WO2024200420A1

WO2024200420A1 - Microglial endocytic receptors for use in the treatment of neurodegenerative disease

Info

Publication number: WO2024200420A1
Application number: PCT/EP2024/058078
Authority: WO
Inventors: Andreas BJÖREFELDT; Rebecca RIISE; John Henrik ZETTERBERG; Eric Hanse
Original assignee: Micthera AB
Current assignee: Micthera AB
Priority date: 2023-03-28
Filing date: 2024-03-26
Publication date: 2024-10-03
Anticipated expiration: 2025-09-28
Also published as: SE2350348A1

Abstract

There is provided a protein comprising an amino acid sequence selected from SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 77, SEQ ID NO 84, SEQ ID NO 91 or SEQ ID NO 33, or a protein comprising an amino acid sequence with at least 90% identity to one of said sequences, the protein in addition comprising an extracellular binding domain that is capable of binding a neurodegenerative disease antigen. The protein can be used for the treatment of neurodegenerative disease.

Description

Microglial endocytic receptors for use in the treatment of neurodegenerative disease

FIELD OF THE INVENTION

This invention generally involves the treatment of neurogenerative diseases, such as Alzheimer's disease, using novel chimeric membrane proteins.

BACKGROUND

Neurodegenerative disease (NDD) is the collective name for a range of disorders where a progressive loss of nerve cells (neurons) and chemical contacts (synapses) in the central nervous system (CNS) is a characteristic feature of the pathology. A majority of NDDs (e.g. Alzheimer's disease, Parkinson's disease, Lewy body disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease) are further defined as proteinopathies, involving the aggregation and/or misfolding of specific protein species in the intra- and/or extracellular space (Wilson et al, Cell, 86(4), 693-714, 2023). A prototypical NDD is Alzheimer's disease (AD), the most common cause of dementia, where a dysregulated brain response toward the protein amyloid beta (A3) is believed to underlie disease development.

A key regulator of the brain's response to aggregated, misfolded or toxic proteins is the CNS-resident immune cell microglia who may protect neurons and synapses by endocy- tosing and degrading harmful proteins.

In AD, microglial dysfunction is strongly implicated, and evidence suggest that deficits in endocytic and lysosomal function may underlie or contribute to disease development and/or progression (Podlesny-Drabiniok et al., Trends Neurosci, 43(12), 965-979, 2020). As the number of individuals suffering from AD and other dementias is projected to increase substantially in the next decades ( Nandi et al., eClinicalMedicine, 51, 101580, 2022), the development of new therapeutic strategies will be imperative to reduce human suffering and lighten the burden on health care systems. Hence, there is an urgent need for efficacious treatments against Alzheimer's and other neurodegenerative diseases. The present disclosure describes strategies for selectively increasing microglial endocytosis of a toxic NDD protein (also referred to as a disease antigen) as exemplified by A|3.

Morrissey et al. (Elife, 7, e36688, 2018) reports engineered Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. However, preliminary data show that the approach used by Morrissey et al., involving the construction of phagocytic receptors using CD8 domains, produced very weak expression in microglia.

Hence, there is a need for a microglia-specific approach to construction and delivery of receptors to enhance endocytosis in these cells.

This invention solves this and other problems.

SUMMARY OF INVENTION

In a first aspect of the invention there is provided a protein comprising an amino acid sequence selected from SEQ ID NO 33-37 or SEQ ID NO 77, 84 or 91 or an amino acid sequence with at least 90% identity to one of SEQ ID NO 33-37 or SEQ ID NO 77, 84 or 91 , the protein in addition comprising an extracellular binding domain that is capable of binding a neurodegenerative disease antigen.

In various embodiments, the protein comprises the amino acid sequence of one of SEQ ID NO 34, 35, 36 or 37; or an amino acid sequence with at least 90% identity to one of SEQ ID NO 34, 35, 36 or 37. In various embodiments the protein comprises the amino acid sequence of SEQ ID NO 34 or an amino acid sequence with at least 90% identity to SEQ ID NO 34.

In various embodiments the protein comprises a single intracellular endocytosis triggering domain.

In various embodiments the binding domain comprises SEQ ID NO 32 or an amino acid sequence with at least 90% identity to SEQ ID NO 32.

In various embodiments the protein comprises SEQ ID NO 22, 23, 24, 25 or 26 or 78, 85, 92, 98, 98 or a sequence which is at least 90% identical to one of the sequences SEQ ID NO 22, 23, 24, 25 or 26 or 78, 85, 92, 98, 98

In one embodiment the sequence is SEQ ID NO 23 or a sequence which is at least 95% identical to SEQ ID NO 23.

In second aspect of the invention there is provided polynucleotide encoding a protein according to the first aspect of the invention.

In third aspect of the invention there is provided a vector comprising a polynucleotide according to the second aspect of the invention.

In various embodiments the vector may comprise a promoter active in microglia cells. In various embodiments the promoter is specific for microglia cells.

In a fourth aspect of the invention there is provided a virus particle comprising a polynucleotide of the second aspect of the invention or a vector according to the third aspect of the invention. The virus particle may be a recombinant adeno-associated viral (rAAV) particle. In a fifth aspect of the invention there is provided a host cell comprising a protein according to the first aspect, a polynucleotide according to the second aspect, a vector according to the third aspect, or a virus particle according to the fourth aspect of the invention. The host cell is preferably a microglial cell. The host cells preferably exhibit endocytic activity towards the neurodegenerative disease antigen.

In a sixth aspect of the invention there is provided a population of host cells according to the fifth aspect of the invention.

In a seventh aspect of the invention there is provided a pharmaceutical composition comprising a protein according to the first aspect, a polynucleotide according to the second aspect, a vector according to the third aspect of the invention, a virus particle according fourth aspect, a host cell according to the fifth aspect of the invention or a population of host cells according the sixths aspect of the invention; the composition further comprising a pharmaceutically acceptable excipient.

In an eight aspect of the invention there is provided a method of treating a subject comprising the administration of a protein according to the first aspect, a polynucleotide according to the second aspect, a vector according to the third aspect of the invention, a virus particle according fourth aspect, a host cell according to the fifth aspect of the invention or a population of host cells according the sixths aspect of the invention, or a pharmaceutical composition according to the eight aspect of the invention.

In various embodiments the disease is one of Alzheimer's disease, Parkinson' s disease, fronto-temporal dementia, Huntington's disease, motor neuron disease, multiple system atrophy (MSA), Creutzfeldt-Jakob disease, vascular dementia, Lewy body dementia, hippocampal sclerosis, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).

In a ninth aspect of the invention there is provided a protein according to the first aspect, a polynucleotide according to the second aspect, a vector according to the third aspect of the invention, a virus particle according fourth aspect, a host cell according to the fifth aspect of the invention or a population of host cells according the sixths aspect of the invention, or a pharmaceutical composition according to the eight aspect of the invention for use in the treatment of a disease.

In a tenth aspect of the invention there is provided a method of preparing a host cell or a population of host cells comprising contacting at least one cell with a polynucleotide of the second aspect of the invention or a vector according to the third aspect of the invention or a virus particle according to the fourth aspect of the invention, to obtain a host cell. The host cell is preferably a microglia cell. In one embodiment the step of contacting is performed in vivo. In one embodiment the step of contacting is performed ex vivo.

In an eleventh aspect of the invention there is provided the use of a protein according to the first aspect, a polynucleotide according to the second aspect, a vector according to the third aspect of the invention, a virus particle according fourth aspect, a host cell according to the fifth aspect of the invention or a population of host cells according the sixths aspect of the invention, or a pharmaceutical composition according to the eight aspect of the invention for use in the manufacture of a pharmaceutical.

DRAWINGS

The accompanying drawings form a part of the specification and schematically illustrate preferred embodiments and principles of the invention.

FIG. 1 is a schematic drawing showing the general structure and individual domains of microglial disease antigen internalization receptors (Mic-DAIRs).

FIG. 2 shows a schematic structure comparison of constructs tested in human-derived or mouse microglial cells. SP; signal peptide, ABD; extracellular antigen-binding domain, scFv adu; single-chain fragment variable of aducanumab, scFv lec; single-chain fragment variable of lecanemab, scFv don; single-chain fragment variable of donanemab, scFv pras; single-chain fragment variable of prasinezumab, TMD; transmembrane domain, ICD; intracellular domain comprising or consisting of an endocytosis-triggering domain. As customary, the protein architectures are shown with the N-terminal to the left and the C-ter- minal to the right.

FIG. 3 shows microscopy images of human microglia-like cell cultures, (a) brightfield image of cells in culture (b-f) fluorescence microscopy images showing transgene expression 5 days post viral rAAV transduction. rAAVs encoding EGFP only (b), a human endocytic receptor comprised of the signal peptide, hinge- and transmembrane domain of CD8 (c), a human endocytic receptor comprised of the signal peptide, hinge- and transmembrane domain of CD28 (d), the human microglial disease antigen internalization receptor 3 (Mic- DAIR3) (e) and the human microglial disease antigen internalization receptor 4 (Mic- DAIR4) (f) were delivered to cultures and incubated overnight.

