20 mM Tris-HCl, 1 M NaCl (pH 8). The eluted peak was loaded onto a reversed-phase chromatography column using 99.9% HO, 0.1% TFA as the running buffer. Elution was performed using a 20 CV buffer gradient of 70% ACN, 0.1% TFA. Fractions corresponding to the peak of interest were pooled, and the optical density was measured at 280 nm. The sample was lyophilized overnight under vacuum (LabConco CentriVap Concentrator). Lyophilized samples not used directly 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 loaded onto a Superose 6 Increase column using 20 mM sodium phosphate, 0.2 mM EDTA (pH 7.4) as the running buffer, which was then further stored at −20°C to obtain a homogenous and pure α-syn monomer sample.

[0242] ThT aggregation assay For kinetic experiments, each sample contained 30 μM or 70 μM α-syn monomer in the presence of 20 μM ThT and various concentrations of Bri2 BRICHOS monomer in a 20 mM sodium phosphate, 0.2 mM EDTA, pH 7.4 buffer, or with NaCl added to 154 mM. Two 1 mm diameter glass beads were used in each well. Fluorescence was recorded using a 448-10 nm excitation filter and a 482-10 nm emission filter (FLUOStar Omega, BMG Labtech, Offenberg, Germany) while the plate was shaken at 300 rpm for a 9-minute on, 1-minute off cycle. Each cycle was set to 10 minutes, and the temperature was set to 37°C. For seed preparation, α-syn fibrils were obtained from a previous ThT kinetics experiment using only 70 μM α-syn monomer. These fibrils were then sonicated for 3 minutes using a probe sonicator (SONICS Vibra Cell™) for 2 seconds on, 2 seconds off. For all experiments, aggregation traces were normalized and averaged across six replicates for each sample.

[0243] Analysis of α-syn aggregation kinetics Aggregation traces of 70 μM and 30 μM α-syn monomer at molar ratios of 0–100% and various concentrations of BRICHOS were normalized, averaged over six replicates, and truncated at the plateau of the aggregation reaction. Primary nucleation, secondary nucleation, and elongation were expressed as a combined rate k + k. n The aggregation rate was fitted using the secondary-nucleation-dominated unseeded nucleation model from Amylofit, which includes k and k + k2 as nuclei. Individual fits were first performed to obtain the dependence of these parameters on BRICHOS concentration. Secondary nucleation was then dominated by initial aggregate number and concentration, which allowed for evaluation of the contribution of a single nucleation rate. Two nucleation rate constants were set as global fit parameters, meaning they were restricted to the same value at all BRICHOS concentrations, while one nucleation coefficient was set as an individual fit parameter.

[0244] Highly seeded aggregation kinetics was analyzed using GraphPad. The extension rate was extracted by fitting a straight line to the first 2 hours of the aggregation curve.

[0245] Preparation of fibrils incubated with Bri2 BRICHOS α-syn monomer (70 μM) was incubated similarly to the ThT kinetics experiment, but without ThT. After approximately 100 hours of experimentation, fibrils were spun down at 16,200 × 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).

[0246] transmission electron microscope Five microliters of a diluted solution of α-syn fibrils (approximately 15 μM) was applied to a 200-mesh Formvar-coated nickel grid, and excess solution was removed using blotting paper after 10 minutes of incubation. The grid was washed twice with 10 μl of MQ water and then stained with 1% uranyl formate for 5 minutes. Excess stain was blotted with blotting paper and air-dried. Fibril morphology was analyzed using a transmission electron microscope (FEI Tecnai 12 Spirit BioTWIN, operated at 100 kV) with a 2k × 2k Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH, Muenster, Germany). 15–20 images were randomly recorded for each sample, and fibril diameters were measured using Image J software.

[0247] Naive Page BRICHOS was added to α-syn fibril samples at an equimolar concentration (8 μM). Samples were prepared under non-denaturing conditions and then run on a 7.5% naive gel. BRICHOS was visualized by Coomassie blue staining.

[0248] Oligomer Capture Equimolar concentrations of α-syn monomer (70 μM) with or without BRICHOS were incubated as in the ThT kinetics experiments. Samples were collected at various time points, flash-frozen in liquid nitrogen, and stored at -80°C until use. Samples were prepared under non-denaturing conditions, run on naive PAGE 10%, and stained with Coomassie blue.

[0249] Surface plasmon resonance Binding analyses were performed using a Biacore 3000 instrument at 25° C. Three experiments were performed using different ChiPs: pre-immobilized α-syn fibrils, salted α-syn fibrils, and α-syn monomers.

[0250] The system was primed with the following running buffer: 20 mM sodium phosphate, 0.2 mM EDTA, pH 7.4. Various 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 min. Channels 1 and 3 were left empty and served as references to control for possible nonspecific binding of α-syn. If BRICHOS protein was bound, it was flushed with 20 mM NaOH and buffer, and the chip was allowed to equilibrate for 15 min before injecting the sample again. Each sample was subjected to two replicates.

[0251] NMR experiments 1 H- 15 N HSQC titrations were performed on a Bruker 700 MHz spectrometer at 281 K with either BRICHOS or α-syn sonicated fibrils, respectively, in 20 mM NaP, 0.2 mM EDTA at pH 7.4. 15 N-labeled α-syn or 15 All NMR data were processed with Bruker Topspin and analyzed with Poky.

