SE2350348A1 - Microglial endocytic receptors for use in the treatment of neurodegenerative disease - Google Patents

Abstract

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

Description

I\/|icroglial endocytic receptors for use in the treatment of neurodegenerative disease FIELD OF THE INVENTION This invention generally involves the treatment of neurogenerative disease, such as Alz- heimer's disease, using novel chimeric proteins.

BACKGROUND Neurodegenerative disease (NDD) is the collective name for a range of disorders where progressive deterioration of nerve cells (neurons) and chemical contacts (synapses) in ar- eas ofthe central nervous system (CNS) is a characteristic feature of the pathology. A ma- jority of NDDs (e.g. Alzheimer's disease, Parkinson's disease, Lewy body disease, fronto- temporal dementia, Huntington's disease, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease) are further defined as proteinopathies, involving the aggregation and/or misfold- ing of specific protein species in the intra- and/or extracellular space (Wilson et al 2023). A prototypical NDD is Alzheimer's disease (AD), the most common cause of dementia, where an inability of the brain to clear, and resolve early insults of, toxic beta-amyloid (AB) spe- cies is believed to underlie disease development.

A key player in the early defense against toxic AB is microglia, the brain's resident immune cell, which may protect neurons and synapses from Aß-induced damage by endocytosing and degrading the protein. ln AD, however, microglial endocytic function becomes heavily impaired, and such loss-of- function could underlie or contribute to disease development and/or progression (Zhang et al 2021). As the number of individuals suffering from AD and other dementias is pro- jected to increase substantially in the next decades (Winblad et al 2016), the development of efficacious treatments will be imperative to reduce human suffering and lighten the burden on health care systems. Hence, there is a need for improved treatment of Alz- heimer's and other neurodegenerative diseases. The present disclosure describes strategies for selectively increasing microglial endocytosis of a toxic NDD protein (also re- ferred to as a disease antigen) as exemplified by Aß.

I\/Iorrissey et al. (DOI: https://doi.org/10.7554/eLife.36688.001) reports engineered Chi- meric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf spe- cific targets, including cancer cells. However, preliminary data show that the approach used by Morrissey et al., involving the construction of phagocytic receptors using CD8 do- mains, produced very weak expression in microglia.

Hence, there is a need for a microglia-specific approach to construction and delivery ofre- ceptors 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 se- quence selected from SEQ ID NO 33-37 or an amino acid sequence with at least 90% iden- tity to one of SEQ ID NO 33-37, 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 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 se- quence 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 a se- quence which is at least 90% identical to one of the sequences SEQ ID NO 22, 23, 24, 25 or 26. ln one embodiment the sequence is SEQ ID NO 23 or a sequence which is at least 95% identical to SEQ ID NO 23. ln second aspect ofthe invention there is provided polynucleotide encoding a protein ac- cording to the first aspect of the invention. ln third aspect of the invention there is provided a vector comprising a polynucleotide ac- cording to the second aspect of the invention. ln various embodiments the vector may comprise a promoter active in microglia cells. ln various embodiments the promoter is specific for microglia cells. ln a fourth aspect ofthe invention there is provided a virus particle comprising a polynu- cleotide 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) par- ticle. ln a fifth aspect of the invention there is provided a host cell comprising a protein accord- ing 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. ln a sixth aspect of the invention there is provided a population of host cells according to the fifth aspect of the invention. ln a seventh aspect ofthe invention there is provided a pharmaceutical composition com- prising a protein according to the first aspect, a polynucleotide according to the second as- pect, 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. ln an eight aspect ofthe invention there is provided a method of treating a subject com- prising the administration of a protein according to the first aspect, a polynucleotide ac- cording 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 inven- tion or a population of host cells according the sixths aspect ofthe invention, or a pharma- ceutical composition according to the eight aspect of the invention. ln various embodiments the disease is one of Alzheimer's disease, Parkinson' s disease, fronto-temporal dementia, Huntington's disease, motor neuron disease, Creutzfeldt-Jakob disease, vascular dementia, Lewy body dementia, hippocampal sclerosis, multiple sclerosis (I\/IS) and amyotrophic lateral sclerosis (ALS) . ln a ninth aspect ofthe 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 as- pect of the invention or a population of host cells according the sixths aspect of the inven- tion, or a pharmaceutical composition according to the eight aspect of the invention for use in the treatment of a disease. ln 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 inven- tion 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. ln one embodiment the step of contacting is performed in vivo. ln one embodiment the step of contacting is performed ex vivo.

DRAWINGS The accompanying drawings form a part ofthe 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 mi- croglial disease antigen internalization receptors (I\/|ic-DA|Rs).

Fig.2 shows a schematic structure comparison of constructs tested in human microglia-like cells. SP; signal peptide, ABD; extracellular antigen-binding domain, scFv adu; single-chain fragment variable of aducanumab, TI\/ID; transmembrane domain, ICD; intracellular do- main comprising or consisting of an endocytosis-triggering domain. As customary, the pro- tein architectures are shown with the N-terminal to the left and the C-terminal 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 re- ceptor comprised ofthe 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 (hI\/lic-DAIR3) (e) and the human microglial disease antigen internalization receptor 4 (hI\/lic-DAIR4) (f) were delivered to cultures and incubated overnight.