FIG. 4 shows representative double fluorescence microscopy images of A 1-42 endocyto- sis in human microglia-like cells virally transduced with EGFP only (a-b), human microglial disease antigen internalization receptor 3 (Mic-DAIR3) (c-d) and human microglial disease antigen internalization receptor 4 (Mic-DAIR4) (e-f). White arrows highlight levels of A|3 uptake in transduced cells, grey arrows highlight levels of A|3 uptake by non-transduced neighboring cells. Cultures were incubated with fluorescently labelled A 1-42 for 2 hrs at 37° C 5 days post rAAV transduction.

FIG. 5 is a summary bar graph showing the normalized fluorescence intensity of A 1-42 detected in human microglia-like cells transduced with EGFP (viral control), human endocytic receptor 1-3 (hERl-3, three candidate constructs based on domains from other endogenous endocytic receptors in microglia), human microglial disease antigen internalization receptor 3 (Mic-DAIR3) and human microglial disease antigen internalization receptor 4 (Mic-DAIR4).

FIG. 6 shows representative double fluorescence microscopy images obtained from a separate endocytosis assay using 1 pm fluorescent polystyrene beads coated with A 1-42 or scrambled A|3 peptide. White arrows highlight beads/bead clusters detected in transduced cells, (a-f), endocytosis of Api-42-coated beads by human microglia-like cells expressing EGFP only (a-b), human microglial disease antigen internalization receptor 3 (Mic-DAIR3) (c-d) and human microglial disease antigen internalization receptor 4 (Mic-DAIR4) (e-f). (g- I), endocytosis of scrambled A|3-coated beads by human microglia-like cells expressing EGFP only (g-h), human microglial disease antigen internalization receptor 3 (Mic-DAIR3) (i-j) and human microglial disease antigen internalization receptor 4 (Mic-DAIR4) (k-l). Cultures were incubated with coated beads for 12 hrs at 37° C 5 days post rAAV transduction.

FIG. 7 is a summary bar graph showing the average number of A 1-42 vs scrambled A|3- coated beads endocytosed per cell in human microglia-like cells transduced with EGFP only, human microglial disease antigen internalization receptor 3 (Mic-DAIR3) or human microglial disease antigen internalization receptor 4 (Mic-DAIR4).

FIG. 8 shows the gating strategy used to define single live human microglia-like cells in flow cytometric analyses of endocytosis. (a), cell size and granularity analyzed by forward side scatter-area and height (FSC-A and FSC-H) and side scatter-area (SSC-A) (b). (c), plot showing gating for live nucleated cells using LiveDead and Nuclear Blue dyes.

FIG. 9 a-i shows representative flow cytometry density plots of human microglia-like cells after incubation with fluorescent A 1-42 for non-transduced control (a), mScarlet-trans- duced viral control (b), Mic-DAIRl (c), Mic-DAIR2 (d), Mic-DAIR3 (e), Mic-DAIR4 (f), Mic- DAIR5 (g), Mic-DAIR6 (h) and Mic-DAIR7 (i). Human microglia-like cultures were incubated with fluorescently labelled A 1-42 for 2 hrs at 37° C. The cells were transduced with rAAV 5 days prior to flow cytometric analysis.

FIG. 10 shows summary bar graphs of mean fluorescence intensity data obtained from human microglia-like cells using flow cytometry analysis of mScarlet positive cells (viral control + Mic-DAIRs) (a), and mScarlet negative cells corresponding to non-transduced cells from the same well (b). FIG. 11 shows the effect of replacing the aducanumab scFv with other scFv domains targeting amyloid, or alpha-synuclein. (a) schematic figure shows four different Mic-DAIRs based on the FcRy scaffold (Mic-DAIR3), but with different scFv domains, (b) bar graph showing mean fluorescence intensity data obtained from flow cytometry analysis of mScarlet positive cells (viral control + Mic-DAIRs). (c) bar graph showing mean fluorescence intensity data obtained from mScarlet negative cells, corresponding to non-trans- duced cells from the same well. Human microglia-like cultures were incubated with fluorescently labelled A 1-42 for 2 hrs at 37° C. The cells were transduced with rAAV 5 days prior to flow cytometric analysis.

FIG. 12 shows the effect of replacing the intracellular domain of Mic-DAIR3 with the ITI M- containing intracellular domain of the inhibitory Fc gamma receptor FcyRllb (Mic-DAIR12). (a) schematic showing structural comparison of Mic-DAIR3 and Mic-DAIR12. (b) bar graph showing mean fluorescence intensity data obtained from flow cytometry analysis of mScarlet positive cells (viral control + Mic-DAIRs). (c) bar graph showing mean fluorescence intensity data obtained from mScarlet negative cells, corresponding to non-trans- duced cells from the same well. Human microglia-like cultures were incubated with fluorescently labelled A 1-42 for 2 hrs at 37° C. The cells were transduced with rAAV 5 days prior to flow cytometric analysis.

FIG. 13 shows the mean fluorescent intensity recorded over time for mScarlet (viral control) relative to Mic-DAIR3 in presence or absence of 5 pM cytochalasin d. Human micro- glia-like cultures were incubated with fluorescently labelled A 1-42 for 0.5, 4 and 24 hrs at 37° C. The cells were transduced with rAAV 5 days prior to analysis.

FIG. 14 shows microscopy images of the primary mouse microglial cultures, (a) expression of the microglia-specific marker P2RY12. (b) expression of the microglia marker IBA1. (c) expression of mScarlet after rAAV transduction, (d) magnified image of mScarlet expression. (e) expression of mouse Mic-DAIR3 (mMic-DAIR3) after rAAV transduction, (f) expression of mouse Mic-DAIR4 (mMic-DAIR4) after rAAV transduction. Primary mouse microglial cultures were transduced with rAAV 5 days prior to expression analysis. FIG. 15 shows representative double fluorescence microscopy images of A 1-42 endocy- tosis in mouse primary microglia virally transduced with mScarlet only (a-c), mouse microglial disease antigen internalization receptor 3 (mMic-DAIR3) (d-f) and mouse microglial disease antigen internalization receptor 4 (mMic-DAIR4) (g-i). White arrows in merged images highlight A|3 fluorescence in transduced cells. Cultures were incubated with fluorescently labelled A 1-42 for 2 hrs at 37° C 5 days post rAAV transduction.

FIG. 16 shows representative flow cytometry density plots of primary mouse microglia after incubation with fluorescent A 1-42 for non-transduced control (a), mScarlet-trans- duced viral control (b), cells transduced with mouse microglial disease antigen internalization receptor 3 (mMic-DAIR3) (c) and mouse microglial disease antigen internalization receptor 4 (mMic-DAIR4) (d). The primary cultures were incubated with fluorescently labelled A 1-42, or scrambled A|3 peptide (not shown in plots), for 2 hrs at 37° C. Microglia were transduced with rAAV 5 days prior to flow cytometric analysis.

FIG. 17 shows summary bar graphs of mean fluorescence intensity data obtained from primary mouse microglia using flow cytometry analysis of mScarlet positive cells (viral control + Mic-DAIRs) (a), and mScarlet negative cells corresponding to non-transduced cells from the same well (b).

FIG. 18 shows representative double fluorescence microscopy images of in vivo expression of mouse microglial disease antigen internalization receptor 3 (mMic-DAIR3) and 4 (mMic- DAIR4) in layer 5 parietal cortex of transgenic TMEM119-CreERT2 mice. Images show colocalization of IBA1 reactivity with mMic-DAIR3 (al-a3) and mMic-DAIR4 (bl-b3) expression in microglia. DETAILED DESCRIPTION

As used herein, the terms "binding domain" refers to a polypeptide that possesses the ability to specifically and preferably non-covalently bind to a neurogenerative disease antigen. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or re- combinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain consists of or comprises an antigen-binding domain, such as an antibody or an antibody fragment. The binding domain may be an antigen binding domain. The antigen binding domain may be derived from a monoclonal antibody.

Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g., TNF-a), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for the specific ability to bind to a biological molecule. In a preferred embodiment the binding domain is a single chain variable fragment (scFv). An scFv comprises a single polypeptide comprising the variable domain of the heavy and light chains of a monoclonal antibody with a linker in between the chains. (Munoz-Lopez et al, Cancers (Basel). 2022 Sep; 14(17): 420 and references therein)

An antibody may be a naturally occurring, recombinantly produced, genetically engineered, or modified form of an immunoglobulin, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, ADAPTIR). A monoclonal antibody or a fragment thereof may be non-human, chimeric, humanized, or human, preferably humanized or human. Antibody fragments that have binding capacity are also included. Examples of antibody fragments include, but are not limited to, domain antibodies, sFv, scFv, Fab, Fab', F(ab')2, and Fv fragments, Fab' -SH, F(ab')2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. A "Fab" (fragment antigen binding) is a portion of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain of a monoclonal antibody via an inter-chain disulfide bond. The antibody or fragment thereof may be any antibody derived from a mammal such as mouse, rat, hamster, rabbit, goat, horse and chicken. When the antibody is of non-human origin, it is preferably humanized. The isotype of the antibodies may be any of IgG, IgM, IgE, IgA, IgY and the like.