[0252] ITC 216 μM BRICHOS monomer was injected 18 times with a 2 μl injection syringe into the sample cell containing 20 μM α-syn monomer or a control (iTC200 α-syn Microcal, GE Healthcare) buffer (20 mM NaP, 0.2 mM EDTA, pH 7.4). Experiments were performed at room temperature with a stirring speed of 1000 rpm.

[0253] FIDA Recombinant wild-type human α-syn was expressed in Escherichia coli using the pET11-D construct and purified as described in 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 for 3 hours at 37°C with 900 rpm shaking in a Heating Digital Shaking Drybath (Thermo Scientific), followed by size exclusion using a Superose 6 prep grade XK 26 / 100 column (GE Healthcare). Monomeric and oligomeric fractions were collected and stored at 4°C. Prior to analysis, oligomeric fractions were pooled and concentrated using a 100 kDa spin filter (Amicon Ultra). α-Syn fibrils were prepared by dissolving lyophilized α-syn in PBS and fibrillating the filtered solution at a concentration of 1.1 mg / mL in a 96-well plate with 3 mm glass beads for 3 days at 37°C with shaking at 600 rpm. Fibrils were isolated by centrifugation at 13,000 rpm for 10 min, redissolved in PBS at a concentration of 1 mg / mL, and fragmented by sonication using a Q500 ultrasonicator (Qsonica, Connecticut, USA) fitted with a 2 mm microtip probe at 20% intensity with a setting of 10 s pulses and 10 s pauses for a total of 30 s of sonication. BRICHOS was labeled with Alexa-488 NHS ester (Thermo Scientific) by mixing 43.6 μM BRICHOS in 100 mM bicarbonate buffer (pH 8.3) with Alexa-488 in DMSO at a molar ratio of 1:2, followed by incubation at room temperature for 1 hour. The reaction was quenched by the addition of 100 mM Tris-HCl (pH 7.4), and the labeled BRICHOS was separated from unreacted label using a PD10 column (GE Healthcare) equilibrated with PBS.Protein concentration and labeling efficiency were estimated using a DeNovix DS-11 (DeNovix). Flow-induced dispersion analysis was performed on a FIDA One instrument (FIDA Biosystems) with LED-induced fluorescence detection using an excitation wavelength of 480 nm. A standard capillary (inner diameter: 75 μm, outer diameter: 375 μm, total length: 100 cm, length to detection window: 84 cm) (FIDA Biosystems) was rinsed with 1 M NaOH and equilibrated with PBS at 25 °C. Experiments were performed by priming the capillary with running buffer at a pressure of 3500 mbar. Then, various concentrations of analyte (α-syn monomer, oligomer, or fibril) and 50 nM indicator (BRICHOS-Alexa488) were sequentially injected into the capillary for 20 s at a pressure of 3500 mbar for analyte injection and 50 mbar for indicator injection, respectively. The final injection of analyte was carried out at a pressure of 400 mbar for 180 seconds to allow for the transfer of indicator and analyte to the detector side. The resulting taylograms were plotted as the hydrodynamic radius (R) for each binding reaction. h The resulting isothermal binding curves were fitted to the following equation:

[0254]

number

[0255] In the formula, K D is the dissociation constant, [A] is the analyte concentration, and R I and R IA are the R of the indicator and the complex, respectively h is.

[0256] Electrophysiological experiments For electrophysiological experiments, all chemical compounds used in the extracellular solution were obtained from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Kainic acid (KA) was obtained from Tocris Bioscience (Bristol, UK). α-syn fibrils were prepared by incubating α-syn monomers in PBS, 0.1% sodium azide, pH 7.4, at 37°C and 900 rpm for 7 days. They were then sonicated for 3 minutes using a probe sonicator, 2 seconds on, 2 seconds off.

[0257] We tested the effects of α-syn fibrils in vitro by incubating hippocampal gamma oscillations ex vivo using 4-6 week-old wild-type (WT) mice (n = 12). For brain extraction, mice were deeply anesthetized with isoflurane. Brains were excised and placed in ice-cold modified artificial cerebrospinal fluid (ACSF) containing 80 mM NaCl, 24 mM NaHCO3, 25 mM glucose, 1.25 mM NaH2PO4, 1 mM ascorbic acid, 3 mM sodium pyruvate, 2.5 mM KCl, 4 mM MgCl2, 0.5 mM CaCl2, and 75 mM sucrose, aerated with carbogen (95% O2 and 5% CO2). Horizontal slices (350 μm thick) of the ventral hippocampus of both hemispheres were prepared using a Leica VT1200S vibratome (Leica Microsystems). Immediately after sectioning, slices were transferred to a humidified interface holding chamber containing standard ACSF (ACSF): 124 mM NaCl, 30 mM NaHCO3, 10 mM glucose, 1.25 mM NaH2PO4, 3.5 mM KCl, 1.5 mM MgCl2, and 1.5 mM CaCl2, under a continuous supply of humidified carbogen. The chamber was maintained at 37°C during sectioning and then cooled to room temperature (approximately 22°C) for a minimum of 1 hour. To test the toxic effects of α-syn and BRICHOS R221E monomer on hippocampal gamma oscillations, four conditions were selected: 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 were preincubated for 30 min in an incubation chamber underwater containing ACSF. During incubation, slices were continuously supplied with carbogen gas (5% CO2, 95% O2) aerated with ACSF. After incubation, slices were transferred to an interface-style recording chamber for extracellular recording.