Fig. 4 shows representative double fluorescence microscopy images of Aß1-42 endocytosis in human microglia-like cells virally transduced with EGFP only (a-b), human microglial dis- ease antigen internalization receptor 3 (hI\/lic-DAIR3) (c-d) and human microglial disease antigen internalization receptor 4 (hI\/lic-DAIR4) (e-f). White arrows highlight levels of AB uptake in transduced cells, grey arrows highlight levels of AB uptake by non-transduced neighboring cells. Cultures were incubated with fluorescently labelled Aß1-42 for 2 hrs at 37° C 5 days post rAAV transduction.

Fig. 5 is a summary bar graph showing the normalized fluorescence intensity of Aß1-42 de- tected in human microglia-like cells transduced with EGFP ctrl, human endocytic receptor 1-3 (hER1-3) (the non Fc-constructs of Fig 2), human microglial disease antigen internaliza- tion receptor 3 (hI\/|ic-DA|R3) and human microglial disease antigen internalization recep- tor 4 (hivnc-oAiRzi).

Fig. 6 shows representative double fluorescence microscopy images obtained from a sepa- rate endocytosis assay using 1 pm fluorescent polystyrene beads coated with Aß1-42 or scrambled Aß peptide. White arrows highlight beads/bead clusters detected in transduced cells. (a-f), endocytosis of Aß1-42-coated beads by human microglia-like cells expressing EGFP only (a-b), human microglial disease antigen internalization receptor 3 (hI\/|ic-DA|R3) (c-d) and human microglial disease antigen internalization receptor 4 (hI\/lic-DAIR4) (e-f). (g-l), endocytosis of scrambled Aß-coated beads by human microglia-like cells expressing EGFP only (g-h), human microglial disease antigen internalization receptor 3 (hI\/|ic-DA|R3) (i-j) and human microglial disease antigen internalization receptor 4 (hI\/lic-DAIR4) (k-l). Cultures were incubated with coated beads for 12 hrs at 37° C 5 days post rAAV transduc- tion.

Fig. 7 is a summary bar graph showing the average number of Aß1-42 vs scrambled Aß- coated beads endocytosed per cell in human microglia-like cells transduced with EGFP only, human microglial disease antigen internalization receptor 3 (hMic-DAIR3) and hu- man microglial disease antigen internalization receptor 4 (hMic-DAIR4).

Fig. 8 shows representative double fluorescence microscopy images of Aß1-42 endocytosis in mouse primary microglial monocultures virally transduced with mScarlet only (a-c), mouse microglial disease antigen internalization receptor 3 (mI\/lic-DAIR3) (d-f) and mouse microglial disease antigen internalization receptor 4 (mI\/lic-DAIR4) (g-i). White arrows in merged images highlight Aß fluorescence in transduced cells. Cultures were incubated with fluorescently labelled Aß1-42 for 2 hrs at 37° C 5 days post rAAV transduction.

Fig. 9 shows flow cytometry density plots of primary mouse microglia after incubation with fluorescent Aß1-42 in non-transduced control (a), mScarlet-transduced viral control (b), cells transduced with mouse microglial disease antigen internalization receptor 3 (mI\/lic-DAIR3) (c) and mouse microglial disease antigen internalization receptor 4 (mMic- DAIR4) (d). The primary cultures were incubated with fluorescently labelled Aß1-42, or scrambled Aß peptide (not shown in plots), for 2 hrs at 37° C. I\/|icroglia were transduced with rAAV 5 days prior to flow cytometric analysis.

Fig. 10 is a summary bar graph of data obtained from primary mouse microglia in flow cy- tometry experiments. The graph shows the mean fluorescence intensity of Aß1-42, vs scrambled Aß peptide, collected from a minimum of 2000 primary mouse microglia per group and experiment.

Fig. 11 shows representative double fluorescence microscopy images of in vivo expression of mouse microglial disease antigen internalization receptor 3 (mI\/lic-DAIR3) and 4 (mMic- DAIR4) in layer 5 parietal cortex oftransgenic TI\/|EI\/|119-CreERT2 mice. Images show co- localization of |ba-1-reactivity with mMic-DAIR3 (a1-a3) and mI\/lic-DAIR4 (b1-b3) expres- sion in microglia.

DETAILED DESCRIPTION As used herein, the terms "binding domain," refer to a polypeptide that possesses the ability to specifically and non-covalently bind to a neurogenerative disease antigen. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombi- nantly produced binding partner for a biological molecule or other target of interest. ln some embodiments, the binding domain is an antigen-binding domain, such as an anti- body or an antibody fragment. 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. ln a preferred embodiment the binding domain is a single chain variable fragment (scFv).

An antibody may be a naturally occurring, recombinantly produced, genetically engi- neered, or modified form of an immunoglobulin, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SM|Ps, multispecific antibodies (e.g., bispecific anti- bodies, 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, Fab, Fab', F(ab')2, and Fv fragments, Fab' -SH, F(ab')2, diabodies, linear antibodies, scFv anti- bodies, VH, and multispecific antibodies formed from antibody fragments. A "Fab" (frag- ment 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 via an inter-chain disul- fide 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 lgG, |gM, lgE, |gA, lgY and the like.