The skilled person knows how to generate an antibody or an antibody fragment against a certain antigen. Immunization may be done using the antigen sequences described herein. There are well-known methods for producing, purifying and isolating antibodies and determining their binding capacity. There are also well-known methods for using an antibody to determine the presence of an antigen. It is referred to Current Protocols in Immunology and Current Protocols in Molecular Biology for details. As an example, monoclonal antibodies against a neurodegenerative disease antigen may be generated using the well- known hybridoma technology (Kohler and Milstein, Nature, 256, 495-497, 1975). A single clone can be isolated by limiting dilution analysis, the soft agar assay, a method using a fluorescence activated cell sorter and the like. In the limiting dilution analysis, for example, colonies of the hybridoma are serially diluted to around 1 cell/well in a medium before cultivation to isolate the hybridoma which produces the desired antibody.

Antibody clones may also be generated using other methods, such as, for example, phage display.

A binding domain, such as an antibody, preferably has an affinity towards a neurodegenerative disease antigen. Affinity can be expressed using the dissociation constant or Kd. Preferred binding affinities include those with a dissociation constant (Kd) less than 10“⁶ M, more preferably 5xl0“⁷ M, more preferably 10“⁷ M, more preferably 5xl0“⁸ M, 10“⁸ M, more preferably 5xl0“⁹ M, more preferably 10“⁹ M, more preferably 5xlO“¹⁰ M, more preferably IO^-10 M, more preferably 5xl0^-11 M, more preferably 10^-11 M, more preferably 5xl0“¹² M, more preferably 10“¹² M, even more preferably 5xl0“¹³ M, or and most preferably 10 ¹³ M. The affinity may be de determined using ELISA. As used herein "neurodegenerative disease" refers to: Alzheimer's disease, Parkinson's disease, fronto-temporal dementia, Huntington's disease, motor neuron disease, multiple system atrophy (MSA), Creutzfeldt-Jakob disease, vascular dementia, Lewy body dementia, hippocampal sclerosis, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). In a preferred embodiment, the disease is Alzheimer's disease.

As used herein "neurodegenerative disease antigen" refers to an antigen that is expressed in the CNS, preferably in the brain and associated with a neurodegenerative disease. In certain embodiments a neurodegenerative disease antigen is a protein or peptide that is overexpressed or inappropriately expressed in the CNS. A neurodegenerative disease antigen may be a protein that is unfolded or misfolded. In some embodiments, the neurodegenerative disease antigen is a protein or part thereof that is misfolded, fibrillized or aggregated. The neurodegenerative disease antigen may have a toxic effect on surrounding cells in the CNS. A neurodegenerative disease antigen may be in the form of particles of a size of up to 0.1 pm, 1 pm or 10 pm. In various embodiments, the neurodegenerative disease antigens include antigens that comprise or consist of amyloid beta (SEQ ID NO 52) (Alzheimer's), beta-secretase 1(SEQ ID NO 53) (Alzheimer's), Tau (SEQ ID NO 54) (Alzheimer's), apolipoprotein E4 (ApoE4) (SEQ ID NO 55), alpha-synuclein (SEQ ID NO 56) (Parkinsons disease), a mutated form of huntingtin (HTT) (SEQ ID NO 57) (Huntington's disease), superoxide dismutase 1 (SOD1) (SEQ ID NO 58) (ALS), leucine rich repeat kinase 2 (LRRK2) (SEQ ID NO 59) (Parkinson's disease), prion protein (prP) (SEQ ID NO 60) (Creutzfeldt-Jakob disease) or TAR DNA-binding protein 43 (TDP-43) (SEQ ID NO 61) (frontotemporal dementia or ALS), or parts of these sequences Preferably the binding domain is capable of binding one antigen selected from these antigens.

In a preferred embodiment the neurodegenerative disease antigen is amyloid beta.

As used herein, the term "amyloid beta" (A3) refers to a fragment of amyloid precursor protein (APP) that is produced upon cleavage of APP by p-secretase 1 ("BACE1"), as well as modifications, fragments and any functional equivalents thereof, including, but not limited to, Api-40 peptide, and A|31-42 peptide. A may be a monomer, or may associate to form oligomers or fibril structures. A|3 fibrils may aggregate into amyloid plaques, e.g., such as those found in brains of Alzheimer's disease patients. Amyloid beta may have the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO 52) (A 1-42 peptide).

In a preferred embodiment the disease being treated is Alzheimer's disease.

The term "subject," "patient" and "individual" are used interchangeably herein and are intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs.

"Endocytosis" refers to all kinds of engulfment by cells of antigens or particles. Endocyto- sis may involve phagocytosis which may involve formation of a phagosome which includes the antigen. A phagosome may fuse with a lysosome which causes the breakdown of the antigen. Endocytosis also comprises other processes by which a cell engulfs particles, proteins, peptides or antigens such receptor-mediated endocytosis and pinocytosis.

References to sequences SEQ ID NO 1 to SEQ ID NO 40 and SEQ ID NO 43 and 45 or any other peptide sequence herein in particular SEQ ID NO 72-92, 96, 98 and 100 and 102 may also, in various embodiments, refer to sequences that are substantially identical to the reference sequence. "Substantially identical" shall, for amino acid sequences, mean a sequence that has a percent identity which is at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, or 98% and most preferably at least 99% identical to the reference sequence. Sequence identity is determined using sequence alignment. Percent identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, using BLAST for proteins (BLASTP) (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10. Doi: 10.1016/S0022-2836(05)80360-2. PMID: 2231712.) BLAST is used with default settings (as of March 2023 which are: word size: 3, gap penalty existence: 11, gap penalty extension: 1).

A "signal peptide" is inserted into the membrane of the endoplasmic reticulum during protein synthesis and contributes to positioning of the Mic-DAIR in the cell membrane. The signal peptide is typically a short peptide (usually 15-30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for the secretory pathway. A signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C- terminal region with a cleavage site for the enzyme signal peptidase. In eukaryotes, a signal peptide causes translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved, creating a mature protein which then proceeds to its appropriate destination, which preferably is the outer cell membrane. The signal peptide is typically cleaved after insertion into the membrane, resulting in a mature protein.

"Microglial cells" (or "microglia") are glial cells in the CNS that express markers such as ionized calcium binding adapter molecule 1 (I BAI), CD68, P2RY12, TMEM119 or HEXB. Microglial cells are typically located in the brain and the spinal cord. Microglia cells are the primary immune cells of the CNS and play import roles in development, homeostasis and neuroprotection.

The inventors have surprisingly found that Fc receptors, and certain other glial membrane proteins namely TREM2, DAP12 and SIRPpi (collectively referred to as "scaffold proteins") can be used as suitable scaffolds for fusion proteins (Mic-DAIRs) to be used together with an extracellular binding domain for stimulating endocytosis of neurodegenerative diseases antigens by microglial cells. Not only are the Mic-DAIRs well expressed in microglial cells but they also enable an increased engulfment of neurogenerative disease antigens. Hence the Mic-DAIR is able to induce endocytosis of neurodegenerative disease antigens when transfected in cells. Mic-DAIRs may be used for the treatment of neurodegenerative disease, such as Alzheimer's. Suitable proteins include: Fc receptor gamma chain (FcRy or FCERG), Fc gamma receptor I, (FcyRI or FCGR1), Fc gamma receptor Ila (FcyRlla or FCG2A), Fc gamma receptor lie (FcyRllc or FCG2C), Fc gamma receptor Illa (FcyRllla or FCG3A), TREM2 (Triggering receptor expressed on myeloid cells 2), DAP12 (DNAX adaptor protein 12 or TYROBP) and SIRPpi (Signal regulatory protein beta 1). The proteins are preferably of human origin. However, protein domains from other species can be useful, in particular when treating other species than humans. For example, for use in mice, mouse proteins may be preferred.

The invention relates generally to a protein, which may also be referred to as a "protein", a "fusion protein" or a "chimeric protein", or as a "microglial disease antigen internalization receptor" ("Mic-DAIR") herein.

When the Mic-DAIR is introduced into microglia it enables the cells to engulf the neuro- degenerative disease antigens, thereby preventing disease development or treating the disease by slowing its progression and/or reversing disease pathology. The microglial cells may engulf plaques, protein, protein aggregates, protein monomers, protein oligomers, protein fibrils or cells that are characteristic of neurodegenerative disease.

The general structure of a Mic-DAIR is seen in FIG. 1. In general, the Mic-DAIR comprises a single polypeptide chain comprising an extracellular binding domain capable of binding a neurodegenerative disease antigen, a transmembrane domain, and an intracellular domain comprising or consisting of an endocytosis-triggering domain, where the transmembrane domain is located between the extracellular domain and the intracellular domain. The Mic-DAIR may optionally comprise a hinge domain. The structure arrangement is preferably from the N-terminal to the C-terminal: extracellular binding domain, transmembrane domain, intracellular endocytosis-triggering domain, or extracellular domain - hinge domain -transmembrane domain, intracellular endocytosis-triggering domain. The Mic- DAIR may optionally comprise a signal peptide. The signal peptide is preferably fused to the extracellular binding domain. The Mic-DAIR optionally comprises a hinge domain. In various embodiments, all domains except the extracellular binding domain is from a Fc receptor or other scaffold protein selected from SEQ ID NO 27-31, SEQ ID NO 76, 83 and 90; or the corresponding mouse sequences.

