Compositions and methods for treating neurodegenerative diseases

Abstract

Compositions and methods for the treatment of neurodegenerative diseases are provided. Aspects of the present disclosure provide for a chimeric antigen receptor (CAR) constructs including: an antigen-binding domain; a hinge; a linker; a transmembrane protein (e.g., CD28, CD8); and a phagocytosis-inducing intracellular signaling domain. In some embodiments, the CAR includes a Dectin1 sequence. In some embodiments, the CAR construct includes a Megf10 sequence.

Description

[0001] Docket No.: 019794 / US4

[0002] COMPOSITIONS AND METHODS FOR TREATING NEURODEGENERATIVE DISEASES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority from U.S. Provisional Application Serial No. 63 / 681 ,513 filed 09 August 2024, and from U.S. Provisional Application Serial No. 63 / 777,299 filed 25 March 2025, which are incorporated herein by reference in their entireties.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

[0006] MATERIAL INCORPORATED-BY-REFERENCE

[0007] The Sequence Listing, which is a part of the present disclosure, includes a computer- readable form comprising nucleotide and / or amino acid sequences of the present invention (file name “019794-WO_2025-08-08_Sequence-Listing.xml” created on 06 August 2025; 67,744 bytes). The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

[0008] FIELD

[0009] The present disclosure generally relates to treatment of neurodegenerative diseases, disorders, or conditions using antigen-targeted CAR-expressing constructs.

[0010] SUMMARY

[0011] Use of CARs for the treatment of protein aggregation and neurodegenerative diseases is unexplored to date.

[0012] Among the various aspects of the present disclosure is the provision of compositions and methods for the treatment of neurodegenerative diseases, disorders, and conditions.

[0013] An aspect of the present disclosure provides for a chimeric antigen receptor (CAR) construct comprising or a construct encoding a CAR construct: an antigen-binding domain (e.g., an scFv); optionally, a hinge; optionally, a linker; optionally, a transmembrane protein (e.g., CD28, CD8); or a phagocytosis-inducing intracellular signaling domain. In some embodiments, the antigen-binding domain targets protein aggregates and / or the Docket No.: 019794 / US4 phagocytosis-inducing intracellular signaling domain induce phagocytosis upon oligomerization.

[0014] Another aspect of the present disclosure provides for an expression vector capable of expressing the CAR construct of claim 1 in a host cell.

[0015] An additional aspect of the present disclosure provides for a method of inducing phagocytosis of a protein aggregate (e.g., amyloid-|3 peptides) comprising expressing the protein aggregate-targeting, phagocytosis-inducing CAR in a phagocyte (e.g., microglia, astrocyte). In some embodiments, the phagocyte expressing the protein aggregate-targeting, phagocytosis-inducing CAR is capable of phagocytosing the targeted protein aggregate.

[0016] A further aspect of the present disclosure provides for a method of clearing diseasecausing or pathogenic protein aggregates (e.g., amyloid-[3 peptides) in a subject having a neurodegenerative disease disease-causing or pathogenic protein aggregates (e.g., Alzheimer’s disease (AD) or Cerebral Amyloid Angioapathy (CAA)) comprising expressing the CAR of any one of the preceding claims in a phagocyte or introducing the CAR- expressing phagocyte to a protein aggregate.

[0017] Yet another aspect of the present disclosure provides for a method of clearing amyloid-[3 peptides comprising administering CAR-expressing brain-resident phagocytes to a subject having an A|3 associated disease, disorder, or condition (e.g., AD or Cerebral Amyloid Angioapathy (CAA)). In some embodiments, the CAR is a protein aggregatetargeting, phagocytosis-inducing CAR.

[0018] Yet an additional aspect of the present disclosure provides for a method of treating a neurodegenerative disease associated with accumulation of disease-causing or pathogenic proteins, comprising: administering an antigen-targeted, phagocytosis inducing CAR- expressing brain-resident phagocyte to a subject having a neurodegenerative disease associated with accumulation of disease-causing or pathogenic proteins, or a significant risk of contracting such a disease.

[0019] Yet a further aspect of the present disclosure provides for a method of phagocyte (e.g., microglia, astrocyte) replacement in a subject, comprising: depleting resident phagocytes (e.g., microglia), optionally with an MCSF-R inhibitor (e.g., such as PLX5622); and / or administering an antigen-targeted, phagocytosis inducing CAR transduced brain- Docket No.: 019794 / US4 resident phagocytes (e.g., microglia) to the brain of the subject to repopulate the brain phagocytes.

[0020] Still another aspect of the present disclosure provides for a method of generating phagocytosis-inducing, protein aggregate-targeting chimeric antigen receptor (CAR) phagocyte cells comprising: providing phagocyte cells; and / or transducing a phagocytosisinducing, protein aggregate-targeting CAR via a viral vector or non-viral particle into the phagocyte cells for an amount of time sufficient to virally transduce the phagocytosisinducing, protein aggregate-targeting CAR into the phagocyte cells, resulting in CAR- transduced phagocyte cells.

[0021] Still an additional aspect of the present disclosure provides for a method of generating phagocytosis-inducing, protein aggregate-targeting chimeric antigen receptor (CAR) phagocyte cells comprising: providing phagocyte cells; and / or transducing a phagocytosisinducing, protein aggregate-targeting CAR via a non-viral particle or lipid nanoparticle (LNP) into the phagocyte or phagocyte precursor cells for an amount of time sufficient to transduce the phagocytosis-inducing, protein aggregate-targeting CAR into the phagocyte cells, resulting in CAR-transduced phagocyte cells. In some embodiments, the viral vector comprises a chimeric antigen receptor (CAR) is a CAR lentivirus (LVV) or CAR adeno- associated viral (AAV) vector. In some embodiments, the viral vector is a lentiviral vector (LVV).

[0022] Still a further aspect of the present disclosure provides for a method of administering phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells to a subject in need thereof comprising: isolating phagocyte cells from a subject or a donor; generating phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells according to any one of the disclosed aspects or embodiments; and / or administering a therapeutically effective amount of phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells into the subject. In some embodiments, the CAR construct comprises an antigen binding domain or the antigen-binding domain comprises an A|3 binding domain. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv is derived from (e.g., a truncated version or variant thereof) one or more Amyloid B binding antibodies (e.g., Bapineuzumab, Crenezumab, Lecanemab, among others). In some embodiments, the scFv is derived from (e.g., a truncated version or variant Docket No.: 019794 / US4 thereof) one or more Amyloid beta binding antibodies that bind to Amyloid beta fibril present in CAA(Soederberg et al, Nature, 2024). In some embodiments, the scFv is fused to the hinge or transmembrane domains from stable or non-dimerizing proteins (e.g., CD8, CD28). In some embodiments, the antigen-binding domain is an scFv derived from the antigen binding fragment (Fab) region of Crenezumab,Bapineuzumab or Lecanemab. In some embodiments, the A|3 binding domain comprises at least a portion, a fragment, or a variant of an o-Ap antibody. In some embodiments, the scFv, the hinge (if present), the transmembrane protein (if present), and the phagocytosis-inducing intracellular signaling domain are operably linked. In some embodiments, the scFv comprises a heavy (H) chain Ig domain and / or a light (L) chain Ig domain and / or a linker peptide. In some embodiments, the H or L chain is derived from at least a portion, fragment, or variant of Crenezumab,Bapineuzumab or Lecanemab antigen-binding fragments (Fabs). In some embodiments, at least a portion, fragment, or variant of Crenezumab comprises a portion that interacts with A|3. In some embodiments, the portion or variant thereof of the Crenezumab comprises the heavy chain 13-16 N-terminus. In some embodiments, the Ig domain is truncated at the terminal p-sheet. In some embodiments, the hinge and / or the transmembrane domain, if present, supplies stability constraint. In some embodiments, the hinge and / or the transmembrane domain, if present, are from or derived from stable and / or non-dimerizing proteins (e.g., CD8, CD28). In some embodiments, the intracellular domain is fused or operably linked to a linker, an extracellular domain, or a hinge. In some embodiments, the intracellular domain is a phagocytosis-inducing protein. In some embodiments, the intracellular signaling domain is Mertk, CD3^, MegflO, Dectinl , or CD19 PI3K. In some embodiments, the intracellular signaling domain is phagocytosis-inducing protein, such as AXL, TYRO3, MEGF10, DECTIN1 , MER, CD3 , Fc, CD64, or Fc receptor. In some embodiments, the intracellular signaling domain is Megfl O. In some embodiments, the intracellular signaling domain is Dectinl . In some embodiments, the intracellular signaling domain induces phagocytosis of Ap upon crosslinking (or binding to AP). In some embodiments, the brain-resident phagocyte is a macrophage, microglial cell, or astrocyte. In some embodiments, the brain-resident phagocyte is an autologous microglial cell. In some embodiments, the brain-resident phagocyte is a natural phagocyte, a native phagocyte, or a donor phagocyte. In some embodiments, the protein aggregate is pathological or associated Docket No.: 019794 / US4 with a neurodegenerative disease. In some embodiments, the neurodegenerative disease, disorder, or condition is associated with accumulation of a pathological protein, such as Amyloid B (A|3), hyperphosphorylated Tau, alpha-synuclein, or TDP-43. In some embodiments, the neurodegenerative disease is Alzheimer;s Disease, Parkinson's Disease, dementia with Lewy bodies and / or multiple systems atrophies, or Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the neurodegenerative disease is Cerebral Amyloid Angioapathy (CAA). In some embodiments, the CAR is expressed in a brain-resident phagocyte (e.g., macrophages, astrocytes, microglia).. In some embodiments, the CAR is transduced with a CAR-coding retrovirus (e.g., lentivirus) or an adeno-associated viral (AAV) vector or non-viral particle that genetically codes for a CAR of any one of the preceding claims. In some embodiments, the CAR is transduced with a CAR-coding nucleic acid using a nonviral particle or Lipid Nanoparticle (LNP). In some embodiments, the CAR is transduced with a CAR-coding RNA using a nonviral nanoparticle or Lipid Nanoparticle (LNP). In some embodiments, the CAR is transduced with a CAR-coding DNA using a nonviral nanoparticle or Lipid Nanoparticle (LNP). In some embodiments, the expression of CAR is achieved by lentiviral transduction of brain-resident phagocytes (e.g., macrophages, microglia, astrocytes). In some embodiments, the CAR targets protein aggregates Amyloid B (A|3), hyperphosphorylated Tau, alpha-synuclein, or TDP-43. In some embodiments, the CAR prevents A|3 deposition; prevents Ap-associated pathology; only requires a single dose; binds A|3; Induce phagocytosis of A|3; and / or is expressed permanently by a brain-resident phagocyte. In some embodiments, CAR-dependent clearance promotes phagocytosis through noninflammatory pathways.