A binding domain 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 5><10-7 M, more preferably 10-7 M, more preferably 5><10-8 M, 10-8 M, more preferably 5><10-9 M, more preferably 10-9 M, more preferably 5><10-1° M, more preferably 10-10 M, more pref- erably 5><10-11 M, more preferably 10-11 M, more preferably 5><10-12 M, more preferably 10-12 M, even more preferably 5><10-13 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, Huntingtons disease, motor neuron disease, Creutz- feldt-Jakob disease, vascular dementia, Lewy body dementia, hippocampal sclerosis,multi- ple sclerosis and amyotrophic lateral sclerosis (ALS). ln a preferred embodiment, the dis- ease 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. ln certain embodiments a neurodegenerative disease antigen is a protein or peptide that is overexpressed or inappropriately expressed in the CNS. A neurodegenerative disease anti- gen may be a protein that is unfolded or misfolded. ln some embodiments, the neuro- degenerative disease antigen is a protein or part thereof that is misfolded, fibrillized or ag- gregated. 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 10 um. ln various embodiments, the neurodegenerative disease antigens in- clude antigens from amyloid beta (Alzheimer's), beta-secretase 1 (Alzheimer's), Tau (Alz- heimer's), apolipoprotein E4 (ApoE4), alpha-synuclein (Parkinsons disease), huntingtin (HTT) (Huntington's disease), superoxide dismutase 1 (SOD1) (ALS), leucine rich repeat ki- nase 2 (LRRK2) (Parkinson's disease), prion protein (prP) (Creutzfeldt-Jakob disease). Pref- erably the binding domain is capable of binding one antigen selected from these antigens. ln a preferred embodiment the neurodegenerative disease antigen is amyloid beta. Amy- loid beta may have the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO 52) (Aß1-42 peptide). ln a preferred embodiment the disease being treated is Alzheimer's disease.

As used herein, the term "amyloid beta" (Aß) refers to a fragment of amyloid precursor protein (APP) that is produced upon cleavage of APP by ß-secretase 1 ("BACE1"), as well as modifications, fragments and any functional equivalents thereof, including, but not limited to, Aß1-40 peptide, and Aß1-42 peptide. Aß may be a monomer, or may associate to form oligomers or fibril structures. Aß fibrils may aggregate into amyloid plaques, e.g., such as those found in brains of Alzheimer's disease patients.

The term "subject," "patient" and "individual" are used interchangeably herein and are in- tended 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 ofthe antigen. Endocytosis also comprises other processes by which a cell engulfs particles, pro- teins, peptides or antigens such receptor-mediated endocytosis and pinocytosis.

References to sequences SEO ID NO 1 to SEO ID NO 40 and SEO ID NO 43 and 45 herein may also, in various embodiments, refer sequences that are substantially identical to the reference sequence. "Substantially identical" shall, for amino acid sequences, mean a se- quence 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. Se- quence 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 poly- peptide 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, I\/Iiller W, I\/Iyers EW, Lipman DJ. Basic local alignment search tool.J I\/Iol 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 I\/Iarch 2023 which are: word size: 3, gap penalty exist- ence:11, gap penalty extension: 1).

A "signal peptide" is inserted into the membrane of the endoplasmic reticulum during pro- tein synthesis and contributes to positioning of the I\/Iic-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 11 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. ln eukaryotes, a sig- nal peptide causes translocation ofthe newly synthesized protein to the endoplasmic re- ticulum where it is cleaved, creating a mature protein which then proceeds to its appropri- ate destination. The signal peptide is typically cleaved after insertion into the membrane, resulting in a mature protein.

"Ivlicroglial cells" (or "microglia") are glial cells in the CNS that express markers such as ionized calcium binding adapter molecule 1 (lba-1) TI\/|EI\/| 119 or HEXB. I\/|icroglial cells are typically located in the brain and the spinal cord. I\/|icroglia cells are the primary immune cells ofthe CNS and play import roles in development, homeostasis and neuroprotection.

The inventors have surprisingly found that Fc receptors can be used as suitable scaffolds for fusion proteins (Mic-DA|Rs) to be used together with an extracellular binding domain for stimulating endocytosis of neurodegenerative diseases antigens by microglial cells. Not only are the Mic-DA|Rs well expressed in microglial cells but enable an increased engulf- ment of neurogenerative disease antigens. Mic-DA|Rs may be used for the treatment of neurodegenerative disease, such as Alzheimer's. Suitable Fc receptors include: Fc receptor gamma chain (FcRy or FCERG), Fc gamma receptor I, (FcyRl or FCGR1), Fc gamma receptor ||a (FcyRlla or FCRlla), Fc gamma receptor ||c (FcyRllc or FCRllc) and Fc gamma receptor |||a (FcyRllla or FCRllla). The Fc proteins are preferably of human origin. However, protein do- mains from other species can be useful, in particular when treating other species than hu- mans. 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 internaliza- tion receptors" ("I\/|ic-DA|R") herein.