In various embodiments, the intracellular domain comprising the endocytosis triggering

5 domain has a maximum length (number of amino acid residues). The maximum length of the intracellular domain may be for example at most 120, more preferably at most 100, more preferably at most 80, more preferably at most 70 more preferably at most 60 and most preferably at most 50 amino acid residues. w When expressed in a eucaryotic cell, such as a microglial cell, a population of Mic-DAIR molecules will be produced. The protein is a membrane protein. A population of these molecules will be positioned in the cell membrane with the extracellular domain outside the cell and the intracellular domain in the cytoplasm. Hence at least a part of the Mic- DAIR, in particular the binding domain is located on the cell surface.

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Table 1 shows the various SEQ ID NO:s for the various domains of the human scaffold proteins and Mic-DAIRs.

Adu= aducanumab, Lec=lecanemab, Don=donanemab, Pras= prasinezumab

Table 1

Mic-DAIRs 1-8 uses a scFv based on aducanumab (SEQ ID NO 32) inserted between the sig¬

5 nal peptide and the hinge domain of the respective scaffold protein.

Mic-DAIR9 uses a scFv based on lecanemab (SEQ ID NO 62) inserted between the signal peptide and the hinge domain of FcRy. w MIC-DAIR10 uses a scFv based on donanemab (SEQ ID NO 63) inserted between the signal peptide and the hinge domain of FcRy.

Mic-DAIRll uses a scFv based on prasinezumab (SEQ ID NO 64) inserted between the signal peptide and the hinge domain of FcRy.

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Mic-Dairl2 is a control construct having an inhibitory intracellular domain and used in one of the Examples as described below. SEQ ID NO 22, 23, 24, 25, 26, 78, 85 and 92 represents the sequence of each Fc receptor or other scaffold protein (SEQ ID NO 27, 28, 29, 30, 31, 76, 83 and 90 respectively) where the binding domain of SEQ ID NO 32 has been inserted between the signal peptide and the hinge domain of each Fc receptor.

SEQ ID NO SEQ ID NO 22, 23, 24, 25, 26, 78, 85 and 92 represents SEQ ID NO 33, 34, 35, 36, 37, 77, 84, and 91 with the addition of the SEQ ID NO 32 as the extracellular binding domain and the signal peptides.

SEQ ID NO 96, 98, and 100 and optionally 102 represents various useful Mic-DAIRs as outlined in Table 1.

The intracellular signaling domain, also referred to as the endocytosis triggering domain, preferably comprises or consists of a sequence selected from one of the human Fc receptors (SEQ ID NO SEQ 4, 8, 12, 16, 20, 75, 82 or 89) which are the intracellular domains of the human Fc receptors FcRy, FcyRI, FcyRlla, FcyRllc, FcyRllla, and TREM2, DAP12 and SIRPpi, respectively. The endocytosis triggering domain may be capable of providing an intracellular signal that triggers endocytosis, for example when a neurogenerative disease antigen is bound to the binding domain. The endocytosis triggering domain may be able to interact with various endogenous intracellular signaling partners, such as proteins or small molecules. The endocytosis signaling domain may be able to participate in signal transduction in the cytoplasm of the host cell. There are several different endocytosis systems in the human cell, and the receptor may use any one of them that is capable of triggering endocytosis of neurodegenerative disease antigens. For example, the intracellular signaling domain may comprise one or more immunoreceptor tyrosine-based activation (ITAM) motifs known to trigger endocytosis. This is the case with the intracellular domains of FcRy, FcyRlla and FcyRllc (SEQ ID NO 4, 12 and 16 respectively). However, notably SEQ ID NO 8 does not comprise an ITAM sequence motif, and it is surprising that it can trigger engulf- ment to an extent even larger than SEQ ID NO 4, which does have an ITAM motif, since Mic-DAIR4 which comprises SEQ ID NO 8 is highly efficient in engulfing amyloid beta (see for example figures 4-7, 10, 15 and 17 of the Examples). Without being bound by any theory, this could be due to the dimerization or oligomerization of Mic-DAIR4 with an endogenous protein (e.g. an adapter protein) displaying one or more ITAM motifs.

In various embodiments, the endocytosis triggering domain may bind of soluble or nonsoluble or membrane bound intracellular proteins, for example signal transduction proteins including kinases or other proteins or molecules. In various embodiments the endocytosis triggering domain may induce endocytosis upon binding to Src family- or spleen tyrosine kinases. In various embodiments, one or more amino acid residues or motifs present in the endocytosis triggering domain for example a tyrosine residue, is phosphorylated (for example by a Src or spleen tyrosine kinase) prior to the induction of endocytosis. Hence, the endocytosis trigger domain may comprise a motif that can be phosphorylated. The motif may comprise a tyrosine residue that becomes phosphorylated.

In various embodiments, the Mic-DAIR comprises a two, three or more intracellular endocytosis triggering domains. These may be arranged in any suitable manner in the intracellular domain of the Mic-DAIR, however, it is preferred that they are comprised in the same polypeptide chain. For example, a first intracellular endocytosis triggering domain is arranged proximal to the transmembrane domain and closer to the N-terminal in relation to a second intracellular endocytosis triggering domain. A first endocytosis triggering domain may be fused to a second endocytosis triggering domain. In some embodiments the intracellular domain of a Mic-DAIR comprises or consists of a plurality of intracellular endocyto- sis-triggering domains all selected from the group consisting of SEQ ID NO 4, 8, 12, 16, 20„ 75, 82 and 89, in particular SEQ ID NO 8, 12, 16, 20, 75, 82 and 89 and no further intracellular endocytosis triggering domains. In particular signaling domains from the proteins MRC1, MERTK, Tyro3, Axl or ELMO may be excluded. Hence, in some embodiments the Mic-DAIR does not comprise a intracellular endocytosis triggering domain from one of the proteins MRC1, MERTK, Tyro3, Axl or ELMO.

However, it may be advantageous to use as few intracellular endocytosis-triggering domains as possible. In various embodiments, the Mic-DAIR comprises only one intracellular endocytosis-triggering domain. In various embodiments, the intracellular domain of a Mic-

DAIR comprises or consists of one of SEQ ID NO 4, 8, 12, 16, 20, 75, 82 and 89.

The transmembrane domain is located between the extracellular binding domain and the intracellular endocytosis triggering domain. The transmembrane may be directly fused to the extracellular domain or there may be a domain in between such as a hinge region. The transmembrane domain may be directly fused to the intracellular endocytosis triggering domain or there may be a domain in between. The transmembrane domain spans the lipid bilayer of the cell membrane and may comprise one or more alpha helices. The transmembrane domain is preferably composed of predominantly hydrophobic amino acids. The transmembrane domain may anchor the Mic-DAIR protein in the cell membrane. The transmembrane domain preferably comprises or consists of a sequence selected from one of the human Fc receptors FcRy, FcyRI, FcyRlla, FcyRllc, FcyRllla, or one of TREM2, DAP12 or SIRPpi (SEQ ID NO 3, 7, 11, 15,19, 74, 81 or 88).

The extracellular binding domain is capable of binding a neurodegenerative disease antigen. Binding may be specific for the antigen. Any useful binding domain may be used. The extracellular binding domain may be an antigen binding domain. The antigen binding domain may be derived from a monoclonal antibody.

Examples of a binding domains is SEQ ID NO 32 or a part thereof. SEQ ID No 32 is a single chain fragment of the BIIB037 antibody (aducanumab) (SEQ ID NO 50 and 51). The extracellular domain may be directly fused to the transmembrane domain or there may be a domain in between, in particular a hinge domain.

In various embodiments the disease being treated is Alzheimer's and the neurodegenerative disease antigen is amyloid beta (SEQ ID NO 52) or Tau (SEQ ID NO 54).

In various embodiments, the disease being treated is Parkinson's disease and the neuro- generative disease antigen is alpha-synuclein (SEQ ID NO 56). In various embodiments the disease being treated is Huntington's disease and the neuro- degenerative disease antigen is a mutated form of huntingtin (SEQ ID NO 57).

In various embodiments, the disease being treated is ALS and the neurodegenerative disease antigen is superoxide dismutase (SOD1) (SEQ ID NO 58).

Other useful binding domains include other domains cable of binding a neurodegenerative disease antigen. Binding domains may be derived from the following antibodies: Antibodies against amyloid beta, for example lecanemab (BAN2401), donanemab (LY3OO2813), remternetug (LY3372993), solanezumab (LY2062430), gantenerumab (RO4909832), bapi- neuzumab (AAB-001), crenezumab (MABT5102A), ABBV-916 or ACU193; antibodies against Tau, for example bepranemab (UCB0107), semorinemab (R07105705), E2814, PRX005, or Lu AF87908 (hC10.2); antibodies against alpha-synuclein, for example prasinezumab (PRX002) or cinpanemab (BIIB054); antibodies against mutated huntingtin (mtHTT) for example C6-17 (AFFiRiSAG); and antibodies against superoxide dismutase (SOD1) for example W20. The binding domain may be derived from any of these antibodies or sequences with similar function.

Further examples of useful binding domains include a scFv based on lecanemab (SEQ ID NO 62) a scFv based on donanemab (SEQ ID NO 63) or a scFv based on prasinezumab (SEQ ID NO 64).

The skilled person knows that the so-called CDR sequences of the light and heavy chains of antibodies are particularly important for antigen binding and that binding domains can be designed using the CDR sequences.