[0023] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0024] DESCRIPTION OF THE DRAWINGS

[0025] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0026] FIG. 1 (A-E) is an exemplary embodiment of in vitro CAR transduction promoting potent phagocytosis in accordance with the present disclosure. FIG. 1A: Schematic representation of designed CARs with different intracellular signaling domains. FIG. 1 B: Docket No.: 019794 / US4

[0027] Confocal imaging of THP-1 cells transduced with the CARs in A fused to GFP, nuclei are stained with DAPI. FIG. 1 C: Immortalized CAR astrocytes were incubated with monomeric Ap-Alexa 647. Two color histograms are shown for fluorescence of GFP and A|3-Alexa 647 binding in live cells. FIG. 1 D: Flow cytometry analysis of oligomeric A[342-pHrodo uptake by immortalized astrocytes. FIG. 1 E: Quantifications of the percentage of oligomeric Ap42- pHrodo+ CAR astrocytes at Oh and 24h post four hours of phagocytosis.

[0028] FIG. 2(A-L) is an exemplary embodiment of transcriptomic and cytokine responses of CAR astrocytes post A[3 oligomer phagocytosis in accordance with the present disclosure. FIG. 2(A-C): Immortalized astrocytes from FIG. 2A: Control-GFP, FIG. 2B: CARA-MEGF10, and FIG. 2C: CARA-DECTIN-1 , were analyzed by bulk RNA sequencing 24 hours post phagocytosis of oligomeric A|3 as described in the material and methods. Volcano plots highlight differentially expressed genes between treated and untreated groups. (D: ELISA of immortalized astrocyte cytokine secretion post A|3 phagocytosis. Heatmap shows fold change in cytokine secretion for CAR constructs (GFP, MEGF10, DECTIN 1 ) with and without oAp42 stimulation. FIG. 2 (E-L): Quantification of cytokine concentrations (pg / mL) in response to oA[342 stimulation for different CAR constructs. Shown are FIG. 2E: CCL3, FIG. 2F: CCL5, FIG. 2G: TNFa, FIG. 2H: TNF Rl, FIG. 2I: MCSF, FIG. 2J: IFNy, FIG. 2K: IL-7, and FIG. 2L: IL-15.

[0029] FIG. 3(A-P) is an exemplary embodiment of AAV-CARA treatment significantly preventing amyloidosis and amyloid-associated pathology in vivo in accordance with the present disclosure. FIG. 3A: Schematic representation of Php.EB.AAV-CAR DNA constructs. FIG. 3B: Experimental timeline for AAV injection and pathological assessment in 5xFAD mice. FIG. 3C: Representative X34 staining of amyloid plaques in cortical sections from mice injected with PhpEB.AAV-EGFP, PhpEB.AAV-Adu-Megf10, or PhpEB.AAV-Adu-Dectin1 at 5 months of age. FIG. 3 (D-E): Quantification of X34+ plaque area in FIG. 3D: cortex and FIG. 3E: hippocampus. FIG. 3F: Representative X34 staining of amyloid plaques in sagittal brain sections. FIG. 3G: Quantification of total X34+ plaque area in sagittal sections. FIG. 3(H-I): ELISA results of A 4O / 42 in the FIG. 3H: PBS soluble or FIG. 3I: GdnHCI soluble cortical tissues of treated 5xFAD mice at 5m of age. FIG. 3J: Representative confocal images of X34+ plaques (blue) and LAMP1 staining (white) in cortex and hippocampus. FIG. 3 (K- L): Quantification of total LAMP1 + voxels within 15pm around X34+ plaques (%) in the FIG. Docket No.: 019794 / US4

[0030] 3K: cortex or FIG. 3L: hippocampus of treated 5xFAD mice at 5m of age. FIG. 3M: Representative images and FIG. 3N: quantification of total Synapsin+ voxels within15pm around X34+ plaques (%) in the cortex of treated 5xFAD mice at 5m of age. FIG. 30: Representative images and FIG. 3P: quantification of total PSD95+ voxels within15pm around X34+ plaques (%) in the cortex of treated 5xFAD mice at 5m of age. Data in FIG. 3(D, E, G-l, K, L, N-P) are presented as mean ± SEM. Statistical significance: *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001 , ns: not significant.

[0031] FIG. 4(A-H) is an exemplary embodiment of differential glial cell responses in two CARA treatments in accordance with the present disclosure. FIG. 4A: Representative images and FIG. 4B: quantification of the percentage of GFAP+ voxels within15pm around X34+ plaques (%) in the cortex of treated 5xFAD mice at 5m of age. FIG. 4C: Representative images and FIG. 4D: quantification of the percentage of LAMP1 within GFAP+ voxels in 63x images containing X34+ plaques in the cortex of treated 5xFAD mice at 5m of age. FIG. 4E: Representative images and FIG. 4F: quantification of the percentage of IBA1 + voxels within 15pm around X34+ plaques (%) in the cortex of treated 5xFAD mice at 5m of age. FIG. 4G: Representative images and FIG. 4H: quantification of the percentage of CD68+ voxels within15pm around X34+ plaques (%) in the cortex of treated 5xFAD mice at 5m of age.

[0032] FIG. 5(A-I) is an exemplary embodiment of snRNAseq analysis of pooled samples showed distinct astrocyte and microglia signature in CARA animals in accordance with the present disclosure. FIG. 5A: The LIMAP of pooled samples from AAV-CAR treated 5xFAD mice. N=5 per group. FIG. 5B: Different cell clusters in the UMAP of pooled samples from AAV-CAR treated 5xFAD mice. N=5 per group. FIG. 5C: WPRE+ nuclei indicating AAV- expression in the astrocytes in the UMAP of pooled samples. N=5 per group. FIG. 5D: The volcano plots of DEGs in astrocytes of CAR-Megf10 or FIG. 5E: CAR-Dectin1 vs. EGFP- control mice. FIG. 5F: The UMAP of astrocyte subclusters in AAV-CAR treated 5xFAD mice compared with GFP controls. FIG. 5G: The volcano plots of DEGs in microglia of CAR- Megf10 or FIG. 5H: CAR-Dectin1 vs. EGFP-control mice. FIG. 5I: The UMAP of microglia subclusters in AAV-CAR treated 5xFAD mice compared with GFP controls.

[0033] FIG. 6(A-B) is an exemplary embodiment of live cell imaging of A|3 uptake by CAR receptor in accordance with the present disclosure. Representative images of live cell imaging on oligomeric A[342-594 colocalized with and phagocytosed by the chimeric antigen Docket No.: 019794 / US4 receptor (CAR-Megf10) at different time points as indicated. FIG. 6A shows images without AP42-594. FIG. 6B shows images with A[342-594.

[0034] FIG. 7(A-B) is an exemplary embodiment of switching of scFv from Crenezumab to Aducanumab prior to in vivo experiments in accordance with the present disclosure. FIG. 7A: The mean fluorescence intensity of oligomeric Ap42-pHrodo uptake by Bapineuzumab- based, Crenezumab-based and Aducanumab-based CAR-Megf10 compared to the GFP control at 4h. FIG. 7B: The mean fluorescence intensity of oligomeric Ap42-pHrodo uptake by Bapineuzumab- based, Crenezumab-based and Aducanumab-based CAR-Megf10 compared to the GFP control at 24h vs. Oh.

[0035] FIG. 8(A-I) is an exemplary embodiment of additional characterization of astrocytes and microglial responses in vivo in accordance with the present disclosure. FIG. 8A: Representative images showing GreenLantern-labeled chimeric antigen receptors localized on astrocytic processes near X34+plaques. White arrows pointed to the CAR expressed by astrocytes. FIG. 8B: Representative images and FIG. 8C: quantification of the percentage of GFAP+voxels within 15pm around X34+plaques (%) in the hippocampus of treated 5xFAD mice at 5m of age. FIG. 8D: Representative images and FIG. 8E: quantification of the percentage of IBA1+voxels within15pm around X34+plaques (%) in the hippocampus of treated 5xFAD mice at 5m of age. FIG. 8F: Representative images and FIG. 8G: quantification of the percentage of APOE+voxels within 15pm around X34+plaques (%) in the hippocampus of treated 5xFAD mice at 5m of age. FIG. 8H: Representative images and FIG. 8I: quantification of the percentage of MHC-II+voxels within IBA1+voxels 15pm around X34+plaques (%) in the cortex of treated 5xFAD mice at 5m of age.

[0036] FIG. 9(A-B). is an exemplary embodiment of heatmap data of representative genes in different astrocyte and microglia clusters in the snRNAseq analysis in accordance with the present disclosure. FIG. 9A: Gene signature of astrocyte clusters in the snRNAseq analysis in FIG. 5F. FIG. 9B: Gene signature of microglia clusters in the snRNAseq analysis in FIG. 5I.

[0037] FIG. 10(A-D) is an exemplary embodiment of additional snRNAseq analysis of oligodendrocytes and ligand-receptor analysis in AAV-CAR treated mice compared to GFP controls in accordance with the present disclosure. FIG. 10A: The volcano plots of DEGs in Docket No.: 019794 / US4 astrocytes of CAR-Megf 10 or FIG. 10B: CAR-Dectin1 vs. EGFP-control mice. FIG. 10C: The ligand(astrocyte)-receptor(microglia) analysis in CAR-Megf10 or FIG. 10D: CAR-Dectin1 vs. EGFP- control mice.

[0038] FIG. 11 (A-B) is an exemplary embodiment of a Cre-CD8aTMIong-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 11 A: nucleotide sequence (SEQ ID NO: 3). FIG. 11 B: amino acid sequence (SEQ ID NO: 4).

[0039] FIG. 12(A-B) is an exemplary embodiment of a Adu-CD8aTM-Dectin1 CAR construct in accordance with the present disclosure. FIG. 12A: nucleotide sequence (SEQ ID NO: 5). FIG. 12B: amino acid sequence (SEQ ID NO: 6).