When the I\/lic-DAIR is introduced into microglia it enables the cells to engulf the neuro- degenerative disease antigens, thereby treating the disease slowing its progression and/or 12 reversing disease pathology The microglial cells may engulf protein, protein aggregates or cells that are characteristic of neurodegenerative disease.

The general structure of a I\/lic-DAIR is seen in Fig.1.|n 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 do- main comprising or consisting of an endocytosis-triggering domain, where the transmem- brane domain is located between the extracellular domain and the intracellular domain. The I\/lic-DAIR may optionally comprise a hinge domain. The structure arrangement is pref- erably from the N-terminal to the C-terminal: extracellular binding domain, transmem- brane domain, intracellular endocytosis-triggering domain. The I\/lic-DAIR may optionally comprises a signal peptide. The I\/lic-DAIR optionally comprises a hinge domain. ln various embodiments, all domains except the extracellular binding domain is from a Fc receptor selected from SEQ ID NO 27-31 or the corresponding mouse sequences. ln various embodiments, the intracellular domain comprising the endocytosis triggering 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.

When expressed in a eucaryotic cell, such as a microglial cell, a population of I\/lic-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 I\/|ic- DAIR, in particular the binding domain is located on the cell surface.

Table 1 shows the various SEQ ID NO:s for the various domains of the human Fc receptors and I\/|ic-DA|Rs. 13 Transmem- lntracellu- Fc se- Hinge + Fc+adenuca- FC recep- Signal Hinge brane domain lar ET do- quence TM + ICD numab tor peptide domain (TM) main (ICD) FcERG 1 2 3 4 27 33 221 FCGR1 5 6 7 8 28 34 232 FCRIIa 9 10 11 12 29 35 24 FCRllc 13 14 15 16 30 36 25 FCRllla 17 18 19 20 31 37 26 1 Mic-DAIR3, 2 Mic-DAIR4 Table 1 SEO ID NO 22, 23, 24, 25, and 26 represents the sequence of each Fc receptor (SEO ID NO 27, 28, 29 30 and 31, respectively) where the binding domain of SEO ID NO 32 has been inserted between the signal peptide and the hinge domain of each Fc-receptor.

SEO ID NO SEO ID NO 22, 23, 24, 25, and 26 represents SEO ID NO 33, 34, 35, 36 and 37 with the addition ofthe SEO ID NO 32 as the antigen binding domain and the signal pep- tides.

The intracellular endocytosis triggering domain preferably comprises or consists of a se- quence selected from one of the human Fc receptors (SEO ID NO SEO 4, 8, 12, 16 or 20) which are the intracellular domains of the human Fc receptors FCERG, FCGR1, FCRIIa, FCRllc and FCRllla, respectively. The intracellular endocytosis triggering domain may be ca- pable of providing an intracellular signal that triggers endocytosis, for example when a neurogenerative disease antigen is bound to the binding domain. The endocytosis trigger 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 par- ticipate in signal transduction in the cytoplasm ofthe host cell. There are several different endocytosis systems in the human cell, and the receptor may use any one ofthem that is capable of triggering endocytosis of neurodegenerative disease antigens. For example, the intracellular endocytosis triggering domain may comprise an ITAM motifwhich is a known endocytosis signaling motif. This is the case with the intracellular domains of FCERG, FCRIIa and FCRllc (SEO ID NO 4, 12 and 16 respectively). However, notably SEO ID NO 8 does not comprise an ITAM sequence motif, and it is surprising that it can trigger 14 engulfment to an extent even larger than SEO ID NO 4, which does have an ITAM motif, since I\/Iic-DAIR 4 which comprises SEO ID NO 8 is highly efficient in engulfing amyloid beta (see for example Figs. 5, 7 and 10 of the Examples).

In various embodiments, the I\/Iic-DAIR comprises a two, three or more intracellular endo- cytosis triggering domains. These may be arranged in any suitable manner in the intracel- lular 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 ar- ranged 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 intra- cellular domain of a I\/Iic-DAIR comprises or consists of a plurality of intracellular endocyto- sis-triggering domains all selected from the group consisting of SEO ID NO 4, 8, 12, 16 and 20, in particular SEO ID NO 8, 12, 16 and 20 and no further intracellular endocytosis trig- gering domains. In particular signaling domains from the proteins I\/IRC1, MERTK, Tyro3, Axl or ELMO may be excluded. Hence, in some embodiments the Mic-DAIR does not com- prise a intracellular endocytosis triggering domain from one of the proteins I\/IRC1, MERTK, Tyro3, Axl or ELMO.

However, it may be advantageous to use as few intracellular endocytosis triggering do- main as possible. In various embodiments, the I\/Iic-DAIR comprises only one intracellular endocytosis -triggering domain. In various embodiments, the intracellular domain of a I\/Iic-DAIR comprises or consists of one of SEO ID NO 4, 8, 12, 16 and 20.

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 transmem- brane domain is preferably composed of predominantly hydrophobic amino acids. The transmembrane domain may anchor the I\/Iic-DAIR protein in the cell membrane. The transmembrane domain preferably comprises or consists of a sequence selected from one ofthe human Fc receptors FCERG, FCGR1, FCRlla, FCRllc and FCRllla (SEO ID NO 3, 7, 11, 15, and 19).