[0258] Recordings were made in the hippocampal area CA3 using borosilicate glass microelectrodes filled with ACSF, with resistance set at 3–6 MΩ. Local field potentials (LFPs) were recorded in an interface chamber at 32°C (perfusion rate 4.5 mL per minute). LFP gamma oscillations were induced with kainic acid (100 nM). Oscillations were allowed to stabilize for 20 min before recording. LFP recordings in the interface chamber were performed using a 4-channel amplifier / signal conditioner M102 amplifier (Electronics Lab, University of Cologne, Germany). Signals were sampled at 10 kHz and low-pass filtered at 1 kHz using Hum Bug 50 Hz denoiser (Quest Scientific, North Vancouver, BC, Canada) software, digitized, and stored using a Digidata 1322A and the Clampex 10.4 program (Molecular Devices, CA, USA). Power spectral density plots (from 60-second long LFP recordings) were calculated in 8192-point averaged Fourier segments using AxographX ​​(Kagi, Berkeley, CA, USA). Gamma oscillation power was calculated by integrating the 20–80 Hz power spectral density with results representing average values ​​acquired over 1 minute.

[0259] result BRICHOS efficiently blocks bulk aggregation of α-syn First, we monitored the aggregation rate of 70 μM α-syn in the presence of recombinant human (rh) Bri2 BRICHOS R221E monomer (hereafter simply referred to as BRICHOS) at various concentrations, ranging from 2 to 95% molar equivalent of α-syn monomer, at physiological pH 7.4 under shaking conditions. We observed that α-syn aggregation was delayed in a concentration-dependent manner up to the addition of 95% molar equivalent of BRICHOS. This resulted in a delay in aggregation half-life ranging from 18.3 ± 1.8 h to 53.1 ± 3 h, as determined by sigmoidal fitting to the individual aggregation traces. At higher BRICHOS concentrations, the inhibitory effect appears to saturate, and no further inhibition of aggregation is observed between 25% and 100% BRICHOS.

[0260] To test whether the inhibitory effect could be masked by electrostatic interactions, we performed the same experiment in the presence of physiological concentrations of salt. The presence of salt significantly accelerated the rate of α-syn aggregation, with the aggregation half-life being 9.6 ± 0.5 h compared to 18.3 ± 1.8 h in the absence of salt. Notably, even under these conditions, BRICHOS efficiently blocked aggregation, delaying the aggregation half-time to 12.1 ± 5 h at 90% molar equivalent. Therefore, the inhibitory effect of BRICHOS is specific to α-syn and operates under near-physiological conditions.

[0261] We further aimed to confirm these results by performing the same experiment at an α-syn concentration of 30 μM instead of 70 μM and adding the same molar ratio of BRICHOS. Indeed, we found that BRICHOS also retarded α-syn aggregation at lower α-syn concentrations, both in the presence and absence of salt. These results indicate that the inhibitory effect is specific to BRICHOS, either through strong electrostatic forces or other types of interactions, such as hydrophobic interactions, with α-syn.

[0262] BRICHOS specifically inhibits secondary nucleation and fibril end elongation Amyloid formation can generally be explained by a polymerization mechanism that depends on nucleation. Primary nucleation (k n The formation of nuclei, known as fibril end extension (k +) induces further fibril expansion. Secondary nucleation (k2) or fragmentation (k - ) may also occur. The α-syn aggregation traces can be fitted to these theoretical kinetic models that account for 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 the monomer-dependent secondary nucleation, a nucleation rate constant k related to fibril fragmentation was also observed. - There may be a secondary reaction independent of the monomer, which is explained in . Typically, this process can be associated with agitation during the fibril reaction.

[0263] Previous studies have shown that α-syn aggregation follows an aggregation mechanism dependent on secondary nucleation at physiological pH. By performing a detailed global fit analysis of the aggregation kinetics, we were able to analyze the nucleation rate as affected by BRICHOS. First, we applied a model that included only the monomer-dependent secondary nucleation reaction, and then added primary nucleation and elongation.

[0264] To elucidate the contribution of individual nucleation events, two nucleation rate constants were set as global fit parameters, meaning that they were constrained to be the same at all BRICHOS concentrations, and one nucleation coefficient was set as an individual fit parameter. The global fit that best explained the data was chosen to be either k or k. + are the free fitting parameters, suggesting that BRICHOS inhibits elongation and / or secondary nucleation. The same results were obtained in the presence of salt and with 30 μM α-syn in the presence of salt.

[0265] Then, the monomer-dependent secondary nucleation k2 is ignored, and the fragmentation-related monomer-independent secondary nucleation k - We also tested an alternative nucleation model in which k is replaced by k as a free fitting parameter. - or k +Although an overall good fit was obtained for , the average residual error favors the second-order nucleation model, which is slightly monomer-dependent.