[0040] FIG. 13(A-B) is an exemplary embodiment of a Cre-CD8aTM-MERTK CAR construct in accordance with the present disclosure. FIG. 13A: nucleotide sequence (SEQ ID NO: 7). FIG. 13B: amino acid sequence (SEQ ID NO: 8).

[0041] FIG. 14(A-B) is an exemplary embodiment of a Cre-CD8aTM-Megf10(long) CAR construct in accordance with the present disclosure. FIG. 14A: nucleotide sequence (SEQ ID NO: 9). FIG. 14B: amino acid sequence (SEQ ID NO: 10).

[0042] FIG. 15(A-B) is an exemplary embodiment of a Cre-CD8aTMIong-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 15A: nucleotide sequence (SEQ ID NO: 11 ). FIG. 15B: amino acid sequence (SEQ ID NO: 12).

[0043] FIG. 16(A-B) is an exemplary embodiment of a Cre-CD8aTM-Dectin1 CAR construct in accordance with the present disclosure. FIG. 16A: nucleotide sequence (SEQ ID NO: 13). FIG. 16B: amino acid sequence (SEQ ID NO: 14).

[0044] FIG. 17(A-B) is an exemplary embodiment of a Cre-CD8aTM-CD3^ CAR construct in accordance with the present disclosure. FIG. 17A: nucleotide sequence (SEQ ID NO: 15). FIG. 17B: amino acid sequence (SEQ ID NO: 16).

[0045] FIG. 18(A-B) is an exemplary embodiment of a Adu-CD8aTMIong-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 18A: nucleotide sequence (SEQ ID NO: 17). FIG. 18B: amino acid sequence (SEQ ID NO: 18).

[0046] FIG. 19(A-B) is an exemplary embodiment of a Adu-CD8aTM-Dectin1 CAR construct in accordance with the present disclosure. FIG. 19A: nucleotide sequence (SEQ ID NO: 19). FIG. 19B: amino acid sequence (SEQ ID NO: 20). Docket No.: 019794 / US4

[0047] FIG. 20(A-B) is an exemplary embodiment of a Bap-CD8aTM-Megf10(long) CAR construct in accordance with the present disclosure. FIG. 20A: nucleotide sequence (SEQ ID NO: 21 ). FIG. 20B: amino acid sequence (SEQ ID NO: 22).

[0048] FIG. 21 (A-B) is an exemplary embodiment of a Cre-CD8aTM-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 21 A: nucleotide sequence (SEQ ID NO: 23). FIG. 21 B: amino acid sequence (SEQ ID NO: 24).

[0049] FIG. 22(A-B) is an exemplary embodiment of a Adu-CD8aTM-MERTK CAR construct in accordance with the present disclosure. FIG. 22A: nucleotide sequence (SEQ ID NO: 25). FIG. 22B: amino acid sequence (SEQ ID NO: 26).

[0050] FIG. 23(A-B) is an exemplary embodiment of a Adu-CD8aTM-Megf10(long) CAR construct in accordance with the present disclosure. FIG. 23A: nucleotide sequence (SEQ ID NO: 27). FIG. 23B: amino acid sequence (SEQ ID NO: 28).

[0051] FIG. 24(A-B) is an exemplary embodiment of a Adu-CD8aTMIong-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 24A: nucleotide sequence (SEQ ID NO: 29). FIG. 24B: amino acid sequence (SEQ ID NO: 30).

[0052] FIG. 25(A-B) is an exemplary embodiment of a Adu-CD8aTM-Megf10(short) CAR construct in accordance with the present disclosure. FIG. 25A: nucleotide sequence (SEQ ID NO: 31 ). FIG. 25B: amino acid sequence (SEQ ID NO: 32) .

[0053] FIG. 26(A-B) is an exemplary embodiment of a Adu-CD8oTM-CD3 CAR construct in accordance with the present disclosure. FIG. 26A: nucleotide sequence (SEQ ID NO: 33). FIG. 26B: amino acid sequence (SEQ ID NO: 34).

[0054] FIG. 27(A-D) is an exemplary embodiment of AD statistical data in accordance with the present disclosure. FIG. 27A shows AD risk by age. FIG. 27B shows birth and birth rate data for AD at-risk population. FIG. 27C shows an increasing trend in AD occurrence. FIG. 27D shows an increasing trend in cause of death due to AD.

[0055] FIG. 28(A-B) is an exemplary embodiment of amyloid targeting in accordance with the present disclosure. FIG. 28A illustrates the amyloid hypothesis. FIG. 28B non-amyloidogenic and amyloidogenic targets.

[0056] FIG. 29(A-F) is an exemplary embodiment of amyloid-targeted treatment to date in accordance with the present disclosure. FIG. 29A shows A|3 treatment in mice. FIG. 29B shows A|3 treatment in human samples. FIG. 29C shows worsening primary outcomes in Docket No.: 019794 / US4 some treatment cases. FIG. 29D shows worsening secondary outcomes in some treatment cases. FIG. 29E lists clinical trial antibodies. FIG. 29F shows Ap-targeted therapy challenges.

[0057] FIG. 30(A-B) is an exemplary embodiment of aA|3 CAR properties in accordance with the present disclosure. FIG. 30A shows ideal characteristics of aAp CARs. FIG. 30B shows scFv region design elements.

[0058] FIG. 31 (A-D) is an exemplary embodiment of CAR design embodiment considerations with Crenezumab in accordance with the present disclosure. FIG. 31 A shows Crenezumab Fragment antigen-binding (Fab). FIG. 31 B shows Ig domains were truncated at the terminal P-sheet. FIG. 31 C shows Crenezumab’s heavy and light chain N-terminus. FIG. 31 D shows Crenezumab’s front and back heavy chain N-terminus interaction with A(3.

[0059] FIG. 32 is an exemplary embodiment of phagocytosis induction upon crosslinking in accordance with the present disclosure.

[0060] FIG. 33 is an exemplary embodiment of control of plaque burden in AD by microglia in accordance with the present disclosure.

[0061] FIG. 34 is an exemplary embodiment of a proposed Ap CAR design in accordance with the present disclosure.

[0062] FIG. 35(A-H) is an exemplary embodiment of proper CAR protein expression, folding, and targeting of the membrane in accordance with the present disclosure. FIG. 35A shows Lentiviral infection of murine microglial cells with Ap CARs. FIG. 35B shows Ap CAR expression in murine microglia. FIG. 35C shows intracellular Ap CAR distribution in HEK. FIG. 35D shows additional intracellular Ap CAR distribution in HEK. FIG. 35E shows intracellular Ap CAR distribution in HMC3. FIG. 35F shows additional intracellular Ap CAR distribution in HMC3. FIG. 35G shows CAR protein expression in HEK. FIG. 35H shows CAR 3 GFP-fusion protein confirms membrane localization.

[0063] FIG. 36 is an exemplary embodiment of Crenezumab scFv binds Ap in accordance with the present disclosure.

[0064] FIG. 37(A-E) is an exemplary embodiment of proper CAR treatment resulting in phagocytosis in accordance with the present disclosure. FIG. 37A shows HMC3 CAR 3 phagocytosis. FIG. 37B shows HEK CAR 3 phagocytosis. FIG. 37C shows live-cell imaging Ap uptake. FIG. 37D shows additional live-cell imaging Ap uptake. FIG. 37E shows further live-cell imaging Ap uptake. Docket No.: 019794 / US4

[0065] FIG. 38(A-G) is an exemplary embodiment of additional in vitro, ex vivo, and in vivo testing in accordance with the present disclosure. FIG. 38A shows single intravenous CAR AAV administration at 2m significantly prevents amyloid plaque deposition (5m). FIG. 38B shows region-specific plaque analysis. FIG. 38C shows CAR-7 and CAR-11 promoted astrocyte clustering around plaques. FIG. 38D shows CAR-7 and CAR-11 promoted microglia clustering around plaques. FIG. 38E shows neuronal dystrophy is reduced by CAR- 7 and CAR-11 . FIG. 38F shows astrocyte lysosomal activity is higher in CAR-7 and CAR-11 . FIG. 38G shows improved behavior is associated with CAR treatment.

[0066] DETAILED DESCRIPTION

[0067] The present disclosure is based, at least in part, on the discovery that CARs can be designed to express on a brain-resident phagocyte, target A|3, and induce phagocytosis. As shown herein, Megfl O and Dectinl CARs fold appropriately, are transported to the membrane, and promote phagocytosis.

[0068] It is presently believed that no one has published the use of CARs for the treatment of protein aggregation and neurodegenerative diseases.

[0069] CHIMERIC ANTIGEN RECEPTOR (CAR) CONSTRUCTS

[0070] The present disclosure provides for cells modified with CARs. It is believed that the present disclosure is the first to design these CAR constructs capable of being incorporated into brain-resident phagocytes to target and phagocytose pathological brain protein aggregates such as Amyloid B (A(3) or hyperphosphorylated Tau in Alzheimer’s Disease, alpha-synuclein in Parkinson Disease, dementia with Lewy bodies and multiple systems atrophies, TDP-43 in Amyotrophic Lateral Sclerosis, etc.

[0071] CARs can be designed in a modular fashion that comprises an extracellular target binding domain, a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and / or one or more intracellular domains (or extracellular domain) that transmit activation signals. Depending on the number of costimulatory domains, CARs can be classified into first (CD3z only), second (one costimulatory domain + CD3z), or third generation CARs (more than one costimulatory domain + CD3z). Introduction of CAR Docket No.: 019794 / US4 molecules into brain-resident phagocyte cells successfully redirects the cells with additional antigen specificity and provides the necessary signals to drive full phagocytosis activation.

[0072] Because antigen recognition by brain-resident phagocyte cells (is based on the binding of the target-binding single-chain variable fragment (scFv) to intact surface antigens, targeting of aggregate proteins is not restricted.

[0073] Furthermore, the CAR construct moieties can be operably linked with a linker. A linker can be any nucleotide sequence capable of linking the moieties described herein. For example, the linker can be any amino acid sequence suitable for this purpose (e.g., of a length of 9 amino acids).

[0074] Antigen-binding Domain

[0075] As described herein, the CAR construct can comprise an antigen-binding domain or target aggregates such as Amyloid B (A[3), hyperphosphorylated Tau, alpha-synuclein, or TDP-43.