The extracellular binding domain is capable of binding a neurodegenerative disease anti- gen. Binding may be specific for the antigen. Any useful binding domain may be used.

Examples of a binding domains is SEO ID NO 32 or a part thereof. SEO ID No 32 is a single chain fragment ofthe BIIB037 antibody (aducanumab) (SEO ID NO 50 and 51). The extra- cellular 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 neurodegenera- tive disease antigen is amyloid beta or Tau.

In various embodiments, the disease being treated is Parkinson's disease and the neuro- generative disease antigen is Alpha-synuclein.

In various embodiments the disease being treated is Huntington's disease and the neuro- degenerative disease antigen is mutated huntingtin.

In various embodiments, the disease being treated is ALS and the neurodegenerative dis- ease antigen is superoxide dismutase (SOD1).

Other useful binding domains include other domains cable of binding a neurodegenerative disease antigen. Binding domains may be derived from the following antibodies: Antibod- ies against beta-amyloid, for example lecanemab (BAN2401), donanemab (LY3002813), remternetug (LY3372993), solanezumab (LY2062430), gantenerumab (RO4909832), bapi- neuzumab (AAB-001), crenezumab (MABT5102A), ABBV-916 or ACU193; antibodies against Tau, for example bepranemab (UCB0107), semorinemab (RO7105705), E2814, 16 PRXO05, or Lu AF87908 (hC10.2); antibodies against Alpha-synuclein, for example prasinezumab (PRXOOZ) or cinpanemab (B||BO54); antibodies against mutated huntingtin (mtHTT) for example C6-17 (AFFiRiSAG); and antibodies against superoxide dismutase (SOD1) for example W20.

The I\/Iic-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 re- gion. 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 ofthe I\/Iic- DAIR. Without being bound by any theory, the hinge domain may contribute to position- ing 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 FCERG, FCGR1, FCRIIa, FCRIIc and FCRIIIa (SEO ID NO SEO 2, 6, 10, 14 and 18).

The I\/Iic-DAIR preferably comprises a signal peptide located in the N-terminal of the pro- tein. 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 prefera- bly comprises or consists of a sequence selected from one ofthe human Fc receptors FCERG, FCGR1, FCRIIa, FCRIIc and FCRIIIa (SEO ID NO SEO 1, 5, 9, 13 and 17). Other useful signal peptides include a CD28 signal peptide, a CD8 signal peptide, a TREM2 signal pep- tide (SEO ID NO 38) or a SRIP1B signal peptide (SEQ ID NO 39) or a I\/IERTK signal peptide (SEQ ID NO 40) or a GI\/I-CSF signal peptide.

Preferred I\/Iic-DAIR protein architectures Preferred architectures of I\/Iic-DAIR include those that comprise the hinge domain, trans- membrane domains and intracellular endocytosis triggering domains of FCERG, FCGR1, 17 FCRIIa, FCRllc and FCRIIIa (SEO ID NO 27, 28, 29, 30 and 31). For example, a I\/Iic-DAIR may comprise one of the following domains, from the N-terminal: extracellular binding domain - SEO ID NO 2 - SEO ID NO 3 - SEO ID NO 4, extracellular binding domain - SEO ID NO 6 - SEO ID NO 7 - SEO ID NO 8, extracellular binding domain - SEO ID NO 10- SEO ID NO 11 - SEO ID NO 12, extracellular binding domain - SEO ID NO 14 - SEO ID NO 15 - SEQ ID NO 16 and extracellular binding domain - SEO ID NO 18 - SEO ID NO 19 - SEQ ID NO 20 Hence, preferred embodiments of I\/Iic-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 and Extracellular binding domain - SEQ ID NO 37.

In general, the Mic-DAIR protein may comprise or consist ofthe 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 - one of the transmembrane domains 3, 7, 11, 15, 19 - one of the intracellular endocytosis triggering domains 4, 8, 12, 16, 20.

In one embodiment, the I\/Iic-DAIR comprises the transmembrane domain of SEO ID NO 7 and the intracellular endocytosis triggering domain of SEO ID NO 8.

In various embodiments the binding domain is inserted between amino acids 18 and 19 of SEO ID NO 27, or between amino acids 15-16 of SEO ID NO 28, or between amino acids 33 and 34 of SEQ ID NO 29 or between amino acids 42 and 43 of SEO ID NO 30 or between amino acids 16 and 17 of SEQ ID NO 31. The indicated insertion position may be changed one, two or three amino acid positions towards the N-terminal or the C-terminal of SEO ID NO 27-31. 18 Further preferred I\/Iic-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, with at least 95% identity with a sequence selected from SEO 8, 12, 16 or 20.

It is understood that direct fusion of one domain to another domain of a I\/Iic-DAIR de- scribed herein does not preclude the presence of intervening junction amino acids. For ex- ample, the number ofjunction amino acids may be from 1-to 8 amino acids.