The Mic-DAIR preferably comprises a hinge domain located between the extracellular binding domain and the transmembrane domain. The hinge region may be directly fused to the transmembrane domain or there may be a domain in between such as a hinge region. The hinge region may be directly fused to the extracellular binding domain, or there may be a domain in between. The hinge region may contribute the activity of the Mie- DAI R. Without being bound by any theory, the hinge domain may contribute to positioning the binding domain outside the host cell surface.

The hinge region preferably comprises or consists of a sequence selected from one of the hinge domains from human Fc receptors FcRy, FcyRI, FcyRlla, FcyRllc, FcyRllla, or from one of TREM2, DAP12 and SIRP 1 (SEQ ID NO SEQ 2, 6, 10, 14, 18, 73, 80, or 87).

The Mic-DAIR preferably comprises a signal peptide located in the N-terminal of the protein. The signal peptide is preferably fused to the extracellular binding domain, but in some embodiments, there may be a domain in between them. The signal peptide preferably comprises or consists of a sequence selected from one of the human Fc receptors FcRy, FcyRI, FcyRlla, FcyRllc, FcyRllla and (SEQ ID NO SEQ 1, 5, 9, 13, 17 ). Other useful signal peptides include a CD28 signal peptide, a CD8 signal peptide, a TREM2 signal peptide (SEQ ID NO 38 or72 ) or a SIRP 1 signal peptide (SEQ ID NO 39 or 86) or a DAP12 signal peptide (SEQ ID NO 79) or a MERTK signal peptide (SEQ ID NO 40) or a GM-CSF signal peptide.

Preferred Mic-DAIR protein architectures

Preferred architectures of Mic-DAIR include those that comprise the hinge domain, transmembrane domains and intracellular endocytosis triggering domains of FcRy, FcyRI, FcyRlla, FcyRllc, FcyRllla and (SEQ ID NO 27, 28, 29, 30, 31,) or of TREM2, DAP12 and SIRPpi (SEQ ID NO 76, 83 and 90). In particular the sequences with SEQ ID NO 33, 34, 35, 36, 37, 77, 84 and 91 are suitable. For example, a Mic-DAIR may comprise one of the following domains, from the N-terminal: extracellular binding domain - SEQ ID NO 2 - SEQ ID NO 3 - SEQ ID NO 4, extracellular binding domain - SEQ ID N- 6 - SEQ ID NO 7 - SEQ ID NO 8, extracellular binding domain - SEQ ID NO 10 - SEQ ID NO 11 - SEQ ID NO 12, extracellular binding domain - SEQ ID NO -14 - SEQ ID NO 15 - SEQ ID NO 16, extracellular binding domain - SEQ ID NO - SEQ ID NO 19 - SEQ ID NO 20, extracellular binding domain- SEQ ID NO-73 - SEQ ID NO -74 - SEQ ID NO 75, extracellular binding domain - SEQ ID NQ-80 - SEQ ID NO-81 - SEQ ID NO 82, and extracellular binding domain - SEQ ID NO 87- SEQ ID NO 88 - SEQ ID NO 89.

In various embodiments the hinge region domains (2, 6, 10, 14, 18, 73, 80, or 87) is optional.

Hence, preferred embodiments of Mic-DAIR proteins include, from the N-terminal:

Extracellular binding domain - SEQ ID NO 33,

Extracellular binding domain - SEQ ID NO 34,

Extracellular binding domain - SEQ ID NO 35,

Extracellular binding domain - SEQ ID NO 36,

Extracellular binding domain - SEQ ID NO 37,

Extracellular binding domain - SEQ ID NO 77,

Extracellular binding domain - SEQ ID NO 84, and

Extracellular binding domain - SEQ ID NO 91.

In general, the Mic-DAIR protein may comprise or consist of the architecture, from the N- terminal: optional signal peptide - extracellular binding domain -optionally one of the hinge regions SEQ ID NO 2, 6, 10, 14, 18, 73, 80, or 87- one of the transmembrane domains 3, 7, 11, 15, 19, 74, 81 or 88- one of the intracellular endocytosis triggering domains 4, 8, 12, 16, 20, 75, 82 or 89.

In one embodiment, the Mic-DAIR comprises the transmembrane domain of SEQ ID NO 7 and the intracellular endocytosis triggering domain of SEQ ID NO 8. An example is Mic-

DAIR4. In various embodiments the binding domain is inserted between amino acids 18 and 19 of SEQ ID NO 27, or between amino acids 15 and 16 of SEQ ID NO 28, or between amino acids 33 and 34 of SEQ ID NO 29 or between amino acids 42 and 43 of SEQ ID NO 30 or between amino acids 16 and 17 of SEQ ID NO 31, between amino acids 18 and 19 of SEQ ID NO 76, or between amino acids 21 AND 22 of SEQ ID NO 83 or between amino acids 29 and 39 of SEQ ID NO 90. The indicated insertion position may be changed one, two or three amino acid positions towards the N-terminal or the C-terminal of SEQ ID NO 27-31, 63, 76, 83 and 90.

Further preferred Mic-DAIR embodiments comprises, from the N-terminal: a. an extracellular binding domain that is capable of binding a neurodegenera- tive disease antigen b. a transmembrane domain, c. one intracellular endocytic triggering domain, which is at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identity with a sequence selected from SEQ 8, 12, 16, 20, 75, 82 or 89.

It is understood that direct fusion of one domain to another domain of a Mic-DAIR described herein does not preclude the presence of intervening junction amino acids. For example, the number of junction amino acids may be from 1-to 8 amino acids. Moreover, For example, junction amino acids may be used in the form a glycine-rich linker of 2 to 31 amino acids in length (Reddy Chichili, Protein Sci, 22(2), 153-67, 2013).

In certain embodiments, a Mic-DAIR comprises amino acid sequences derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, transgenic species thereof, or any combination thereof. In certain embodiments, a Mic-DAIR is murine, human, chimeric (with sequences from two or more species) or humanized. The counterpart of Mic-DAIR:s may be based on corresponding Fc sequences for Fc receptors in mice. SEQ ID NO 32 may be used as the binding domain for use in mice. For example, the mouse Mic-DAIR corresponding to Mic- DAIR3 (SEQ ID NO 22) is SEQ ID NO 43, encoded by SEQ ID NO 44. The murine counterpart of Mic-DAIR4 (SEQ ID NO 23) is SEQ ID NO 45, encoded by SEQ ID NO 46.

Function of Mic-DAIR

Any suitable domain architecture may be used as long as it is able to trigger an increase in endocytic response against a neurodegenerative disease antigen in microglial cells.

In various embodiments, introducing the Mic-DAIR in microglial cells will increase endocytic activity of a neurodegenerative disease antigen in a population of cells with at least 25%, more preferably at least 50% more preferably 100%, more preferably at least 150%, more preferably at least 200%, and most preferably at least 300% compared to cells not expressing the Mic-DAIR. Activity is determined as (increase of activity /activity in unmodified cells x 100). Endocytic activity may be determined by assessing the ability of microglial cells to engulf fluorescently labeled antigens. For example, the average pixel intensity derived from fluorescently-labelled antigen inside individual cells as a population mean is determined after a predetermined incubation time. For example, cells grown in vitro, for example on plates or slides, may be incubated with fluorescently labelled A 1-42 (or other relevant antigen) (500 nM) for 2 hours at 37° C. Cells are then washed with PBS, fixated with 4% formaldehyde for 10 minutes and imaged using a fluorescence microscope, as disclosed in the Examples.

Endocytic activity may be specific meaning that microglial cells expressing Mic-DAIRs will have higher endocytic activity towards the neurogenerative disease antigen than for instance a scrambled control peptide. Activity may be for example at least 50% higher, more preferably at least 100% higher, more preferably at least 200% higher, more preferably at least 300% higher and most preferably at least 500% higher towards the neurodegenerative disease antigen. The percentage is determined as (increase of activity /activity towards scrambled peptide x 100). Methods for detecting specific activity towards amyloid beta vs a scrambled peptide are disclosed in the Examples. In some embodiments a plurality of the host cells, preferably at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95% of the host cells, are capable of endocytosis of a neurodegenera- tive disease antigen.

In various aspects of the invention there is provided polynucleotides that encode a Mic- DAIR protein as described herein. The nucleic acids may be in the form of DNA or RNA and may be double stranded or single stranded. Nucleic acids may be produced and modified using various molecular biology techniques as is known to a person skilled in the art. For example, nucleic acids may be produced or manipulated using various technologies that involve the use of restriction enzymes, polymerases, ligases, degradation enzymes, and other enzymes, PCR, primers, oligomers, E.coli bacteria and various viruses. It is referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012 edition, for details and Ausubel et al. Current protocols in Molecular Biology John Wiley & Sons. Nucleic acids may be produced synthetically.

The skilled person is able to derive nucleotide sequences for various amino acid sequences disclosed herein, including nucleotide sequences for particular domains, by using standard molecular biology software tools for translation, reverse translation and alignment.