[0076] The antigen-binding domain can comprise any domain that binds to or has an affinity to a pathological aggregate associated with neurodegenerative diseases, disorders.

[0077] The antigen-binding domain can comprise an scFv, antibody, antibody fragment, or functional fragment or functional variant thereof having a percent identity to an antibody or fragment thereof having or retaining antigen-binding function or activity. For example, antigen-binding domain can comprise about 40%; about 41 %; about 42%; about 43%; about 44%; about 45%; about 46%; about 47%; about 48%; about 49%; about 50%; about 51 %; about 52%; about 53%; about 54%; about 55%; about 56%; about 57%; about 58%; about 59%; about 60%; about 61 %; about 62%; about 63%; about 64%; about 65%; about 66%; about 67%; about 68%; about 69%; about 70%; about 71 %; about 72%; about 73%; about 74%; about 75%; about 76%; about 77%; about 78%; about 79%; about 80%; about 81 %; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91 %; about 92%; about 93%; about 94%; about 95%; about 96%; about 97%; about 98%; about 99%; or about 100% identity to a functional fragment of an antibody targeted to a protein aggregate.

[0078] Single-chain variable fragments (scFvs)

[0079] Here, a single-chain variable fragment (scFv) can be used to bind antigens. Targeting antibody fragments or scFvs, as described herein, can be against any neurodegenerative Docket No.: 019794 / US4 disease-associated antigen (NDAA). An NDAA can be any antigen known in the art to be associated with neurodegenerative diseases, such as those associated with accumulation of Amyloid B (A|3), hyperphosphorylated Tau, alpha-synuclein,, TDP-43 , etc. scFvs are well known in the art to be used as a binding moiety in a variety of constructs (see e.g., Sentman 2014 Cancer J. 20 156-159; Guedan 2019 Mol Ther Methods Clin Dev. 12 145-156). Any scFv known in the art or generated against an antigen using means known in the art can be used as the binding moiety.

[0080] The antigen-binding capability of the CAR is defined by the extracellular scFv. The format of an scFv is generally two variable domains linked by a flexible peptide sequence, either in the orientation VH-linker-VL or VL-linker-VH. The orientation of the variable domains within the scFv, depending on the structure of the scFv, may contribute to whether a CAR will be expressed on the cell surface or whether the CAR-expressing cells target the antigen and signal. In addition, the length and / or composition of the variable domain linker can contribute to the stability or affinity of the scFv.

[0081] The scFv, traditionally a critical component of a CAR molecule, can be carefully designed and manipulated to influence specificity and differential targeting of tumors versus normal tissues.

[0082] The antigen-binding capability of the CAR is defined by the extracellular scFv, not the targeted antigen. The format of an scFv is generally two variable domains linked by a flexible peptide sequence, either in the orientation VH-linker-VL or VL-linker-VH. The orientation of the variable domains within the scFv, depending on the structure of the scFv, may contribute to whether a CAR will be expressed on the cell surface or whether the cells target the antigen and signal. In addition, the length and / or composition of the variable domain linker can contribute to the stability or affinity of the scFv. scFvs are well known in the art to be used as a binding moiety in a variety of constructs (see e.g., Sentman 2014 Cancer J. 20 156-159; Guedan 2019 Mol Ther Methods Clin Dev. 12 145-156). Any scFv known in the art or generated against an antigen using means known in the art can be used as the binding moiety.

[0083] CAR scFv affinities, modified through mutagenesis of complementary-determining regions while holding the epitope constant, or through CAR development with scFvs derived from therapeutic antibodies against the same target, but not the same epitope, can change Docket No.: 019794 / US4 the strength of the phagocytotic signaling and allow phagocytes to differentiate overexpressed antigens from normally expressed antigens. The scFv, a critical component of a CAR molecule, can be carefully designed and manipulated to influence specificity and differential targeting of tumors versus normal tissues.

[0084] The scFvs described herein can be used for pathological aggregates such as A|3, but can also be expanded for use in other neurodegenerative diseases where an scFv can be generated against a target antigen or antigen epitope. For example, the constructs described herein can be used to treat or prevent aggregation of pathological aggregate proteins.

[0085] Antibodies

[0086] As another example, the cell can include an antigen-binding domain which can be an antibody (from human, mouse, or other animal), a humanized antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a camelid antibody, a native receptor or ligand, or a fragment thereof. For example, the antigen-binding domain can be a singlechain variable fragment (scFv) of an antibody. The antigen-binding domain can be directed to various disease-associated proteins, which may include Ap, tau, etc., or other proteins found to be more highly enriched in or in proximity to brain cells in subjects having a neurodegenerative disease, disorder, or condition than normal tissues.

[0087] TYPES OF ANTIBODY FRAGMENTS

[0088] Neurodegenerative disease-targeting antibody functional fragments, variants, or recombinant proteins thereof can be made and used clinically by methods known in the art (see e.g., Adam Bates and Christine A. Power, Review, David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments, Antibodies 2019, 8, 28) and can be designed to have the desired function or activity as discovered herein. Antigen-binding domains, as described herein, can comprise an antibody fragment or variant (e.g., a fusion protein, scFv, peptide, recombinant proteins, diabodies, unibodies, etc., or a functional fragment, variant, or mutant (e.g., addition, insertion, deletion, substitution)) that have anti- pathological protein aggregate binding activity.

[0089] F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. These antigen-binding fragments can vary in size (MW), valency, or Fc content. These and several additional unique fragment structures can be Docket No.: 019794 / US4 generated from pentameric IgM, including an "lgG"-type fragment, an inverted 5 "lgG"-type fragment, and a pentameric Fc fragment.

[0090] Scheme 1. The names (nomenclature) and structures of some typical IgG fragments are illustrated in the following diagram and summarized below.

[0091] F(ab')2 fragments

[0092] 10 F(ab')2 (110,000 daltons) fragments contain two antigen-binding regions joined at the hinge through disulfides. This fragment is void of most, but not all, of the Fc region.

[0093] Fab' fragments

[0094] Fab' (55,000 daltons) fragments can be formed by the reduction of F(ab')2 fragments. The Fab' fragment contains a free sulfhydryl group that may be alkylated or

[0095] 15 utilized in conjugation with an enzyme, toxin, or other protein of interest. Fab' is derived from F(ab')2; therefore, it may contain a small portion of Fc.

[0096] Fab fragments

[0097] Fab (50,000 daltons) is a monovalent fragment that is produced from IgG and IgM, consisting of the VH, CH1 , and / or VL, CL regions, linked by an intramolecular disulfide

[0098] 20 bond.

[0099] Fv fragments

[0100] Fv (25,000 daltons) is the smallest fragment produced from IgG and IgM that contains a complete antigen-binding site. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab. The VH and VL chains of

[0101] 25 the Fv fragments are held together by non-covalent interactions. These chains tend to dissociate upon dilution, so methods have been developed to cross-link the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker.

[0102] "rlgG" fragments

[0103] "rlgG" refers to reduced IgG (75,000 daltons) or half-IgG. It is the product of selectively reducing just the hinge-region disulfide bonds. Although several disulfide bonds occur in IgG, those in the hinge region are the most accessible and easiest to reduce, especially with mild reducing agents like 2-mercaptoethylamine (2-MEA). HalflgG can be prepared for the purpose of targeting the exposing hinge-region sulfhydryl groups that can be targeted for conjugation, either antibody immobilization or enzyme labeling.

[0104] Fc fragments Docket No.: 019794 / US4

[0105] Fc (50,000 daltons) fragments contain the CH2 and CH3 region and part of the hinge region held together by one or more disulfides and noncovalent interactions. Fc and Fc5p fragments are produced from fragmentation of IgG and IgM, respectively. The term Fc is derived from the ability of these antibody fragments to crystallize. Fc fragments are generated entirely from the heavy chain constant region of an immunoglobulin. The Fc fragment cannot bind antigen, but it is responsible for the effector functions of antibodies, such as complement fixation.

[0106] Transmembrane (TM) Domain

[0107] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. As described herein, the CAR constructs can comprise a transmembrane domain. The transmembrane domain can functionally link the intracellular domain to the extracellular domain or antigen-binding domain and anchors the CAR to the cell membrane. The transmembrane domain can be derived from a receptor that is found naturally on the surface of a cell, such as brain-resident phagocyte or other cell. The TM domain can be derived from any known or predicted TM domain that effectively allows the receptor to span the cell membrane. For example, the TM domain can be a sequence associated with or be derived from (i.e. , comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the CD3, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD48, CD64, CD80, CD86, CD134, CD137, Fc receptors, or CD154. In some embodiments, the transmembrane domain can be derived from the cell-surface protein cluster of differentiation 8 (CD8) or CD28.

[0108] Intracellular Domain

[0109] Various intracellular domains have different functions in different cell types. The present disclosure provides for an intracellular signaling domain useful in brain-resident phagocytes. As described herein, intracellular domains can be effective at generating functional programmable CAR-expressing cells.

[0110] For example, for Alzheimer’s disease (AD) and Amyloid B (A|3), the present disclosure provides for development of a single-chain fragment variable (scFv) derived from Amyloid B binding antibodies (e.g., Bapineuzumab, Crenezumab and Lecanemab, among others) and fusing it to the hinge and transmembrane domains from stable and non-dimerizing proteins Docket No.: 019794 / US4 such as CD8 or CD28. Intracellularly, the aforementioned domains can be fused to phagocytosis-inducing proteins, such as AXL, TYR03, MEGF10, DECTIN1 , MER, CD3 , Fc, CD64, or Fc receptors.

[0111] In some embodiments, the CAR-expressing cells can join the properties of different intracellular domains in one single brain-resident cell by combining two or more intracellular domains in a CAR. For example, such combinations can include one intracellular domain from a first family and one intracellular domain from another family, resulting in the simultaneous activation of different signaling pathways. These are considered costimulatory domains. Each costimulatory domain can have unique properties.

[0112] Hinge (Spacer)

[0113] The hinge, also referred to as a spacer, is in the extracellular structural region of the CAR that separates the binding units from the transmembrane domain. The hinge can be any moiety capable of ensuring proximity of the cell to the target (e.g., CD8-based hinge). With the exception of CARs based on the entire extracellular moiety of a receptor, the majority of CAR (such as CAR T) cells are designed with immunoglobulin (Ig)-like domain hinges or CD8 hinges, but any protein sequence that proves a space between the transmembrane domain and target-binding domain may function as an effective hinge.