[0266] To infer whether BRICHOS preferentially affects elongation, secondary nucleation, or both, highly seeded kinetic analyses were performed. At high seed concentrations, numerous free fibril ends are available, resulting in a state in which the kinetics of aggregation initiation are dominated solely by fibril elongation. Therefore, the elongation rate could be extracted by analyzing the slope of the first 2 hours of the kinetic trace. The initial slope was found to depend on BRICHOS concentration, indicating that BRICHOS inhibits the elongation of α-syn fibrils. The combination of fitting parameters k from the global fit analysis + The dependency of k2 is + By comparing with the initial slope from the highly seeded aggregation kinetic analysis, which is proportional to k + The adjustment of k + It turns out that the large effect observed for k2 cannot be explained solely by the + In addition, k2 is also significantly reduced by the presence of BRICHOS. Therefore, these results indicate that BRICHOS inhibits both secondary nucleation and fibril end elongation.

[0267] There may also be fragmentation-related processes, but k - Models including both k and k2 exhibit several combined fitting parameters that increase the risk of overfitting. The slightly better fit of k2, combined with the mechanistic understanding of the inhibition of secondary nucleation at the fibril surface by BRICHOS binding to α-syn fibrils, provides a reductionist explanation for the mechanism by which BRICHOS blocks monomer-dependent secondary nucleation and fibril end elongation.

[0268] BRICHOS has a reduced inhibitory effect on the aggregation of C-terminally truncated α-syn variants We investigated the effect of BRICHOS on C-terminal truncated variants of α-syn at positions 110 and 121 (hereafter referred to as α-syn110 and α-syn121, respectively). These variants have been shown to have an accelerated tendency toward aggregation (Farzadfard et al., Commun Biol, 5(1):123, 2022). Our findings revealed that BRICHOS exhibited only a limited effect on α-syn121 and a weak inhibitory effect on α-syn110, respectively, which could not be fitted to the data. These results suggest that the presence of the C-terminus is essential for optimal efficacy of BRICHOS in preventing α-syn aggregation. Considering that secondary nucleation involves interactions between the C-terminal segment of α-syn fibrils and the N-terminal segment of α-syn monomers (Kumari et al., Proc Natl Acad Sci USA, 118(10), 2021), the limited effect of BRICHOS on the aggregation rate of C-terminally truncated variants suggests that BRICHOS binds to the C-terminus of α-syn, competing with α-syn monomers in the secondary nucleation process.

[0269] BRICHOS does not affect the morphology of α-syn fibrils To investigate whether BRICHOS modulates α-syn fibril morphology, we collected electron microscopy images of α-syn fibrils in the presence and absence of 100% molar equivalents of BRICHOS during the aggregation reaction. Analysis of the diameters of 100 fibrils selected under these conditions revealed that first-generation BRICHOS-cocultured α-syn fibrils had a diameter of approximately 10.32 ± 2.07 nm, while α-syn fibrils alone had a diameter of 11.52 ± 1.10 nm (Figure 13). This is consistent with the published 3D structure of in vitro α-syn fibrils, which is approximately 10 nm, corresponding to two bundled protofilaments.

[0270] Figure 13 shows that BRICHOS does not affect the morphology of α-syn fibrils. TEM images of α-syn fibrils after fibrillation in the absence (a) or presence of 100% (b) of 70 μM molar equivalent of BRICHOS. (c) Diameter of α-syn fibrils incubated with or without BRICHOS.

[0271] In conclusion, BRICHOS does not affect the diameter of α-syn fibrils when coincubated with α-syn monomers.

[0272] BRICHOS binds to α-syn fibrils but not to monomers To evaluate the binding ability of various α-syn and BRICHOS, we used various biophysical techniques, including isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence intensity distribution analysis (FIDA). SPR experiments confirmed the binding of α-syn fibrils to BRICHOS.

[0273] To determine the amount of BRICHOS that binds to α-syn fibrils, co-cultured BRICHOS-α-syn fibril samples were analyzed by naive PAGE. These experiments suggested that only 9 ± 4% of BRICHOS molecules bind to fibrils when incubated at equimolar concentrations. This result is consistent with the results of sonicated α-syn fibrils. 15 N-labeled BRICHOS was added to 2D 1 H- 15 This was confirmed by solution NMR experiments recording N HSQC spectra. Due to the dynamic nature of BRICHOS, only some of the expected resonances were observed in the spectra, and due to the large size of α-syn fibrils, bound BRICHOS would not be expected to be observed in the solution NMR spectra. Addition of equimolar concentrations of α-syn fibrils was found to result in approximately 10 ± 10% signal loss, indicating that only a small fraction of BRICHOS was bound. Furthermore, similar signal loss was observed upon addition of half or double the molar concentrations of fibrils.

[0274] Fluorescent in-capillary FIDA experiments were performed using fluorescently labeled BRICHOS-Alexa488 to investigate the binding affinity to α-syn fibrils and monomers. Although BRICHOS-Alexa488 did not bind to α-syn monomers, high molecular weights were detected in the presence of fibrils, indicating binding between BRICHOS and Alexa488. Furthermore, no observable binding was observed between BRICHOS and α-syn monomers. 15 NMR HSQC experiments using N-labeled α-syn were performed to verify these findings. This lack of interaction was further supported by ITC experiments, which showed that BRICHOS did not bind to α-syn monomers and that the observed signal was solely due to the heat of dilution of the BRICHOS sample injected into the cell.