Examples of useful polynucleotides include: SEQ ID NO 41 encoding SEQ ID NO 22 (Mic- DAIR3), SEQ ID NO 42 encoding SEQ ID NO 23 (Mic-DAIR4), SEQ ID NO 47 encoding SEQ ID NO 24 (Mic-DAIR5), SEQ ID NO 48 encoding SEQ ID NO 25 (Mic-DAIR8), SEQ ID NO 49 encoding SEQ ID NO 26 (Mic-DAIR6), , SEQ ID NO 93 encoding 78 (Mic-DAIRl), SEQ ID NO 94 (Mic-DAIR2)encoding SEQ ID NO 85, SEQ ID NO 95 encoding SEQ ID NO 92 (Mic-DAIR7), and SEQ ID NO 97 encoding SEQ ID NO 96 (Mic-DAIR9), SEQ ID NO 99 encoding SEQ ID NO 98 (mic-Dair 10) SEQ ID NO 101 encoding SEQ ID NO 100 (Mic-DAIR 11) and parts of these polynucleotide sequences. The skilled person understands how to derive nucleotide sequences for any other amino acid sequences herein, including amino acid sequences for protein domains disclosed herein, starting from the nucleotide sequences disclosed herein or from the amino acid sequences themselves, for example by using standard bioinformatics tools and/or standard molecular biology methods.

The nucleotide sequences provided herein should be seen as examples only since the genetic code is what is referred to as "degenerated".

The polynucleotide may be operatively linked to one or more expression control sequence such as a promoter, enhancers, and the like as is known in the art. The polynucleotide sequence may, for example, comprise an appropriate sequence for inducing transcription of mRNA from DNA in a eucaryotic cell. In some embodiments expression of a microglia-unspecific control sequence is able to induce expression of the Mic-DAIR in microglial cells. An example of such an expression control sequence is the CD68 promotor, AIF-1/IBA1, CX3CR1, MerTK, CD11 b/c, F4/80. In some embodiments an expression control sequence is specific for glial cells. Examples of such expression control sequences are control sequences for P2RY12, TMEM119 and HEXB.

A polynucleotide encoding a Mic-DAIR can be provided in a vector such as a viral vector, a non-viral bacterial cloning vector or an expression plasmid for use in rAAV packaging. A plasmid may comprise sequences for facilitating replication of the plasmid in bacteria, sequences for antibiotic resistance or sequences for inducing expression of Mic-DAIR proteins such as expression control sequences, and the like, as is known in the art of molecular biology. It is referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012 edition, for details and Ausubel et al. Current protocols in Molecular Biology John Wiley & Sons.

Suitable plasmids for bacterial cloning and rAAV packaging may include pUC19, pAAV- SSFV-GOI-WPRE-bGH pA (GOI = gene of interest) and rAAV2/cMG. polynucleotide may be provided in a virus. The virus may be a virus that is suitable for gene therapy.

The virus may be provided as a polynucleotide or as virus particles comprising a polynucleotide.

There are a large number of available viral vectors suitable for use with the compositions of this disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2: 177, 2001). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus derived vectors, for example Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors or HIV-l-derived vectors.

Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).

Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes are known in the art and have been previous described, for example, in U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18: 1748, 2010; Verhoeyen et al., Methods Mol.. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirusbased vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky etal., Gene Ther. 5: 1517, 1998). Other vectors include those derived from baculoviruses and a-viruses. (Jolly, DJ. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors). Further useful methods involving adenovirus are provided in Lin et al., Nat Methods, 19(8), 976-985, 2022, and in Young et al., Proc Natl Acad Sci USA, 120(35), e2302997120, 2023.

Host cells

By introducing a polynucleotide, a viral or non-viral vector comprising a polynucleotide into microglial cells or their precursors, the cells can be made to express the disclosed Mic-DAIR proteins (host cells).

At least one host cell is obtained, however, this disclosure is also related to a population of host cells. The population may be, for example at least 100, more preferably at least 1000 cells or at least 100 000 cells. The cell or the population of host cells may be isolated. For example, the cell or the population of host cells may be cultured or contained in vitro. In a population of host cells, preferably at least 50%, more preferably at least 70% more preferably at least 80% and most preferably at least 90% or 95% of the host cells express the Mic-DAIR. This may be determined for example by studying a sample of host cells using immunofluorescence microscopy and staining for an antibody that is specific for the Mic- DAIR protein such as for example an antibody against the binding domain or, against a tag in the protein, such as a Flag tag.

The host cells or population of host cells preferably has in vitro or in vivo, or ex vivo endo- cytic activity as described herein. The endocytic activity may be determined as described herein.

A microglia cell or a precursor thereof may be contacted with a polynucleotide, a vector or a virus particle in order to obtain a host cell. Microglial cells may be contacted with the polynucleotide, vector or virus particle in vitro, ex vivo or in vivo. Delivery of a polynucleotide or vector to a cell or a population of cells may be carried out using well-known protocols including physical and chemicals methods, such as Lipofec- tamine™, electroporation, magnetoporation, sonoporation, optoporation. Gene editing tools such as CRISPR/Cas9 may be used.

In some embodiments, starting cells are differentiated to microglia cells. Starting cells are preferably isolated from the subject to be treated. A starting cell, for example a human pluripotent, hematopoietic or embryonic stem cell, a precursor cell, or any somatic cell type that does not display microglial identity can be induced to pluripotency and/or differentiated to show microglia-like phenotype. Starting cells may be isolated from a subject with methods known in the art.

Induction of pluripotency may be achieved by ectopic expression of genes encoding a defined set of transcription factors (e.g. OCT4, KLF4, SOX2, cMYC) in the starting cell. The starting cell may be an induced pluripotent stem cell (iPSC) (Bodda et al., J Exp Med, 217(7), e20191422, 2020). In some embodiments the starting cell is a monocyte.

Differentiation of starting cells into microglia cells or microglia-like cell, such as a microglial precursor cell, may involve the use of various combinations of growth factors (e.g.

TGF-pi, GM-CSF, M-CSF, IL-34, cholesterol, CD200, CX3CR1), media supplements (e.g. B27, N-2) and coating substrates (e.g. fibronectin, geltrex, collagen).

A cell population derived from starting cells may be genetically modified to produce a Mic- DAIR at any stage of differentiation using any of the above-mentioned methods.

In some embodiments, microglia cells or precursors thereof, such as starting cells, are isolated from a patient and then contacted with the polynucleotide, vector or virus particle to obtain host cells. The host cells are then administered to the patient, for example by introducing the cells into the CNS, for example by injecting them into the CNS, for example, by injecting the cells into the brain of the patient. In some embodiments a polynucleotide, a vector or a virus particle is delivered to the patient, preferably to the glia cells of the patient to make the cells of the patient express the Mic-DAIR. For example, a formulation comprising a polynucleotide, a vector or a virus particle is injected into the brain of the patient.

Pharmaceutical composition

A pharmaceutical composition may comprise a Mic-DAIR protein, polynucleotide, a vector, virus particles, an enveloped delivery vehicle utilizing cell-specific targeting molecules, or host cells and in addition a pharmaceutically acceptable excipient. The composition may further involve a strategy for microglia-specific targeting using these approaches, e.g. via extracellular display of an antibody or antibody-derived fragment. Various suitable pharmaceutical compositions are known in the art. The pharmaceutical composition is adapted to its contents, such as a polynucleotide, a viral or non-viral vector, virus particles or host cells. The pharmaceutical composition may be adapted to the mode of administration. Various modes of administration are described herein. Suitable formulations for delivery of polynucleotides, viral and non-viral vectors, virus particles and cells are known.

The pharmaceutical composition may comprise or consist of adeno-associated viral particles dissolved in sterile phosphate buffer and administered to patient via a single-dose intravenous infusion (Gowda et al., Lancet Reg., 37, 100817, 2024).

In another embodiment, the pharmaceutical composition may comprise or consist of a cell suspension of Mic-DAIR-expressing microglia or precursor cells dissolved in sterile phosphate buffer. The cells may be administered to patient via intracerebroventricular injection after surgical insertion of a catheter (Leone et al., Cell Stem Cell, 30:12, 1597-1609, 2023). The pharmaceutical composition may be adapted for delivery to the cells, tissues, organs, or body of a subject in need thereof. The pharmaceutical composition may be adapted to be administered to cells such as microglia cells or precursors of microglia cells in vitro, ex vitro or in situ, such as for example to the tissues of the CNS.

The pharmaceutical composition may be water based. In some embodiments the pharmaceutical composition may be desiccated. Examples of excipients include diluents, adjuvants, carriers, stabilizers or vehicles such as for example buffers, salts, nutrients, proteases, preservatives, antibiotics, fungicides, stabilizers, glycerol, dextrose, PEG, lipids, liposomes, microspheres, lipid or non-lipid nanoparticles or nanospheres

Treatment

A Mic-DAIR protein, a polynucleotide, a viral or non-viral vector, a virus particle, a host cell, a population of host cells or a pharmaceutical composition may be used for the treatment of a neurodegenerative disease. Hence a method of treatment may comprise administering any of the above to a subject in need thereof. An effective amount or dose of a protein, a polynucleotide, a vector a virus, a host cell or a population of host cells is administered to a subject. The exact dose may be found with dose-finding as is known in the art. However, the number of cells administered to a patient, may, in some embodiments vary between from 10⁵ to IO¹⁰ cells.

The subject may be a person suffering from a neurodegenerative disease, which, in a preferred embodiment, is Alzheimer's disease. Treatment includes alleviation of a disease or one or more disease symptoms or slowing the progress of a disease that would otherwise progress in a patient.