[0114] Hinges generally supply stability for efficient CAR expression and activity. The hinge (also in combination with the transmembrane domain), can also ensure proper proximity to a target.

[0115] The hinge also provides flexibility to access the targeted antigen. The optimal spacer length of a given CAR can depend on the position of the targeted epitope. Long spacers can provide extra flexibility to the CAR and allow for better access to membrane proximal epitopes or complex glycosylated antigens. CARs bearing short hinges can be more effective at binding membrane-distal epitopes. The length of the spacer can be important to provide adequate intercellular distance for immunological synapse formation.

[0116] As such, hinges may be optimized for individual epitopes accordingly.

[0117] Here, the hinge can be operably linked to the transmembrane domain.

[0118] Extracellular Signaling Domain Docket No.: 019794 / US4

[0119] Optionally, an extracellular signaling domain can be incorporated into the CAR construct to propagate signaling. The extracellular signaling domain can be cloned into the hinge region, but can also be chosen based on the target.

[0120] Constructs and methods of making traditional CAR T constructs, such as intracellular domains are well known; see e.g., Feins S, Kong W, Williams EF, Milone MC, Fraietta JA. An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer. Am J Hematol. 2019;94(S1 ):S3-S9; Rafiq, S., Hackett, C.S. & Brentjens, R.J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17, 147-167 (2020). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0121] NEURODEGENERATIVE DISEASES, DISORDERS, OR CONDITIONS

[0122] The compositions and methods described herein can be used to treat a neurodegenerative disease, disorder, or condition. The treatments described herein can be effective against many neurodegenerative diseases and can be used prophylactically to improve neuro-function or delay aging. As an example, the neurodegenerative disease, disorder, or condition can be associated with accumulation of a pathological protein, such as Amyloid B (A|3), hyperphosphorylated Tau, alpha-synuclein, or TDP-43. As another example, a neurodegenerative disease, disorder, or condition can be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexander disease, Alpers' disease, Alpers Huttenlocher syndrome, alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Arts syndrome, ataxia neuropathy spectrum, ataxia (e.g., with oculomotor apraxia, autosomal dominant cerebellar ataxia, deafness, and narcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay, Batten disease, beta-propeller protein associated neurodegeneration, Cerebral Amyloid Angioapathy (CAA), Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Corticobasal Degeneration, CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN6 disease, CLN7 disease, CLN8 disease, cognitive dysfunction, congenital insensitivity to pain with anhidrosis, dementia, familial encephalopathy with neuroserpin inclusion bodies, familial British dementia, familial Danish dementia, fatty acid hydroxylase-associated neurodegeneration, GerstmannStraussler- Scheinker Disease, GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., with retinitis Docket No.: 019794 / US4 pigmentosa), Huntington's disease (HD), infantile neuroaxonal dystrophy, infantile-onset ascending hereditary spastic paralysis, Huntington’s disease (HD), infantile-onset spinocerebellar ataxia, juvenile primary lateral sclerosis, Kennedy's disease, Kuru, Leigh's Disease, Marinesco-Sjdgren syndrome, Mild Cognitive Impairment (MCI), mitochondrial membrane protein-associated neurodegeneration, Monomelic Amyotrophy, Motor neuron diseases (MND), Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension (Shy-Drager Syndrome), multiple sclerosis, multiple system atrophy, neurodegeneration in Down’s syndrome (NDS), neurodegeneration of aging, Neurodegeneration with brain iron accumulation, neuromyelitis optica, pantothenate kinase- associated neurodegeneration, Opsoclonus Myoclonus, prion disease, Progressive Multifocal Leukoencephalopathy, Parkinson's disease (PD), PD-related disorders, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, prion disease, progressive external ophthalmoplegia, riboflavin transporter deficiency neuronopathy, Sandhoff disease, Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA), Striatonigral degeneration, Transmissible Spongiform Encephalopathies (Prion Diseases), or Wallerian-like degeneration.

[0123] MOLECULAR ENGINEERING

[0124] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0125] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0126] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid," as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a Docket No.: 019794 / US4 naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0127] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0128] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of a significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.

[0129] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0130] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes. Docket No.: 019794 / US4

[0131] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates a target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0132] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0133] For a gene to be expressed, its DNA sequence must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0134] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0135] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0136] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the Docket No.: 019794 / US4 recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0137] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN- 10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0138] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein-encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0139] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to Docket No.: 019794 / US4 regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0140] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0141] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0142] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0143] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, genespecific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process. Docket No.: 019794 / US4

[0144] "Wild-type" refers to a virus or organism found in nature without any known mutation. Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0145] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. Docket No.: 019794 / US4

[0146] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0147] So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art- known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0148] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA: DNA sequence can be determined using the Docket No.: 019794 / US4 following formula: Tm = 81.5 °C + 16.6(log [Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1 -1 ,5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0149] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile- mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome. Docket No.: 019794 / US4 Docket No.: 019794 / US4

[0150] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0151] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial Docket No.: 019794 / US4 and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0152] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147- 173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 - 423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK- iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of 5 the 3' overhangs.

[0153] A vector can be used to introduce a gene that expresses CAR on a phagocyte. Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof. Docket No.: 019794 / US4

[0154] As described herein, viral-like particles (VLPs) are self-assembled, non-infectious structures that resemble viruses but lack viral genetic material. Because they mimic the outer structure of real viruses, VLPs can interact with the immune system similarly to actual viruses. The following are key features of VLPs: 1 . Non-infectious - They do not contain viral genetic material, so they cannot replicate.2. Structurally Similar to Viruses - They are composed of viral proteins that spontaneously assemble into particles that resemble real viruses in shape and size. 3. Immunogenic - They can stimulate immune responses, making them valuable for vaccine development. 4. Drug Delivery - Engineered VLPs can transport therapeutic molecules into cells.

[0155] As described herein, lipid nanoparticles (LNPs) are nanoscale delivery vehicles made of lipids, used for drug and gene delivery, especially for mRNA-based vaccines and therapeutics. They are used to encapsulate and protect nucleic acids (e.g., mRNA, siRNA) Docket No.: 019794 / US4 or small-molecule drugs, facilitating their delivery into cells. Key Features of LNPs include: 1. Biodegradable & Biocompatible - Made from lipids that can be naturally broken down in the body. 2. Efficient Delivery - Protects nucleic acids from degradation and enhances cellular uptake. 3. Non-infectious 4. Targeted Drug Delivery - Can be modified with surface ligands for tissue-specific targeting.

[0156] Genetic modification can be performed either ex vivo or in vivo. The ex vivo strategy is based on the modification of cells in culture and transplantation of the modified cell into a patient. Cells that are most commonly considered therapeutic targets for monogenic diseases are stem cells. Advances in the collection and isolation of these cells 15 from a variety of sources have promoted autologous gene therapy as a viable option.

[0157] FORMULATION

[0158] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. 20 Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0159] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0160] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used. Docket No.: 019794 / US4

[0161] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0162] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0163] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0164] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition. Docket No.: 019794 / US4

[0165] THERAPEUTIC METHODS

[0166] Also provided is a process of treating, preventing, or reversing a neurodegenerative disease, disorder, or condition in a subject in need of administration of a therapeutically effective amount of phagocytes expressing CAR or a vector capable of expressing a CAR construct, so as to enhance clearance of protein accumulation associated with neurodegeneration.

[0167] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neurodegenerative disease, disorder, or condition. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0168] Generally, a safe and effective amount of a CAR phagocyte cell therapy is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a CAR phagocyte cell therapy described herein can substantially inhibit protein accumulation, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition, or reduce protein aggregation.

[0169] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0170] When used in the treatments described herein, a therapeutically effective amount of a CAR phagocyte therapy can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to substantially inhibit protein accumulation, slow the progress of a neurodegenerative disease, Docket No.: 019794 / US4 disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition, or reduce protein aggregation.

[0171] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0172] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0173] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw- Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total Docket No.: 019794 / US4 daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0174] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0175] Administration of a CAR phagocyte therapy can occur as a single event or over a time course of treatment. For example, a CAR phagocyte therapy can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0176] Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for a neurodegenerative disease, disorder, or condition.

[0177] A CAR phagocyte therapy can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a CAR phagocyte therapy can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a CAR phagocyte therapy, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a CAR phagocyte therapy, an antibiotic, an anti-inflammatory, or another agent. A CAR phagocyte Docket No.: 019794 / US4 therapy can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a CAR phagocyte therapy can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.

[0178] CELL THERAPY

[0179] Cells generated according to the methods described herein can be used in cell therapy. Cell therapy (also called cellular therapy, cell transplantation, or cytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting phagocyte cells capable of clearing protein aggregates can be used in the course of treating a neurodegenerative disease, disorder, or condition.

[0180] Cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.

[0181] Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogenic strategy is that unmatched allogenic cell therapies can form the basis of "off the shelf" products.

[0182] Autologous cell therapy or autologous transplantation uses cells that are derived from the subject’s own tissues. It could also involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.

[0183] Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig-derived cells can be transplanted into humans. Xenogeneic cell therapies can involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.

[0184] Cells that can be used to express CARS, as described herein can be brain cells such as any phagocyte, microglial cells, which are phagocytosing macrophages, or astrocytes. Microglial cells can help clean up the central nervous system stroma and parenchyma by phagocytosing apoptotic cells, debris, and inactive synapses. Gene mutations in these cell types are associated with up to 3X risk of developing AD. Docket No.: 019794 / US4

[0185] ADMINISTRATION

[0186] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0187] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0188] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0189] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In Docket No.: 019794 / US4 addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0190] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo', prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.

[0191] SCREENING

[0192] Also provided are screening methods.

[0193] The subject methods find use in the screening of a variety of different candidate molecules (e g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals.

[0194] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or Docket No.: 019794 / US4 heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0195] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0196] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0197] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0198] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict Docket No.: 019794 / US4 the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0199] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and 0 atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0200] KITS

[0201] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to expression vectors, CAR constructs, components of CAR constructs, or cells, such as cells expressing CAR constructs, transduced cells, etc. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0202] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral nonreacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a Docket No.: 019794 / US4 bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.

[0203] Removable membranes may be glass, plastic, rubber, and the like.