[0275] BRICHOS binds to α-syn oligomers We further investigated the ability of BRICHOS to bind to α-syn oligomers. We faced challenges arising from the complexity of α-syn oligomer formation, in which various forms of oligomers with distinct characteristics and diverse structures emerge under various environmental conditions. In our experiments, α-syn oligomers were prepared by incubating α-syn monomers at 37°C for 5 hours, followed by centrifugation to remove large aggregates, and then isolating the oligomers by size-exclusion chromatography. These oligomers appear to be stable, possess β-sheet structures, and are capable of kinetically trapping and permeabilizing vesicles in vitro. In FIDA experiments, we observed that BRICHOS-Alexa488 can bind to these α-syn oligomers. Simulations using kinetic parameters from experimental data indicate that the presence of BRICHOS reduces the rate of secondary nucleation and, therefore, reduces oligomer formation.

[0276] BRICHOS prevents α-syn neurotoxicity Next, we evaluated the effect of BRICHOS on α-syn aggregation, which leads to α-syn neurotoxicity. We performed electrophysiological assays on hippocampal slices from wild-type mice. γ oscillations are a key indicator of neuronal activity and neurotoxicity of added compounds. γ oscillations were recorded after exposure to control buffer (PBS, 0.1% sodium azide, pH 7.4) or sonicated α-syn fibrils alone or in combination with equimolar concentrations of BRICHOS (Figure 14, a–c).

[0277] FIG. 14 shows that BRICHOS monomers prevent the neurotoxic effects of sonicated α-syn fibrils. (a) Schematic of the experiment measuring toxicity to hippocampal slices from mouse brain. (b) Example γ oscillation traces for control conditions (gray), 500 nM sonicated α-syn fibrils, 1 μM sonicated α-syn fibrils, and after incubation of 1 μM sonicated α-syn fibrils with equimolar concentrations of BRICHOS. (c) Example power spectra of γ oscillations for control conditions, 500 nM sonicated α-syn fibrils, 1 μM sonicated α-syn fibrils, and after incubation of 1 μM sonicated α-syn fibrils with equimolar concentrations of BRICHOS.

[0278] Sonicated α-syn fibrils showed a concentration-dependent effect on the power of gamma oscillations. At a fibril concentration of 500 nM, the gamma oscillation power did not decrease significantly, but at a concentration of 1 μM, the gamma oscillation power decreased significantly, indicating a pronounced neurotoxic effect. Interestingly, co-incubation of 1 μM sonicated α-syn fibrils with equimolar concentrations of BRICHOS did not affect the power of gamma oscillations, suggesting that the presence of BRICHOS effectively prevented the neurotoxicity of α-syn fibrils.

[0279] Itemized List of Embodiments 1. An isolated recombinant protein selected from the group consisting of proteins comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) or an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; The method does not include a step of subjecting the mammalian tissue to ultrasound treatment. Isolated recombinant protein. 2. An isolated recombinant protein selected from the group consisting of proteins comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO:2) and proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs:5, 6, 7, 8, 9, and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO:3) and an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO:4); - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; Isolated recombinant protein. 3. A combination of an isolated recombinant protein according to any one of the preceding clauses and lipid microbubbles and / or nanodroplets, - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; The method does not include a step of subjecting the mammalian tissue to ultrasound treatment. 4. A combination of an isolated recombinant protein according to any one of the preceding clauses and lipid microbubbles and / or nanodroplets, - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated recombinant protein to said mammal; for use in a method of treating Alzheimer's disease in a mammal, comprising: The combination, wherein said isolated recombinant protein is not contained within said microbubbles and / or said nanodroplets. 5. The isolated recombinant protein or combination of any one of the preceding clauses, wherein the isolated recombinant protein and the microbubbles and / or nanodroplets are administered intravenously. 6. The isolated recombinant protein or combination according to any one of paragraphs 1 to 5, wherein a therapeutically effective amount of at least 1 mg / kg, such as at least 5 mg / kg, more preferably at least 10 mg / kg, of said isolated recombinant protein is administered. 7. The isolated recombinant protein or combination according to any one of paragraphs 1 to 5, wherein a therapeutically effective amount of the isolated recombinant protein is administered of less than 50 mg / kg, such as less than 30 mg / kg, more preferably less than 20 mg / kg. 8. The isolated recombinant protein or combination according to any one of paragraphs 1 to 7, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, identity to the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 9. The isolated recombinant protein or combination of paragraph 8, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 10. The isolated recombinant protein or combination of paragraph 9, wherein the isolated recombinant protein is selected from the group consisting of a protein comprising an amino acid sequence having at least 90%, preferably at least 95%, preferably at least 99% identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and a protein comprising an amino acid sequence having at least 90% identity to the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 11. The isolated recombinant protein or combination of paragraph 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). 12. The isolated recombinant protein or combination of any one of paragraphs 1 to 7, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 13. The isolated recombinant protein or combination of paragraph 12, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 14. The isolated recombinant protein or combination of paragraph 13, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 15. The isolated recombinant protein or combination of paragraph 14, wherein the isolated recombinant protein is selected from the group consisting of any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 16. The isolated recombinant protein or combination according to any one of items 1 to 15, wherein the amino acid residue corresponding to position 221 of SEQ ID NO: 1 in the isolated recombinant protein is selected from Glu and Asp. 17. The isolated recombinant protein or combination of paragraph 16, wherein the amino acid residue corresponding to position 221 of SEQ ID NO: 1 in the isolated recombinant protein is Glu. 18. The isolated recombinant protein or combination according to any one of paragraphs 1 to 17, wherein the number of amino acid residues in the isolated recombinant protein is 200 or less, such as 150 or less. 19. The isolated recombinant protein or combination according to any one of paragraphs 1 to 18, wherein the number of amino acid residues of the isolated recombinant protein is 90 or more. 20. The isolated recombinant protein or combination of any one of the preceding clauses, wherein the microbubbles and / or nanodroplets are lipid coated. 21. The isolated recombinant protein or combination of any one of the preceding clauses, wherein the microbubbles and / or nanodroplets contain a gas core of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)2000), and perfluorobutane. 22. The isolated recombinant protein or combination according to any one of paragraphs 1 to 21, wherein the microbubbles and / or nanodroplets contain sulfur hexafluoride, polyethylene glycol (PEG, macrogol), distearylphosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol sodium, and palmitic acid. 23. The isolated recombinant protein or combination according to any one of the preceding clauses, wherein the diameter of the microbubbles and / or nanodroplets is between 1 and 8 μm, such as between 2 and 6 μm, for example between 4 and 5 μm. 24. The isolated recombinant protein or combination of any one of paragraphs 1 to 23, wherein the treatment is selected from the group consisting of preventative treatment, palliative treatment, and definitive treatment.