In some embodiments, a polynucleotide, a vector, virus or host cell, or a population of host cells is delivered to the CNS of the patient. Delivery of a polynucleotide, vector, virus particle or host cell to the CNS may be done using intraparenchymal, intracerebroventricu- lar, intrathecal or intra cisterna-magna administration or via administration into the local or systemic circulation (intravenously, intraperitoneally or intranasaly). The protein may be selectively targeted to microglia/macrophages through the use of microglia specific expression control sequences such as microglia-specific promoters as described herein.

In some embodiments, cells are first isolated from the subject as described herein and then modified to obtain at least one host cell or a population of host cells, which are then administered to the subject. As described above, administration may be done using various routes.

It is realized that everything which has been described in connection to one embodiment is fully applicable to other embodiments, as compatible. Hence, the invention is not limited to the described embodiments, but can be varied within the scope of the enclosed claims. While the invention has been described with reference to specific exemplary embodiments, the description is in general only intended to illustrate the inventive concept and should not be taken as limiting the scope of the invention. The invention is generally defined by the claims.

EXAMPLES

EXAMPLE 1: GENERATION OF MICROGLIAL DISEASE ANTIGEN INTERNALIZATION RECEPTORS 3 AND 4

To generate microglial disease antigen internalization receptors (Mic-DAIRs), amino acid sequences from extracellular, transmembrane- and intracellular domains of endogenous human and mouse membrane receptors associated with microglial endocytosis of A|3 were assembled and back-translated into codon-optimized polynucleotide sequences. To confer antigen-specificity toward the A|3 protein, a single-chain fragment variable (scFv) domain (SEQ ID NO 32) derived from the monoclonal anti-AP antibody aducanumab (BIIBO37, SEQ ID NO 50 and 51) was used.

Mic-DAIR3 (SEQ ID NO 22) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 1) and hinge domain (SEQ ID 2) of the Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEQ ID NO 3 ) and intracellular (SEQ ID NO 4) domains of FcRy (SEQ ID NO 27) were used.

Mic-DAIR4 was constructed by inserting an scFv derived from aducanumab (SEQ ID NO 32) in between the signal peptide (SEQ ID NO 5) and a truncated segment of the extracellular domain (SEQ ID NO 6) of Fc gamma receptor I (FcyRI) (SEQ ID NO 28) (FIG. 2). The transmembrane (SEQ ID NO 7) and intracellular (SEQ ID NO 8) domains of FcyRI (SEQ ID NO 28) were used.

Polynucleotide sequences encoding Mic-DAIR3 (SEQ ID NO 41) and Mic-DAIR4 (SEQ ID NO 42) were synthesized and cloned into expression plasmids for packaging of genes in recombinant adeno-associated virus (rAAV) particles.

Mic-DAIRl (SEQ ID NO 78) was constructed by inserting the scFv (SEQ ID NO 32) of aducanumab in between the signal peptide (SEQ ID NO 72) and hinge domain (SEQ ID 73) of TREM2 (FIG. 2). The transmembrane (SEQ ID NO 74) and intracellular (SEQ ID NO 75) domains of TREM2 (SEQ ID NO 76) was used.

Mic-DAIR2 (SEQ ID NO 85) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 79) and hinge domain (SEQ ID 80) of DAP12 (FIG. 2). The transmembrane (SEQ ID NO 81) and intracellular (SEQ ID NO 82) domains of DAP12 (SEQ ID NO 83) was used.

Mic-DAIR5 (SEQ ID NO 24) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 9) and hinge domain (SEQ ID 10) of FcyRlla (FIG. 2). The transmembrane (SEQ ID NO 11) and intracellular (SEQ ID NO 12) domains of FcyRlla (SEQ ID NO 29) was used.

Mic-DAIR6 (SEQ ID NO 26) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 17) and hinge domain (SEQ ID 18) of FcyRllla (FIG. 2). The transmembrane (SEQ ID NO 19) and intracellular (SEQ ID NO 20) domains of Fcllla (SEQ ID NO 31) was used.

Mic-DAIR7 (SEQ ID NO 92) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 86) and hinge domain (SEQ ID 87) of SIRP 1 (FIG. 2). The transmembrane (SEQ ID NO 88) and intracellular (SEQ ID NO 89) domains of SRIPbl (SEQ ID NO 90) was used.

Mic-DAIR8 (SEQ ID NO 25) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 13) and hinge domain (SEQ ID 14) of FcyRllc (FIG. 2). The transmembrane (SEQ ID NO 15) and intracellular (SEQ ID NO 16) domains of Fcllc (SEQ ID NO 30) was used.

Mic-DAIR9 (SEQ ID NO 96) was constructed by inserting the scFv of lecanemab (SEQ ID NO 62) between the signal peptide (SEQ ID NO 1) and the hinge domain (SEQ ID NO 2) of the Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEQ ID NO 3 ) and intracellular (SEQ ID NO 4) domains of FcRy (SEQ ID NO 27) was used.

Mic-DAIR10 (SEQ ID NO 98) was constructed by inserting the scFv of donanemab (SEQ ID NO 63) between the signal peptide (SEQ ID NO 1) and the hinge domain (SEQ ID NO 2) of the Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEQ ID NO 3 ) and intracellular (SEQ ID NO 4) domains of FcRy (SEQ ID NO 27) were used.

Mic-DAIRll (SEQ ID NO 100) was constructed by inserting the scFv of praseinezumab (SEQ ID NO 64) between the signal peptide (SEQ ID NO 1) and the hinge domain (SEQ ID NO 2) of the Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEQ ID NO 3 ) and intracellular (SEQ ID NO 4) domains of FcRy (SEQ ID NO 27) were used.

Mic-DAIR12 (SEQ ID NO 102) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEQ ID NO 1) and hinge domain (SEQ ID 2) of the Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEQ ID NO 3) of FcRy (SEQ ID NO 27) was used. The intracellular domain of FcyRllb was used (SEQ ID NO 68).

EXAMPLE 2: EXPRESSION OF MICROGLIAL DISEASE ANTIGEN INTERNALIZATION RECEPTORS IN HUMAN MICROGLIA-LIKE CELLS

To visualize transgene expression of Mic-DAIRs in cells a gene encoding a fluorescent tag (e.g. EGFP, mScarlet) was added as part of the single open reading frame downstream of a self-cleaving 2A peptide sequence. rAAV2 particles containing transgenes were applied overnight to human-derived microglia-like cells (MGLCs). Transgene expression was evaluated 5 days post viral transduction using fluorescence microscopy (FIG. 3) and compared to constructs composed of CD28 and CD8 transmembrane and extracellular domains (FIG. 2). Results showed that rAAV encoding these two Mic-DAIRs, but not receptors constructed from CD8- and CD28-based constructs, were able to generate transgene expression in cultures.

EXAMPLE 3: TESTING OF ENDOCYTIC PERFORMANCE IN HUMAN MICROGLIA-LIKE CELLS

MGLCs differentiated from human induced pluripotent stem cells (iPSCs) (Bodda et al., 2020) were seeded at 3 x 10⁴ cells per well in standard 96-well tissue culture plates and maintained as a monoculture in serum-free medium supplemented with human IL-34 and GM-CSF. Transgene expression was obtained by incubating cultures with rAAV2 particles as per example 2. rAAV particles encoding EGFP/mScarlet alone was used as viral control. The ability of Mic-DAIR-expressing cells to endocytose A 1-42 was tested 5 days post transduction as compared to viral control and three candidate constructs based on domains from other endogenous endocytic receptors in microglia (FIG. 2, FIG. 4-5). To visualize endocytosis, cells were incubated with 500 nM fluorescent A 1-42 for 2 hours at 37° C, washed with PBS, fixated with 4% formaldehyde for 10 minutes and imaged using a fluorescence microscope.

To test the specificity of endocytosis, carboxyl-modified fluorescent polystyrene beads (1 pm diameter) were coated with A 1-42, or a scrambled A|3 peptide, for 2 hours in 2.5x excess of the calculated monolayer. Preparations were centrifuged, washed and applied to human MGLCs (3 x 10⁴ cells per well) expressing EGFP, Mic-DAIR3 and Mic-DAIR4 (FIG. 6- 7) at 1 x 10⁶ beads per well in 96-well plates and incubated overnight at 37° C. Following PBS wash and formaldehyde fixation the number of endocytosed beads per EGFP+ cell was examined using a fluorescence microscope. In summary, results from the two separate assays show that Mic-DAIR3 and 4 facilitated an increased Api-42-specific endocytosis by human MGLCs.

EXAMPLE 4: FLOW CYTOMETRY ANALYSIS OF ENDOCYTIC PERFORMANCE IN HUMAN MICROGLIA-LIKE CELLS

To support a more quantitative analysis of endocytic performance, human MGLCs (see EXAMPLE 3 for culture protocol) were transduced using an rAAV capsid (cMG) with improved microglial tropism (Lin et al., 2022). rAAV particles carrying DNA encoding mScarlet (viral control) and MicDAIRsl-7 were applied overnight and allowed 5 days to express the protein. Expressing cultures were incubated with fluorescent A 1-42 (500 nM) for 2 hours at 37° C. Cells were then washed, trypsinized and resuspended in FACS buffer for flow cytometry analysis using a BD LSR Fortessa instrument. Endocytosis of fluorescent A 1-42 was analyzed in single cells (> 2000 cells per sample) as the mean fluorescence intensity using a multicolor panel for detection of living cells (Live/Dead 633), microglial nuclei (Nuclear blue 350), mScarlet expression (viral control + Mic-DAIRs, PE) and A 1-42 (FITC) (FIG. 8-10). EXAMPLE 5: TESTING OF ANTIGEN SPECIFICITY, DEPENDENCE ON THE INTRACELLULAR

DOMAIN, AND ACTIN POLYMERIZATION

We conducted a series of validation tests in human MGLCs (see EXAMPLE 3 for culture protocol) using flow cytometric analysis (parameters in EXAMPLE 4) and Mic-DAIR3 for comparison. rAAV-cMG particles carrying DNA encoding mScarlet (viral control) and Mic- DAIRs were applied overnight and allowed 5 days to express the protein. Expressing cultures were incubated with fluorescent A 1-42 (500 nM) or scrambled fluorescent A|3 peptide (500 nM) for 0.5-24 hours at 37° C (FIG. 11-13).