[0204] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic- readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0205] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sam brook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0206] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0207] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending Docket No.: 019794 / US4 upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0208] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0209] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0210] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language Docket No.: 019794 / US4 in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0211] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0212] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0213] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0214] EXAMPLES

[0215] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. Docket No.: 019794 / US4

[0216] EXAMPLE 1: Chimeric Antigen Receptors for the Treatment of Protein Aggregation and Neurodegenerative Diseases

[0217] This example describes the application of Chimeric Antigen Receptors for the treatment of protein aggregation and neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease, among others. Herein is disclosed:

[0218] (1) A chimeric antigen receptor (CAR) developed to induce phagocytosis and clearance of disease-causing protein aggregates in neurodegenerative diseases. The CAR- dependent clearance also promotes phagocytosis through noninflammatory pathways.

[0219] (2) Methods development of individual CARs to target aggregates such as Amyloid B (A(3), hyperphosphorylated Tau, alpha-synuclein, or TDP-43.

[0220] (3) For Alzheimer’s disease and Amyloid B, development of a single-chain variable fragment (scFv) from Amyloid B binding antibodies (e.g., Bapineuzumab, Crenezumab, and Lecanemab, among others) and fusing it to the hinge and transmembrane domains from stable and non-dimerizing proteins such as CD8 and CD28. Intracellularly, the aforementioned domains can be fused to phagocytosis inducing proteins such as AXL, TYR03, MEGF10, DECTIN1 , MER, CD3 , Fc, CD64, or Fc receptors.

[0221] (4) Expression of the CARs is achieved by lentiviral transduction of microglia and astrocytes, which are natural brain-resident phagocytes. The resident microglia will be pharmacologically depleted with MCSF-R inhibitors such as PLX5622. Transduced microglia will then be administered to repopulate the brain. Using a different approach, astrocytes will be transduced with AAVs that genetically code for the CARs.

[0222] (5) The present disclosure demonstrates successful development of an scFv from the Crenezumab antibody. In some exemplary embodiments, the scFv were fused to a CD8 hinge and transmembrane domain and used MEGF10 for the intracellular domain. The data shows that the CAR is expressed and folded properly and it is appropriately targeted to the cell membrane. A CAR transduced human microglial cell line shows increased and potent phagocytosis of Amyloid B. Using pH-sensitive labeling, the data also demonstrates that the phagocytosed Amyloid B is targeted to the intended acidic vesicular compartment. Live cell imaging demonstrates that Amyloid B particles induce clustering of GFP-fused CARs in the cell membrane, an event which is required for MEGF10 signaling and phagocytosis. These Docket No.: 019794 / US4 data also show that the designed Crenezumab-based scFv has appropriate affinity for Amyloid B.

[0223] This present disclosure provides the unique benefit of clearing protein aggregates in neurodegenerative diseases with a single treatment, in contrast to currently available conventional and trial treatments. To date, all treatments aimed towards the clearance of neurodegenerative protein aggregates have failed. Even so, due to their nature, continued administration of the drug for the patient’s lifetime would be required. Considering the prevalence of these diseases and that most, if not all, of these investigative drugs are antibody based, the economic burden posed by prolonged treatment (more than 20 years in case of Alzheimer’s) is not sustainable.

[0224] In some exemplary embodiments, the constructs and / or methods disclosed herein comprise at least one nucleotide sequence selected from Megfl O CAR and Dectinl CAR. In other exemplary embodiments, constructs and methods of the present disclosure comprise at least one protein sequence encoded by MegflO CAR nucleotide described herein and / or at least one protein sequence encoded by Dectinl CAR nucleotide described herein, see MegflO CAR (SEQ ID NO: 1 ) and Dectinl CAR (SEQ ID NO: 2) .

[0225] EXAMPLE 2: Targeting Amyloid-fi Pathology by Chimeric Antigen Receptor Astrocyte (CARA) Therapy

[0226] This example describes CAR constructs and methods of use thereof, including CARA constructs and methods of targeting, reducing, and / or clearing amyloid-p using the disclosed constructs. Alzheimer’s disease (AD) is the most common cause of dementia, progressing through a cascade from amyloid buildup to tau-mediated neurodegeneration. Despite the success of recent anti-amyloid therapies, limitations remain, highlighting the need for novel treatments. Here, new designs are proposed for anti-amyloid chimeric antigen receptor expressed in astrocytes (CAR-A) and confirm their functionality in vitro. Additionally, herein demonstrates the in vivo effectiveness of two designs in preventing amyloid plaques accumulation and related pathologies in a mouse model of amyloidosis. The single nuclei analysis on pooled samples showed that CAR-A treatment induced a unique glial cell response towards the amyloid pathology. These findings provide in vivo evidence supporting the potential of CAR-A based cell or gene therapies for treating AD. Docket No.: 019794 / US4

[0227] Introduction

[0228] Alzheimer’s disease (AD) is the most common cause of dementia worldwide, with its prevalence rising due to an aging population. AD follows a distinctive pathological cascade: amyloid-p (A|3) deposits accumulate in the extracellular space, leading to the hyperphosphorylation of intraneuronal tau proteins, eventually forming tau-associated paired helical filaments (PHF) and causing significant neurodegeneration.

[0229] Although the amyloid cascade theory's ability to fully explain AD is debated, the latest generation of anti-Ap monoclonal antibodies, such as Aducanumab, Lecanemab, and Donanemab, has shown promising results. Notably, both Lecanemab and Donanemab have received FDA approval, supporting the effectiveness of anti-amyloid-p therapies in treating AD. However, these treatments require very high doses to penetrate the central nervous system (CNS). The high cost of long-term treatment with these therapies becomes a concern. Furthermore, the potential side effects necessitate continuous monitoring of patients receiving these antibodies, complicating their clinical use. Currently used treatments carry potential side effects including amyloid-related imaging abnormalities (ARIA). Another challenge is determining the optimal treatment window. Early intervention to remove amyloid deposits may prevent AD progression, but issues regarding the cost and feasibility remain unresolved. While anti-amyloid-p (Ap) antibodies primarily function through antibodydependent cellular phagocytosis (ADCP), targeting Ap aggregates via microglia-mediated clearance, this approach faces significant limitations for long-term management of Alzheimer's disease (AD). The extended preclinical phase of AD, spanning up to two decades before symptom onset, renders chronic antibody administration impractical for secondary prevention, due to issues of patient adherence, healthcare resource allocation, and economic feasibility. Therefore, there is an urgent need for new therapeutic approaches that offer improved efficacy, safety, and convenience for AD treatment.

[0230] To address these challenges, a paradigm shift was proposed in AD therapeutics through the development of chimeric antigen receptors (CARs). By engineering CARs that combine an anti- Ap single-chain variable fragment (scFv) with a phagocytic receptor, a self- sustaining therapeutic system was created within the brain's cellular milieu. This innovative approach circumvents the need for repeated interventions, offering a potential one-time treatment that could provide sustained therapeutic action throughout the course of the Docket No.: 019794 / US4 disease. Moreover, unlike antibody-based therapies that primarily target formed plaques, CAR-expressing cells have the potential to intervene at earlier stages of A|3 aggregation, potentially halting the pathological cascade before significant neuronal damage occurs. Secondly, CARs allow for precise engineering of the cellular response upon A|3 engagement. Unlike antibodies, CARs can be tailored to elicit specific intracellular signaling cascades. This level of control over the phagocytic process and subsequent cellular activation is unprecedented in AD therapeutics. This strategy not only addresses the limitations of current therapies but also paves the way for highly targeted, customizable interventions in AD, particularly during the critical window for secondary prevention.

[0231] The CAR approach has been pioneered in cancer immunology, where novel phagocytic CARs specific for tumor antigens have been designed and primarily deployed in monocytes / macrophages. However, due to the blood-brain barrier and the self-sustaining nature of microglia already residing in the CNS, efficient methods to replace microglia with engineered peripheral monocytes or other myeloid cells are still in development. Moreover, the survival of engineered myeloid cells in the face of massive amyloid deposition in the brain remains a challenge. For example, an Fey receptor-based CAR macrophage injected intracranially into the hippocampus has shown limited survival and therapeutic effects in removing amyloid pathology in vivo. Direct viral infection to engineer endogenous microglia through noninvasive gene delivery approaches is also in its preliminary stages. Consequently, CAR macrophage-based therapies specifically for AD still have significant development ahead.

[0232] The present disclosure explored whether another type of phagocytic glial cell, the astrocyte, could be leveraged to overcome the challenges mentioned above. Anti-A|3 chimeric antigen receptors (CARs) were introduced into astrocytes using the Adeno- associated virus (AAV), creating CAR astrocytes (CAR-A) that could act as superphagocytes, specifically targeting and removing extracellular A|3 aggregates. Newly designed CAR-A are herein reported that significantly boosted astrocytic phagocytosis of Ap oligomers in vitro. Additionally, two of these CAR-A were delivered into CNS astrocytes in vivo through peripheral noninvasive gene delivery using the AAV.PHP.eB strain with a GFAP-promoter. This approach resulted in a significant reduction in amyloid pathology in vivo. Overall, these results demonstrated the effectiveness of the anti-Ap CAR-A concept and highlighted the Docket No.: 019794 / US4 significant therapeutic potential of CAR-A for treating AD.

[0233] Results

[0234] In the CNS, astrocytes and microglia act as phagocytes equipped with receptors capable of directly engulfing cells, synapses, particles, or debris without the need for opsonization. Additionally, microglia can phagocytose cells or particles that are coated with antibodies through Fc receptors. To develop anti-A|3 CARs, receptors capable of direct phagocytosis were leveraged. The anti-Ap scFv was linked to the intracellular domain of one of three receptors that mediate phagocytosis in development, homeostasis, and disease: MEGF10, expressed by astrocytes; Dectinl (also known as CLEC7A), expressed by microglia; and MERTK, expressed by both. In one additional CAR, the anti-Ap scFv was linked to the signaling domain of CD3^, which is well-established for CAR-T signaling and has been recently deployed to design an anti-tumor CAR macrophage. The anti-Ap scFv was linked to these intracellular domains with the transmembrane domain of CD8 adopted from CAR-T constructs. However, since the scFv-CD8-MERTK chimeric construct was stuck on the cell membrane, the CD8 transmembrane region was replaced with that of the MERTK (FIG. 1A). For the initial in vitro screening, the anti-Ap scFv was derived from the sequence of Crenezumab due to the availability of its structure in complex with the Ap42 peptide. Using this X-ray crystal structure, a suitable (GSSS)n linker was designed from the C-terminal of the heavy chain to the N-terminal of the light chain to maintain antigen binding ability (data not shown). All CAR constructs were tagged with a bright GFP (GreenLantern) at the C- terminal.