[0280] 25. A method of treating Alzheimer's disease in a mammal, including a human, in need thereof, comprising: - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; - administering to the mammal an isolated recombinant protein selected from the group consisting of proteins comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) or an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); Including, the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; The method does not include a step of subjecting the mammalian tissue to ultrasound treatment. method. 26. A method of treating Alzheimer's disease in a mammal, including a human, in need thereof, comprising: - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; - administering to the mammal an isolated recombinant protein selected from the group consisting of proteins comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively), wherein the protein does not comprise an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO: 3) or an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); It consists of the isolated recombinant protein is not contained within the microbubbles and / or nanodroplets; method. 27. The method of paragraph 25 or 26, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to the BRICHOS domain of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 28. The method of paragraph 27, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 29. The method of paragraph 28, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 30. The method of paragraph 29, wherein the isolated recombinant protein is selected from the group of proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 31. The method of paragraph 25 or 26, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 32. The method of paragraph 31, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 33. The method of paragraph 31, wherein the isolated recombinant protein is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably 95%, preferably 99% identity to residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 34. The method of paragraph 33, wherein the isolated recombinant protein is selected from the group consisting of residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 35. The method according to any one of items 25 to 34, wherein the amino acid residue corresponding to position 221 of SEQ ID NO: 1 is selected from Glu and Asp. 36. The method of paragraph 35, wherein the amino acid residue corresponding to position 221 of SEQ ID NO: 1 is Glu. 37. The method according to any one of items 25 to 34, wherein the lipid microbubbles and / or nanodroplets are those according to any one of items 20 to 23.

[0281] 38. (i) a first protein portion selected from the group consisting of a protein comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively); and (ii) a second protein or polypeptide portion, preferably of at least 50 amino acid residues; 1. An isolated protein having the formula: the isolated protein does not contain an amino acid sequence having at least 70% identity to residues 1-89 of human Bri2 (SEQ ID NO:3); the isolated protein does not contain an amino acid sequence having at least 70% identity to human ABri23 (SEQ ID NO: 4); Isolated proteins. 39. The isolated protein of paragraph 38, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 40. The isolated protein of paragraph 39, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 41. The isolated protein of paragraph 40, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 42. The isolated protein of paragraph 41, wherein the first protein portion is selected from the group of proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively). 43. The isolated protein of paragraph 38, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 44. The isolated protein of paragraph 43, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 45. The isolated protein of paragraph 44, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 46. ​​The isolated protein of paragraph 45, wherein the first protein portion is selected from the group consisting of residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5). 47. The isolated protein according to any one of paragraphs 38 to 46, wherein the amino acid residue of the first protein portion corresponding to position 221 of SEQ ID NO: 1 is selected from Glu and Asp. 48. The isolated protein of paragraph 47, wherein the amino acid residue in the first protein portion corresponding to position 221 of SEQ ID NO:1 is Glu. 49. The isolated protein according to any one of paragraphs 38 to 48, wherein the number of amino acid residues in the first protein portion is 200 or less, for example, 150 or less. 50. The isolated protein according to any one of items 38 to 48, wherein the number of amino acid residues in the first protein portion is 90 or more. 51. The isolated protein according to any one of paragraphs 38 to 50, wherein the second protein or polypeptide portion has 50 to 2000 amino acid residues, for example, 50 to 1000, for example, 50 to 500, for example, 50 to 100. 52. The isolated protein of any one of paragraphs 38 to 51, wherein the size of the second protein or polypeptide portion is 5 to 200 kDa, such as 5 to 100 kDa, such as 5 to 50 kDa, such as 5 to 10 kDa. 53. The isolated protein of any one of paragraphs 38 to 52, wherein the first protein moiety is linked directly or indirectly to the amino terminus or carboxy terminus of the second protein or polypeptide moiety. 54. The isolated protein of any one of paragraphs 38 to 53, wherein the second protein or polypeptide portion constitutes the amino terminus and / or carboxy terminus of the protein. 55. The isolated protein according to any one of items 38 to 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. 56. The isolated protein of paragraph 55, wherein the second protein or polypeptide moiety is an antibody. 57. The isolated protein of paragraph 55, wherein the second protein or polypeptide moiety 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. 58. The isolated protein of paragraph 55, wherein the second protein or polypeptide moiety is selected from the group consisting of α-L-iduronidase, iduronate sulfatase, N-acetylgalactosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, α-galactosidase, α-glucosidase, β-glucocerebrosidase, and / or lysosomal acid lipase. 59. The isolated protein of any one of paragraphs 38 to 58, wherein the isolated protein is a recombinant fusion protein. 60. The isolated protein of any one of paragraphs 38 to 58, wherein the first protein moiety is chemically conjugated to the second protein or polypeptide moiety.