We tested the antigen specificity of endocytosis by replacing the aducanumab scFv (used in Mic-DAIR3) with scFv:s from two other A|3-targeting antibodies in clinical use (lecanemab: Mic-DAIR9, donanemab: Mic-DAIRIO), as well as an alpha-synuclein-targeting scFv (prasinezumab: Mic-DAIRll) (FIG. 11). Mean fluorescent intensity values for A 1-42 were robustly increased for all A|3-targeting Mic-DAIRs versus control, but not for the al- pha-synuclein-targeting Mic-DAIR. However, no increase in mean fluorescence intensity values were observed for any Mic-DAIRs after application of scrambled A|3 peptide. This together shows that Mic-DAIRs enhance endocytosis specifically for its disease antigen.

To test the dependence of Mic-DAIR-mediated endocytosis on intracellular signaling, we replaced the FcRy-derived ITAM-containing ICD used in Mic-DAIR3 with an ITIM-containing ICD derived from the inhibitory Fc gamma receptor lib (FcyRllb, Mic-DAIR12) (FIG. 12). Application of fluorescent A 1-42 for 2 hours generated a robust increase in mean fluorescence intensity for Mic-DAIR3 relative to control as in previous experiments. Consistent with a role of ITAM signaling in triggering endocytosis, the ITIM-containing Mic-DAIR12 showed a marked reduction in A 1-42 uptake.

Lastly, to examine a broader time range of Mic-DAIR-mediated endocytosis and the dependence on actin polymerization, MGLCs expressing mScarlet (viral control) or Mic-DAIR3 were incubated with fluorescent A 1-42 for 0.5, 4 and 24 hours in the presence or absence of cytochalasin d (CytoD, 5 pM). The mean fluorescence intensity increased exponentially over the first 4 hours for both groups, and was maintained at the 24 hour time point (FIG. 13). Mean fluorescence intensity showed a strong dependence on the presence of cytochalasin d in both groups, consistent with this signal representing intracellular uptake (rather than e.g. passive membrane attachment) of A|31-42.

EXAMPLE 6: TESTING OF ENDOCYTIC PERFORMANCE IN PRIMARY MOUSE MICROGLIA

Primary mouse microglia isolated from C57/BL6 mice at postnatal day 2 were seeded at 1.5-2.5 x 10⁴ cells per well in standard 96-well plates and maintained as monocultures in TIC medium (Bohlen et al., Neuron, 94(4), 759-773, 2017). The rAAV-cMG capsid (Lin et al., 2022) was used to efficiently deliver genes encoding murine versions of Mic-DAIR3 (SEQ ID NO 43 and 44) and Mic-DAIR4 (SEQ ID NO 45 and 46) to primary cells alongside viral control (mScarlet) overnight (FIG. 14). The ability of Mic-DAIRs to increase endocytosis of A|3 was tested 5 days post transduction by application of fluorescent labelled A 1-42 (500 nM) 2 hours at 37° C (FIG. 15). Following incubation, the cells were washed with PBS, fixated with 4% formaldehyde for 10 minutes and imaged using a fluorescence microscope.

To test the specificity of A|3 endocytosis in primary microglia, cells were incubated with either fluorescent-labelled A 1-42 or scrambled A|3 peptide (both at 500 nM concentration) for 2 hours at 37° C. Microglia were then washed, trypsinized and resuspended in FACS buffer for flow cytometry analysis using a BD LSR Fortessa instrument. Uptake of A 1-42 vs scrambled control was analyzed in single cells (> 2000 cells per sample) as mean fluorescence intensity in samples consisting of non-transduced control cells, mScarlet (viral controls), Mic-DAIR3 and Mic-DAIR4 (FIG. 16-17). In conclusion, results obtained in primary mouse microglia suggest that mic-DAIR3 and 4 promote enhanced selective endocytosis of A 1-42.

EXAMPLE 7: MICROGLIAL EXPRESSION OF DISEASE ANTIGEN INTERNALIZATION RECEPTOR 3 AND 4 IN THE MOUSE BRAIN To target delivery microglia in the live mouse brain, genes encoding mScarlet, Mic-DAIR3 and Mic-DAIR4 were cloned into a FLEx-On (Cre recombinase-dependent) rAAV expression vector and packaged using a newly developed capsid for improved in vivo transduction (Lin et al., 2022). Transgenic mice expressing inducible Cre-recombinase under the micro- glia-specific TMEM119 promoter (TMEM119-CreERT2, JAX stock #031820) were used to achieve selective gene recombination in microglia. TMEM119-CreERT2 mice were bilaterally injected with rAAV in parietal cortex under isoflurane anesthesia using a stereotaxic instrument. 3-7 days post-surgery mice were administered tamoxifen (100 mg/kg, i.p) for 4 consecutive days to induce Cre recombinase activity. Fixation of mouse brains by transcardiac perfusion with 4% formaldehyde was performed 10-14 days following tamoxifen treatment. 30 pm coronal sections were cut on a vibratome and immunostained with IBA1 to confirm that expression of Mic-DAIR occurred in microglia (FIG. 18).

EXAMPLE 8: USE OF MIC-DAIR AS TREATMENT FOR ALZHEIMER'S DISEASE

For use in the treatment of Alzheimer's disease, a Mic-DAIR equipped with an anti-amyloid or anti-tau scFv can be provided as gene therapy medicinal product using a delivery vector suitable for clinical use. The gene therapy is composed by a non-replicating recombinant adeno-associated virus serotype 9 (AAV9) based vector containing DNA encoding the human Mic-DAIR under control of the CD68 promoter. The vector will be administered into a CNS compartment (intrathecal, intraventricular, intracisternal) by, or under supervision of, a neurosurgeon. To reduce the risk of severe adverse events during and after the administration of the gene therapy, immunosuppressant drugs such as corticosteroids, may be used to dampen inflammation associated with viral delivery.

Treatment outcome will be determined by PET scan analysis of amyloid plaques, monitoring of amyloid/tau blood biomarkers and cognitive performance testing.

Claims

1. A protein comprising an amino acid sequence selected from SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 77, SEQ ID NO 84, SEQ ID NO 91 or SEQ ID NO 33, or a protein comprising an amino acid sequence with at least 90% identity to one of said sequences, the protein in addition comprising an extracellular binding domain that is capable of binding a neurodegenerative disease antigen.

2. The protein of claim 1 where the neurodegenerative disease antigen is amyloid beta.

3. The protein according to claim 1 or 2 where the protein comprises the amino acid sequence of one of SEQ ID NO 34, 35, 36 or 37; or an amino acid sequence with at least 90% identity to one of SEQ ID NO 34, 35, 36 or 37.

4. The protein according to any one of claims 1 to 3 where the protein comprises the amino acid sequence of SEQ ID NO 34 or an amino acid sequence with at least 90% identity to SEQ ID NO 34.

5. The protein of any one of claim 1 or 2 where the protein comprises SEQ ID NO 23, 24, 25 or 26, 78, 85, 92, 98, 98 or 22 or a sequence which is at least 90% identical to one of the sequences SEQ ID NO 23, 24, 25 or 26, 78, 85, 92, 98, 98 or 22.

6. The protein of claim 5 where the protein comprises SEQ ID NO 23, 24, 25 or 26 or a sequence which is at least 90% identical to one of the sequences SEQ ID NO 23, 24, 25 or 26.

7. The protein according to claim 6 where the sequence is SEQ ID NO 23 or a sequence which is at least 95% identical to SEQ ID NO 23.

8. The protein according to any one of claims 1 to 7 comprising a single intracellular endocytosis triggering domain.

9. A polynucleotide encoding a protein according to any one of claims 1 to 8.

10. A vector comprising a polynucleotide according to claim 9.

11. A virus particle comprising the polynucleotide of claim 6 or the vector of claim 10.

12. A host cell comprising a protein according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, or a virus particle according to claim 11.

13. A population of host cells according to claim 12.

14. A method of treating a subject comprising administering, to a subject, protein according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, or a virus particle according to claim 11 or a host cell according to claim 12 or a population of host cells according to claim 13.

15. The method of treatment according to claim 14 where the disease Alzheimer's disease.

16. A protein according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, or a virus particle according to claim 11 or a host cell according to claim 12 or a population of host cells according to claim 13 for use in treatment.

17. The protein according to any one of claims 1 to 8, a polynucleotide according to claim 9, a vector according to claim 10, or a virus particle according to claim 11 or a host cell according to claim 12 or a population of host cells according to claim 13 for use in treatment of Alzheimer's disease.