In certain embodiments, a I\/Iic-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 I\/Iic-DAIR is murine, human, chimeric (with sequences from two or more species) or humanized. The counterpart of I\/Iic-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 I\/Iic-DAIR corresponding to Mic- DAIR3 (SEO ID NO 22) is SEO ID NO 43, encoded by SEQ ID NO 44. The murine counterpart of Mic-DAIR4 (SEO ID NO 23) is SEO ID NO 45, encoded by SEO ID NO 46.

Function of I\/Iic-DAIR Any suitable domain architecture may be used as long 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 endo- cytic 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 19 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 in- tensity 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 Aß1-42 (500 nM) for 2 hours at 37° C. Cells are then washed with PBS, fixated with 4% formalde- hyde for 10 minutes and imaged using a fluorescence microscope, as disclosed in the Ex- amples.

Endocytic activity may be specific meaning that microglial cells expressing Mic-DA|Rs will have higher endocytic activity towards the neurogenerative disease antigen than for in- stance 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 neurodegenera- tive disease antigen. The percentage is determined as (increase of activity /activity to- wards scrambled peptide x 100). I\/|ethods for detecting specific activity towards amyloid beta vs a scrambled peptide are disclosed in the Examples. ln some embodiments a plurality of the host cells, preferably at least 50%, more prefera- bly 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. ln various aspects of the invention there is provided polynucleotides that encode a I\/|ic- 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. lt 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 Molecu- lar Biology John Wiley & Sons. Nucleic acids may be produced synthetically.

The ski||ed 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: SEO ID NO 41 encoding SEO ID NO 22 (I\/Iic- DAIR3), SEQ ID NO 42 encoding SEQ ID NO 23 (Mic-DAIR4), SEQ ID NO 47 encoding SEQ ID NO 24, SEQ ID NO 48 encoding SEQ ID NO 25 and SEQ ID NO 49 encoding SEQ ID NO 26 and parts of these polynucleotide sequences.

The nucleotide sequences provided herein should be seen as examples only since the ge- netic 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 se- quence 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-un- specific control sequence is able to induce expression ofthe I\/Iic-DAIR in microglial cells. An example of such an expression control sequence is the CD68 promotor, AIF-1/Iba-1, 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 se- quences for TM EM 119 and HEXB.

A polynucleotide encoding a I\/Iic-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, se- quences for antibiotic resistance and sequences for inducing expression of I\/Iic-DAIR pro- teins such as expression control sequences, and the like, as is known in the art of molecu- lar biology. It is referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold 21 Spring Harbor Laboratory Press, 2012 edition, for details and Ausubel et al. Current proto- cols 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.

A 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 polynucle- otide.

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 I\/|oloney murine leukemia virus (I\/ILV)-derived vectors, and include more complex retrovi- rus-derived vectors, e.g., lentivirus-derived vectors or HIV-1-derived vectors.

Other examples include lentivirus vectors derived from H|V-2, FIV, equine infectious ane- mia 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. lmmunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 1411155, 2003; Frechaet al., I\/|ol. Ther. 18: 1748, 2010; Verhoeyen et al., I\/|ethods Mol.. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and ex- pression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus- based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from 22 herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky etal., Gene Ther. 5: 1517, 1998). Other vectors include those de- rived 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 R, Zhou Y, Yan T, Wang R, Li H, et al. 2022. Directed evolution of adeno-associated virus for efficient gene delivery to microglia. Nat Methods 19: 976-85.

Host cells By introducing a polynucleotide, a vector or a virus comprising a polynucleotide into mi- croglial cells or their precursors, the cells can be made to express the disclosed I\/lic-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. ln a population of host cells, preferably, preferably at least 50%, more preferably at least 70% more preferably at least 80% and most preferably at least 90 % or 95 % ofthe host cells express the I\/lic-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 I\/lic-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. 23 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. I\/|icroglial 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 CRlSPR/Cas9 may be used. ln 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 differ- entiated to show microglia-like phenotype. Starting cells may be isolated from a subject with methods known in the art. lnduction of pluripotency may be achieved by ectopic expression of genes encoding a de- fined set oftranscription factors (e.g. OCT4, KLF4, SOX2, cMYC) in the starting cell. The starting cell may be an induced pluripotent stem cell (iPSC) (Bodda C, Reinert LS, Fruh- wurth S, Richardo T, Sun C, et al. 2020. HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.J Exp I\/|ed 217). ln some embodiments the starting cell is a monocyte.

Differentiation of starting cells into microglia cells or microglia-like cell, such as a micro- glial precursor cell, may involve the use of various combinations of growth factors (e.g. TGF-ßl, GI\/I-CSF, I\/I-CSF, IL-34, cholesterol, CDZOO, 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. 24 ln some embodiments, microglia cells or precursors thereof, such as starting cells, are iso- lated 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 in- troducing the cells introduced into the CNS, for example by injecting them into the CNS, for example, by injecting the cells into the brain of the patient. ln some embodiments a polynucleotide, a vector or a virus particle is delivered to the pa- tient, preferably to the glia cells of the patient to make the cells of the patient express the l\/lic-DAIR.

Pharmaceutical composition A pharmaceutical composition may comprise a I\/lic-DAIR protein, polynucleotide, a vector, virus particles, or host cells and in addition a pharmaceutically acceptable excipient. Vari- ous suitable pharmaceutical compositions are known in the art. The pharmaceutical com- position is adapted to its contents, such as a polynucleotide, a vector, virus particles or host cells. The pharmaceutical composition is adapted to the mode of administration. Var- ious modes of administration are described herein.