[0282] 61. A combination of an isolated protein according to any one of paragraphs 38 to 60 and a plurality of lipid microbubbles and / or nanodroplets, wherein the isolated protein is not contained within the microbubbles and / or nanodroplets. 62. A kit comprising an isolated protein according to any one of paragraphs 38 to 60 and a plurality of lipid microbubbles and / or nanodroplets, wherein the isolated protein is not contained within the microbubbles and / or nanodroplets. 63. The combination or kit according to item 61 or 62, wherein the lipid microbubbles and / or nanodroplets are those according to any one of items 20 to 23. 64. A method for transporting the isolated protein according to any one of paragraphs 38 to 60 across the blood-brain barrier in a mammal, including a human, in need thereof, comprising: - administering a plurality of lipid microbubbles and / or nanodroplets to said mammal; - administering said isolated protein to said mammal; A method consisting of: 65. The method of claim 64, wherein the method does not include a step of ultrasonic treatment of the mammalian tissue. 66. The method according to item 64 or 65, wherein the lipid microbubbles and / or nanodroplets are those according to any one of items 20 to 23. 67. An isolated protein according to any one of paragraphs 38 to 60, - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated protein to said mammal; for use in a treatment comprising transporting said isolated protein across the blood-brain barrier in said mammal, The isolated protein is an isolated protein that is not contained within the microbubbles and / or nanodroplets. 68. The isolated protein described in paragraph 67, wherein the method does not include an ultrasonic treatment step for the mammalian tissue. 69. An isolated protein according to any one of paragraphs 38 to 60, - administering a plurality of lipid microbubbles and / or nanodroplets to a mammal, including a human, in need thereof; - administering said isolated protein to said mammal; for use in therapy comprising transporting said isolated protein across the blood-brain barrier in said mammal, The isolated protein is an isolated protein that is not contained within the microbubbles and / or nanodroplets. 70. The isolated protein according to any one of paragraphs 67 to 69, wherein the lipid microbubbles and / or nanodroplets are those according to any one of paragraphs 20 to 23. 71. A method for transporting the isolated protein according to any one of paragraphs 38 to 60 across the blood-brain barrier in a mammal, including a human, in need thereof, comprising: - administering said isolated protein to said mammal; A method comprising: 72. A method for transporting an isolated protein according to any one of paragraphs 38 to 60 across the blood-brain barrier in a mammal, including a human, in need thereof, comprising: - administering said isolated protein to said mammal; A method consisting of: 73. A method of treating a disease in a mammal, including a human, in need thereof, comprising: - administering to said mammal the isolated protein according to any one of paragraphs 55 to 60; A method comprising: 74. A method of treating a disease in a mammal, including a human, in need thereof, comprising: - administering to said mammal the isolated protein according to any one of paragraphs 55 to 60; A method comprising: 75. An isolated protein according to any one of paragraphs 55 to 60 for use as a pharmaceutical. 76. - Administering the isolated protein according to any one of paragraphs 55 to 60 to a mammal, including a human, in need thereof; 61. The isolated protein according to any one of Items 55 to 60, for use in treating a disease in said mammal, comprising: 77. - Administering the isolated protein according to any one of paragraphs 55 to 60 to a mammal, including a human, in need thereof; 61. The isolated protein according to any one of Items 55 to 60, for use in treating a disease in a mammal, comprising:

Claims

1. (i) a first protein portion selected from the group consisting of a protein comprising an amino acid sequence having at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2); and a protein comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse, and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively); and (ii) a second protein or polypeptide moiety, preferably of at least 50 amino acid residues, 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; 1. An isolated protein having the formula: the isolated protein does not contain an amino acid sequence having at least 70% identity with residues 1-89 of human Bri2 (SEQ ID NO:3); the isolated protein does not contain an amino acid sequence having at least 70% identity with human ABri23 (SEQ ID NO: 4); - an isolated protein for use in a method of treating a disease in a mammal, including a human, in need thereof, the method comprising administering said isolated protein to said mammal without the use of lipid microbubbles or nanodroplets.