[0235] First a THP-1 cell line was transfected with different CAR constructs and examined their expression and subcellular localization of the receptors using confocal microscopy. Although some proteins were enriched in the endoplasmic reticulum (ER) near the nuclei, the majority of the receptors were expressed on the plasma membranes (FIG. 1 B), suggesting proper protein folding and trafficking. Based on this expression screen, all constructs were further expressed in an immortalized mouse astrocyte cell line SV40T to evaluate the functional capacity of CAR-expressing astrocytes. To verify the functionality of the CAR receptors, either constant dyes such as Alexa-594 and Alexa-647 or a pH-sensitive dye, pHrodo, were conjugated to monomeric Ap42 peptides and oligomerized the dye- conjugated Ap42 peptides. Firstly, CAR-expressing astrocytes were incubated with Docket No.: 019794 / US4 monomeric AP42-647 on ice to verify the surface binding by flowcytometry (FIG. 1 C), where only GFP-expressing control showed no binding signal. For further verification, Crene- Megfl O was used as an example and incubated cells expressing it with Alexa-594 labeled oA[342 (oA[342-A594) for live cell imaging (FIG. 6(A-B)). Clear cellular internalization of Crene-Megf10 and OAP42-A594 was observed after their colocalization on the cell surface, demonstrating Ap-specific phagocytosis driven by Crene- Megfl O. When challenged with oligomeric A[342-pHrodo (oA[342-pHrodo), the CAR-expressing astrocyte cells phagocytosed more oA[342-pHrodo in 4 hours compared to GFP-transfected cells (FIG. 1 D and FIG. 1 E). The degradation of phagocytosed oA|342-pHrodo was next compared across the four different CARs by monitoring the decay of intracellular oA[342 signals after 24 hours (FIG. 1 D). Following an enhanced phagocytosis of oA[342-pHrodo compared to GFP- transfected astrocytes, CAR-expressing astrocytes managed to degrade a significant amount of oAp42- pHrodo after 24 hours (FIG. 1 E). However, perhaps due to rapid phagocytosis, Crene- MertK and Crene-CD3 expressing astrocytes exhibited more stress granules and sometimes underwent cell death, as determined by flow cytometry (data not shown). Additionally, a high- phagocytosed population of CAR astrocytes was consistently observed which persisted with high-level of oA[342 even after 24 hours, particularly in the Crene-CD3^ expressing astrocytes. These observations may indicate a reduced degradation of oA[342 if initial phagocytosis level was too high. Therefore, one astrocyte-expressing receptor, Megfl O- and one microglia-expressing receptor, Dectinl -based CARs were chosen for further experiments.

[0236] Previous clinical trials of anti-Ap therapies targeting monomeric A|3 peptides have shown limited effectiveness in amyloid removal. This is partially due to the excessive abundance of antigens. Therefore, to enhance the therapeutic effect of anti-Ap CAR on amyloid pathology and potentially as a preventive therapy, the scFv of Aducanumab, which targets all Ap oligomers, was chosen over the scFv of Crenezumab or other FDA-approved anti-Ap antibodies like Lecanemab and Donanemab. This decision was based on three reasons: A) Aducanumab targets Ap oligomers that may appear earlier than pro-fibrils or plaque-associated pyro-glutamine present in advanced plaques. B) The ADCP-independent phagocytosis of Ap oligomers by astrocytes does not require proximity to Ap plaques in contrast to the ADCP-dependent phagocytosis. C) Focusing on Ap oligomers rather than Docket No.: 019794 / US4 monomers may address the ineffectiveness observed in early anti-A|3 therapeutic studies, such as those involving solanezumab. To verify the efficacy of the Aducanumab scFv constructs, the Aducanumab scFv was compared with two scFvs derived from different anti- A|3 antibodies using Megfl 0 based CAR as a model (FIG. 7(A-B)). The Aducanumab scFv- Megfl O (Adu-Megf10) CAR showed superior phagocytosis of oAp42 compared to the Bapineuzumab scFv- and Crenezumab scFv- Megfl O. While beginning at different levels, all three scFv-Megf10 CAR astrocytes managed to digest phagocytosed oAp42 aggregates after 24h (FIG. 7B). Before conducting in vivo tests, it was examined whether the enhanced phagocytosis of Ap by Adu-Megf 10 and Adu-Dectin1 would cause signs of cell stress in astrocytes (FIG. 2(A-C)). To do this, the transcriptome profiles were analyzed of astrocytes expressing Adu-Megf10, Adu-Dectin1 , or GFP controls using RNA-seq after 24 hours of exposure to oAp42. Long-term oAp42 treatment induced upregulation of many stressresponse genes including I gf bp7 , Rnf183, Acp5, Enpp5, Hsbpl 11 , Red 14 and Mir125a (FIG. 2A). Gene expression changes were also observed in metabolism related pathways including Apom, Apobec2, Cdh6, Slc27a6, etc., which suggested a cellular stress induced by longterm oAp42 treatment. Adu-Megf10- and Adu-Dectin1 -expressing astrocytes showed fewer DEG profiles observed in the GFP control astrocytes upon the long-term oAp42 treatment (FIG. 2B and FIG. 2C), except individual genes such as Ifi44. This indicates that Adu-Megf10 and Adu-Dectin1 astrocytes maintain a homeostatic profile while engulfing oAp42 in vitro. Given transcriptom ic analysis may not fully capture cytokine released by astrocytes, the media was also collected and sent for multiplex ELISA analysis of cytokines by Raybiotech Cytokine Array (FIG. 2(E-M)). The fold change was analyzed of each cytokine concentration in the media from an average of technical triplicates of every condition (FIG. 2E). Upon the 24h oAp42 challenge, GFP-expressing astrocytes released more pro inflammatory cytokines including BLC, IFNy, IL-5, IL-7, IL-15, and MCP-5 while downregulated anti-inflammatory cytokines such as IL-6. However, the expression of Adu-Megf10 appeared to downregulate all cytokines. Interestingly, the expression of Adu-Dectin1 increased the release of IL-7, IL- 15, MCSF and TIMP-1 at baseline (FIG. 2E), while the release of TNFo, TNF Rl and MCSF was elevated (FIG. 2(H-J)) and IL-7 and IL-15 released were suppressed by oA|342 challenge (FIG. 2(L-M)). In addition, cytokines of CCL3 and CCL5 were only upregulated by Adu- Dectinl astrocytes in response to oA|342 challenge (FIG. 2(F-I)). These results suggested an Docket No.: 019794 / US4 opposite trend of cytokine releases between Adu-Megf10 and Adu-Dectin1 by astrocytes regardless their enhanced phagocytosis against Ap aggregates, which led us to introduce both constructs in vivo and compare with GFP controls.

[0237] Therefore, Adu-Megf10, Adu-Dectin1 , and GFP control CARs were inserted into a GFAP-promoter-based AAV.PHP.eB expression system to achieve the expression of CARs in astrocytes. Inserting the Adu-Megf10 CAR into the AAV vector required truncating the GfaABCI D promoter due to length constraints. The viruses were injected intravenously at 2.5 months of age in 5xFAD mice, before they exhibit Ap deposition (FIG. 3A and FIG. 3B). All mice were examined at 5 months of age, once Ap plaques had formed. In the collected cohort, overall amyloid pathology was first assessed across the hemibrain using X34 staining of Ap plaques (FIG. 3(C-G)). In the cortex and hippocampus, both AAV. PHP. eB-Adu-Megf10 and AAV.PHP.eB-Adu-Dectin1 treatment groups showed a similar drastic reduction of Ap plaques in (FIG. 3(C-E)). Across the entire hemibrain, AAV.PHP.eB-Adu-Megf10 and AAV.PHP.eB- Adu-Dectin1 treatments significantly reduced amyloid plaque formation 2.5 months post-injection (FIG. 3F and FIG. 3G), with the Adu-Megf10 group showing almost no amyloid plaques. The anti-Ap40 / 42 ELISA demonstrated a similar trend of reduced Ap42 in the PBS-insoluble but GdnHCl-soluble fraction of the cortical tissues in the AAV.PHP.eB- Adu-Megf10 and AAV.PHP.eB-Adu-Dectin1 treatment groups, with no detectable insoluble Ap42 observed in the Adu-Megf10 group (FIG. 3H and FIG. 3I). Residual Ap plaques in the AAV.PHP.eB-Adu-Dectin1 group were mostly localized in the midbrain (FIG. 3C and FIG. 3F).

[0238] The impact on dystrophic neurites labeled by LAMP1 staining was further assessed around the remaining plaques in the cortex and hippocampus (FIG. 3J). Due to the lack of Ap plaques in the AAV.PHP.eB-Adu-Megf10 and AAV.PHP.eB-Adu-Dectin1 treatment groups, only three plaques per hippocampus per brain slice were sampled for this analysis (FIG. 3 and FIG. 3L), also FIG. 8(B-E)). In line with the dramatic reduction of amyloid deposition in the brain, the total volume of dystrophic neurites around X34+Ap plaques was significantly lower in both CAR-introduced groups compared to the control (FIG. 3K and FIG. 3L). Consistently, Ap-plaque-induced local synaptic loss was alleviated in both CAR- introduced groups, as quantified by a pre-synaptic marker, Synapsin (FIG. 3M and FIG. 3N), and a post-synaptic marker, PSD95 (FIG. 30 and FIG. 3P). Adu-Dectin1 showed a trend of Docket No.: 019794 / US4 even less neuronal dystrophy and synaptic loss compared to Adu-Megf10, although this was not statistically significant, contrary to its lesser pathology reduction as measured by X34+A|3 plaques. Overall, it was concluded that early injection with either AAV.PHP.eB-Adu- Megfl O or AAV.PHP.eB-Adu-Dectin1 at 2.5 months of age can significantly prevent amyloid pathology and amyloid-pathology-associated neuronal dystrophy and synaptic loss for 2.5 months in the 5xFAD model.