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. ln some embodiments the pharma- ceutical composition may be desiccated. Examples of excipients include diluents, adju- vants, carriers, stabilizers or vehicles such as for example buffers, salts, nutrients, prote- ases, preservatives, antibiotics, fungicides, stabilizers, glycerol, dextrose, PEG, lipids, lipo- somes, microspheres, nanoparticles or nanospheres.

Treatment A I\/lic-DAIR protein, a polynucleotide, a vector a virus particle, a host cell, a population of host cells or a pharmaceutical composition may be used for the treatment of a neuro- degenerative 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 polynu- cleotide, a vector a virus, a host cell or a population of host cells is administered to a sub- ject. 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 1010 cells.

The subject may be a person suffering from a neurodegenerative disease, which, in a pre- ferred 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. ln 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, intraperitoneallyor intranasaly). The protein may be selectively targeted to microglia/macrophages through the use of microglia specific ex- pression control sequences such as microglia specific-promoters as described herein. ln 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 vari- OUS FOUteS. lt 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 lim- ited to the described embodiments, but can be varied within the scope ofthe enclosed 26 claims. While the invention has been described with reference to specific exemplary em- bodiments, the description is in general only intended to illustrate the inventive concept and should not be taken as limiting the scope ofthe invention. The invention is generally defined by the claims.

EXAl\/I PLES EXAl\/IPLE 1: GENERATION OF l\/IICROGLIAL DISEASE ANTIGEN INTERNALIZATION RECEPTORS 3 AND 4 To generate microglial disease antigen internalization receptors (I\/Iic-DAIR) amino acid se- quences from extracellular, transmembrane- and intracellular domains of human and mouse activating fragment crystallizable (Fc) receptors were back-translated into codon- optimized polynucleotide sequences. To confer antigen-specificity toward the beta-amy- loid (AB) protein, a single-chain fragment variable (scFv) domain (SEO ID NO 32) derived from the monoclonal anti-AB antibody Aducanumab (BIIBO37, SEO ID NO 50 and 51) was used. I\/Iic-DAIR3 (SEO ID NO 22) was constructed by inserting the scFv of aducanumab in between the signal peptide (SEO ID NO 1) and hinge domain (SEO ID 2:) ofthe common Fc receptor gamma chain (FcRy) (FIG. 2). The transmembrane (SEO ID NO 3 :) and intracellu- lar (SEO ID NO 4) domains of FcRy (SEO ID NO 27) were used. Mic-DAIR4 was constructed by inserting an scFv derived from aducanumab (SEO ID NO 32) in between the signal pep- tide (SEO ID NO 5) and a truncated segment ofthe extracellular domain (SEO ID NO 6) of Fc gamma receptor I (FcyRI) (SEO ID NO 28) (FIG. 2). The transmembrane (SEO ID NO 7) and intracellular (SEO ID NO 8) domains of FcyRI (SEO ID NO 28) were used. Polynucleotide sequences encoding I\/Iic-DAIR3 (SEO ID NO 41) and Mic-DAIR4 (SEO ID NO 42) were syn- thesized and cloned into expression plasmids for packaging of genes in recombinant adeno-associated virus (rAAV) particles. 27 EXAl\/IPLE 2: EXPRESSION OF l\/IICROGLIAL DISEASE ANTIGEN INTERNALIZATION RECEPTORS IN CELL CULTURES To visualize transgene expression of Mic-DAIRs in cells a gene encoding a fluorescent tag (e.g. EGFP, mScarlet) was added as part ofthe single open reading frame downstream of a self-cleaving 2A peptide sequence. rAAV particles containing transgenes were applied overnight to human-derived microglia-like (I\/IGL) cells. Transgene expression was evalu- ated 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 I\/Iic-DAIRs, but not receptors constructed from CD8- and CD28-based constructs, were able to generate transgene expression in cultures.

EXAl\/IPLE 3: TESTING OF ENDOCYTIC PERFORMANCE IN HUl\/IAN l\/IICROGLIA-LIKE CELLS MGL cells 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 GI\/I-CSF. Transgene expression was obtained by incubating cultures with rAAV particles as per example 2. rAAV particles encoding EGFP/mScarlet alone was used as viral control. The ability of Mic-DAIRs to endocytose Aß1-42 was tested 5 days post transduction as compared to viral control and three candidate constructs based on domains from other endogenous phagocytic receptors in microglia (FIG. 2, FIG. 4-5). To visualize endocytosis, cells were incubated with 500 nM fluorescent Aß1-42 for 2 hours at 37° C, washed with PBS, fixated with 4% formaldehyde for 10 minutes and imaged using a fluorescence micro- scope.