2. - administering said isolated protein to said mammal without the use of lipid microbubbles or nanodroplets.

3. 3. The isolated protein of claim 1 or 2, wherein the second protein or polypeptide moiety is an antibody.

4. The isolated protein of claim 3 , wherein the antibody is a monoclonal antibody.

5. 5. The isolated protein of claim 4, wherein the monoclonal antibody is selected from aducanumab, gantenerumab, 3D6 (bapineuzumab), m266 (solanezumab), donanemab, and lecanemab.

6. 6. The isolated protein of claim 5, wherein the monoclonal antibody is selected from aducanumab, gantenerumab, 3D6 (bapineuzumab), and m266 (solanezumab).

7. The isolated protein of claim 5, wherein the antibody is donanemab.

8. The isolated protein of claim 5, wherein the antibody is lecanemab.

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

11. 4. The isolated protein of any one of claims 1 to 3, wherein the second protein or polypeptide moiety is effective in treating Parkinson's disease.

12. 12. The isolated protein of claim 11, wherein the second protein or polypeptide moiety is selected from glucocerebrosidase, progranulin, prosaposin, cathepsin D, and an antibody.

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

14. 13. The isolated protein of any one of claims 1 to 12, wherein the first protein moiety is chemically conjugated to the second protein or polypeptide moiety.

15. 15. The isolated protein of any one of claims 1 to 14, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

16. The isolated protein of claim 15, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

17. The isolated protein of claim 16, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

18. The isolated protein of claim 17, wherein the first protein portion is selected from the group of proteins comprising any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively).

19. 15. The isolated protein of any one of claims 1 to 14, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 70% identity with residues 113 to 231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

20. 20. The isolated protein of claim 19, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 80%, preferably at least 85%, identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

21. 21. The isolated protein of claim 20, wherein the first protein portion is selected from the group of proteins comprising an amino acid sequence having at least 90%, preferably at least 95%, identity to residues 113-231 of human Bri2 (SEQ ID NO: 2) and the BRICHOS domain of human Bri2 (SEQ ID NO: 5).

22. 22. The isolated protein of claim 21, wherein the first protein portion 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).

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

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

25. 25. The isolated protein of any one of claims 1 to 24, wherein the number of amino acid residues in the first protein portion is 200 or less, such as 150 or less.

26. 26. The isolated protein of any one of claims 1 to 25, wherein the number of amino acid residues in the first protein portion is 90 or more.

27. 27. The isolated protein of any one of claims 1 to 26, wherein the second protein or polypeptide moiety has between 50 and 2000 amino acid residues, such as between 50 and 1000, such as between 50 and 500, such as between 50 and 100.

28. 28. The isolated protein of any one of claims 1 to 27, wherein the size of the second protein or polypeptide moiety is between 5 and 200 kDa, such as between 5 and 100 kDa, for example between 5 and 50 kDa, such as between 5 and 10 kDa.

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

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

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

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

33. 33. The isolated protein of claim 31 or 32 for use as a medicament.

34. 33. An isolated protein according to claim 31 or 32 for use in treating Parkinson's disease in a mammal, including a human, in need thereof.

35. An isolated protein having an amino acid sequence which has at least 70% identity to residues 113-231 of human Bri2 (SEQ ID NO: 2), and a protein having an amino acid sequence which has at least 70% identity to any of the BRICHOS domains of human, chimpanzee, bovine, porcine, mouse and rat Bri2 (SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively), for use in treating Parkinson's disease in a mammal, including a human in need thereof.

36. 36. The isolated protein of claim 35, wherein the isolated protein is further defined as being specified with respect to a protein portion according to any one of claims 15 to 26.

37. 5. The isolated protein of claim 4.

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

39. 39. The isolated protein of claim 37 or 38, wherein the antibody is donanemab.

40. 39. The isolated protein of claim 37 or 38, wherein the antibody is lecanemab.

41. 10. A method for transporting an isolated protein according to any one of claims 1 to 30 across the blood-brain barrier in a mammal, including a human, in need thereof, comprising: - administering said isolated protein to said mammal; Including, The method, wherein the administering step is performed without the use of lipid microbubbles or nanodroplets.

42. 1. A method of treating a disease in a mammal, including a human, in need thereof, comprising: - administering to said mammal an isolated protein according to any one of claims 1 to 30, Including, The method, wherein the administering step is performed without the use of lipid microbubbles or nanodroplets.

43. 1. A method of treating Parkinson's disease in a mammal, including a human, in need thereof, comprising: - administering to said mammal an isolated protein according to claim 31 or 32, A method comprising:

44. 1. A method for slowing alpha-synuclein accumulation in a mammal, including a human, in need thereof, comprising: - administering to said mammal an isolated protein according to claim 31 or 32, A method comprising:

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

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

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