To test the specificity of endocytosis, carboxyl-modified fluorescent polystyrene beads (1 pm diameter) were coated with Aß1-42, or a scrambled Aß peptide, for 2 hours in 2.5x ex- cess of the calculated monolayer. Preparations were centrifuged, washed and applied to human iPSC-derived microglia (3 x 104 cells per well) expressing EGFP, I\/Iic-DAIR3 and I\/Iic- DAIR4 (FIG. 6-7) at 1 x 106 beads per well in 96-well plates and incubated overnight at 37° 28 C. Following PBS wash and formaldehyde fixation the number of endocytosed beads per EGFP+ cell was examined using a fluorescence microscope. ln summary, results from the two separate assays show that I\/lic-DAIR3 and 4 facilitated an increased Aß1-42-specific endocytosis by human MGL cells.

EXAl\/IPLE 4: TESTING OF ENDOCYTIC PERFORMANCE IN PRll\/IARY l\/IOUSE l\/IICROGLIA 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 2017). A newly developed rAAV capsid (Lin et al 2022) was used to efficiently deliver genes encoding murine versions of I\/lic-DAIR3 (SEQ ID NO 43 and 44) and Mic-DAIR4 (SEQ ID NO 45 and 46) to primary cells alongside viral control (mScarlet) overnight. The ability of Mic-DA|Rs to increase endocytosis of Aß was tested 5 days post transduction by application of fluorescent labelled Aß1-42 (500 nM) 2 hours at 37° C (FIG. 8). 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 Aß endocytosis in primary microglia, cells were incubated with ei- ther fluorescent-labelled Aß1-42 or scrambled Aß peptide (both at 500 nM concentration) for 2 hours at 37° C. I\/|icroglia were then washed, trypsinized and resuspended in FACS buffer for flow cytometry analysis using a BD LSR Fortessa instrument. Uptake of Aß1-42 vs scrambled control was analyzed in single cells (2 2000 cells per sample) as mean fluo- rescence intensity in samples consisting of non-transduced cells, mScarlet viral controls, I\/lic-DAIR3 and Mic-DAIR4 (FIG. 9-10). ln conclusion, results obtained in primary mouse microglia show that mic-DAIR3 and 4 promote an increase in Aß1-42-specific endocytosis.

EXAl\/IPLE 5: l\/IICROGLIAL EXPRESSION OF DISEASE ANTIGEN INTERNALIZATION RECEPTOR 3 AND 4 IN THE l\/IOUSE BRAIN To target delivery microglia in the live mouse brain, genes encoding mScarlet, I\/lic-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 29 (Lin et al 2022). Transgenic mice expressing inducible Cre-recombinase under the micro- glia-specific TM EM 119 promoter (TI\/|EI\/|119-CreERT2, JAX stock #031820) were used to achieve selective gene recombination in microglia. TI\/|EI\/|119-CreERT2 mice were bilater- ally 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 trans- cardiac perfusion with 4% formaldehyde was performed 10-14 days following tamoxifen treatment. 30 um coronal sections were cut on a vibratome and immunostained with Iba- 1 to confirm that expression of I\/lic-DAIR occurred in microglia (FIG. 11).

Bodda C, Reinert LS, Fruhwurth S, Richardo T, Sun C, et al. 2020. HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection. J Exp Med 217 Bohlen CJ, Bennett FC, Tucker AF, Collins HY, I\/lulinyawe SB, Barres BA. 2017. Diverse Requirements for I\/|icroglial Survival, Specification, and Function Revealed by Defined-Medium Cultures. Neuron 94: 759-73 e8 Lin R, Zhou Y, Yan T, Wang R, Li H, et al. 2022. Directed evolution of adeno-associated virus for efficient gene delivery to microglia. Nat Methods 19: 976-85

Claims (9)

Claims

1. A protein comprising an amino acid sequence selected from SEO ID NO 33-37 or an amino acid sequence with at least 90% identity to one of SEO ID NO 33-37, the pro- tein in addition comprising an extracellular binding domain that is capable of bind- ing a neurodegenerative disease antigen. 2. The protein according to claim 1 where the protein comprises the amino acid se- quence of SEO ID NO 34 or an amino acid sequence with at least 90 % identity to

2. SEQID NO

3. The protein according to claim 1 or 2 comprising a single intracellular endocytosis triggering domain.

4. The protein according to any one of claims 1 to 3 where the binding domain com- prises SEQ ID NO 32 or an amino acid sequence with at least 90% identity to SEO ID NO

5. The protein of any one of claim 1 to 4 where the protein comprises SEO ID NO 22, 23, 24, 25 or 26 or a sequence which is at least 90% identical to one of the se- quences SEQ ID NO 22, 23, 24, 25 or

6. The protein according to claim 5 where the sequence is SEQ ID NO 23 or a se- quence which is at least 95% identical to SEQ ID NO

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

8. A vector comprising a polynucleotide according to claim

9. A virus particle comprising the polynucleotide of claim 6 or the vector of claimA host cell comprising a protein according to any one of claims 1 to 6, a polynucle- otide according to claim 7, a vector according to claim 8, or a virus particle accord- ing to claim A population of host cells according to claim A method oftreating a subject comprising administering, to a subject, protein ac- cording to any one of claims 1 to 6, a polynucleotide according to claim 7, a vector according to claim 8, or a virus particle according to claim 9 or a host cell according to claim 10 or a population of host cells according to claim The method of treatment according to claim 12 where the disease Alzheimer's dis- 6856.