[0239] To compare the response of CAR-expressing astrocytes to those in the control group, the distribution, morphology, and activation of glial cells was analyzed around X34+Ap plaques using confocal microscopy, as described in previous literature. Three brain sections from each mouse were analyzed: one was stained with X34 / GFAP / IBA1 , one with X34 / APOE / CD68, and one with X34 / IBA1 / MHC-II to comply with antibody isotypes. Firstly, GFP labeling was observed in astrocytic processes attached to the X34+A plaques, primarily in the Adu-Megf10 and Adu-Dectin1 groups, under 63x high-resolution microscopy (FIG. 8A). Secondly, quantification of total GFAP voxels around X34+plaques showed a significant increase in astrocyte clustering around Ap plaques in the Adu-Dectin1 group (FIG. 4A and FIG. 4B, FIG. 8B and FIG. 80).

[0240] However, both Adu-Megf10 and Adu-Dectin1 expressing astrocytes exhibited a significant increase in LAMP1 expression co-localized with GFAP+cells (FIG. 4C and FIG. 4D), indicating enhanced lysosomal activity in CAR astrocytes around Ap plaques. Microglia clustering around each X34+plaque, assessed by total IBA1 voxels, was significantly increased in the Adu-Dectin1 group only (FIG. 4E and FIG. 4F, FIG. 8D and FIG. 8E), similar to the astrocyte clustering. The increased microglia clustering in Adu-Dectin1 group might be correlated with increased MCSF and proinflammatory cytokines such as TNFa, CCL3, CCL5 and TNF Rl as observed only in Adu-Dectin1 in vitro (FIG. 2(A-L)). The lack of difference in glial cell clustering around plaques in the Adu-Megf10 group compared to the GFP control might be due to insufficient amyloid burden or an overall silent cytokine profile (FIG. 2(A-L)) by how this CAR receptor drives glial cell responses. Total plaque-associated microglial lysosomal activity, indicated by the microglia-specific lysosome marker CD68, was significantly reduced in the Adu-Megf10 group compared to the control (FIG. 4G and FIG. 4H), with Adu-Dectin1 showing a similar trend. Two selected markers of microglial activation, Docket No.: 019794 / US4

[0241] APOE and MHC-II, showed no difference across the three groups (FIG. 8(F-I)). In summary, in line with the differences in cytokine releases by two CAR-expressing astrocytes, differences were also observed in glial response towards remaining plaques between two CARs, although both CAR treatments generally enhanced the astrocyte involvement of plaque removal and reduced microglial lysosomal burden.

[0242] Lastly, single nuclei RNA sequencing analysis was performed using pooled posterior cortical samples from five randomly selected mice (FIG. 5A and FIG. 5B, FIG. 9(A-B), FIG. 10(A-D). Astrocytic expression was confirmed of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) from the AAV.PHP.eB constructs (FIG. 5C). Many differentially expressed genes (DEGs) were observed in CAR-treated groups compared to controls in astrocytes (FIG. 5D and FIG. 5E). A consistent upregulation of Cst3 and Wdr17 in both CAR groups was also observed, an upregulation of Malatl only in the CAR-Megf10 astrocytes and an upregulation of Fth 1 only in the Adu-Dectin1 astrocytes (FIG. 5D and FIG. 5E). Genes including Nkain2, Pde4b and DockIO were consistently downregulated in CAR-astrocytes, suggesting an altered astrocyte activity compared to GFP controls (FIG. 5D and FIG. 5E). Interestingly, by unbiased clustering of astrocyte cluster, a unique shift of astrocyte subclusters in the two CAR-treated groups was clearly observed, where the astrocyte subcluster 4 was mostly abundant in GFP controls but almost absent in both CAR- treated groups (FIG. 5F). Although the gene signature of that subcluster 4 might not be representative, perhaps due to the limit number of astrocyte nuclei, the enriched subclusters 0 and 1 in two CAR-treated groups appeared to possess most genes associated with reactive astrocyte discovered in other CNS diseases, like Nrg1 , Unc13c, Kcnq3, Esrrg and Cntnl , or glial cell differentiation such as Fgf13, Fgf14, Rcan2 (FIG. 9A). On the other hand, the Cst3 gene, which encodes cystatin C, was also significantly upregulated in microglia in both CAR-treated groups compared to the control (FIG. 5G and FIG. 5H). Cystatin C plays a crucial role in maintaining protease activity balance and has been reported as an AD-protective gene according to previous studies. While it is predominantly expressed in microglia and astrocytes, further investigations are needed to understand its potential role in CAR astrocyte protection against amyloid pathology. Both CAR-treated microglia downregulated genes like Pcdh9, Tmeff2 and St18, which might be associated with less pathology. Interestingly, the distinct upregulation of disease-associated microglia genes or Docket No.: 019794 / US4 microglial activation genes was observed such as Csmd3, C1qc, Mef2a and Picalm in Adu- Dectinl group only (FIG. 5G and FIG. 5H), which may be consistent with previous histochemistry results where microglial clustering was only enhanced in Adu-Dectin1 mice (FIG. 4E and FIG. 4F). It is consistent in snRNAseq analysis where microglia cluster 3 was only induced in the Adu-Megf10 mice but not in the Adu-Dectin1 treated mice compared to GFP controls (FIG. 5I). Together with the data from astrocytes (FIG. 5D and FIG. 5E), this difference suggested that Adu-Dectin1 treatment led to cell autonomous changes in astrocytes while Adu-Megf10 treatment somehow induced enhanced microglia response with DAM signatures. It is worth noting that oligodendrocyte in both CAR-treated groups showed high levels of expression of Pip 1 and Cldnl 1 , which has been shown to be indicative of a robust myelin function in different disease context. In the 5xFAD mouse model, this could be correlated with drastic reduction in amyloidosis in the CAR-treated groups.

[0243] Discussion

[0244] As disclosed herein, it was demonstrated that two CARs, selected from several original constructs based on phagocytic receptors, effectively prevented amyloid deposition in vivo. Given that astrocytes act as "phagocytes" alongside microglia in health and disease, their functionality in vitro was validated using an immortalized astrocyte cell line. Utilizing astrocytic phagocytosis offers three potential advantages: 1 ) CAR astrocytes might help share the lysosomal burden of microglia dealing with amyloid aggregates, as indicated by the reduced expression of the lysosomal marked CD68. 2) Astrocytic phagocytosis is not impacted by peripheral mechanisms that affect microglia replacement approaches, which can be interfered with by peripherally infiltrated myeloid cells or recovering endogenous microglia. 3) Instead of the intracranial injection used for microglia transplantation, a CNS- penetrating AAV strain, AAV. PHP. eB, was employed to deliver the disclosed CAR into astrocytes in vivo non-invasively. A single intravenous injection of the AAV.PHP.eB-CAR constructs dramatically reduced amyloid plaque formation for 2.5 months post-injection. This significant reduction in amyloid burden was accompanied by a rescue of amyloid-induced dystrophic neurites and synapse loss around A|3 plaques. Additionally, astrocytic lysosomal activity increased, while microglial lysosomal activity around A[3 plaques decreased in both CAR-treated animals. Single nuclei analysis further indicated strong associations between reduced pathology and altered glial cell activations. These results demonstrate that anti Docket No.: 019794 / US4 amyloid CARs, with appropriate designs and delivery methods, can effectively remove amyloid burden in the brain in vivo. Furthermore, they show the potential of leveraging astrocytes to support the microglial response to amyloid pathology in AD treatment.

[0245] Few differences were observed between the two selected CARs, Adu-Megf10 and Adu-Dectin1. Adu-Megf10 showed better removal of amyloid plaques, while Adu-Dectin1 offered better protection against amyloid-induced neuronal dystrophy and glial cell shielding of Ap plaques. Although the major downstream intracellular domains of Megfl O and Dectinl converge into the Syk-dependent signaling pathway, differences in the downstream signaling and cytokine releases activated by Adu-Megf10 versus Adu-Dectin1 intracellular domains may result in different glial cell clustering phenotypes and gene signatures. Additionally, endogenous Megfl O expressed by astrocytes may cluster with Adu-Megf10, potentially impacting downstream signaling. Investigation of the precise signaling pathways involved in anti-Ap phagocytosis is contemplated herein.

[0246] Unlike all currently FDA-approved antibody treatments, the disclosed results showed that boosting cellular phagocytosis specifically against Ap using CARs could dramatically prevent amyloid deposition with a single injection before plaque formation. Moreover, contrast to the Fc- receptor-dependent signaling required for ADCP, more diverse signaling pathway and consequent cellular responses could be leveraged by CAR-based treatment, such as the CARA- Adu-Megf10 and CARA-Adu-Dectin1 herein. Developing successful engineering and CNS delivery methods for other CAR cells, such as CAR-microglia and CAR- T cells, remains challenging. The findings of the present disclosure suggest that all phagocytosis CAR designs can be effective, provided the receptor expression is successfully achieved in the brain via AAV.PHP.eB. Accordingly, comparing the efficiency of different approaches for AD-targeting gene therapies or cell therapies is important.

[0247] Exemplary embodiments of CAR constructs in accordance with the present disclosure are shown in SEQ ID NOs: 3-34; see also FIG. 11 (A-B) through FIG. 26(A-B). Expression- optimal CAR construct embodiments used in vivo (tag removed) are shown in SEQ ID NOs: 3-6; FIG. 11 (A-B) and FIG. 12(A-B). CAR construct embodiments used in vitro (tag removed) include Crenezumab scFv CARs (SEQ ID NOs: 7-16; FIG. 13(A-B) through FIG. 17(A-B)), Aducanumab scFv CARs (SEQ ID NOs: 17-20; FIG. 18(A-B) and FIG. 19(A-B)), and Bapinezumab scFv CARs (SEQ ID NOs: 21 -22; FIG. 20(A-B)). Additional derivative Docket No.: 019794 / US4 embodiments of CAR constructs (tag removed) are shown in SEQ ID NOs: 23-34; see also FIG. 21 (A-B) through FIG. 26(A-B).

[0248] EXAMPLE 3: Chimeric Antigen Receptors for the treatment of Alzheimer’s Disease

[0249] This example describes CAR constructs for Alzheimer's treatment including data showing that intravenous CAR administration significantly prevents amyloid plaque deposition.

[0250] Successful treatment of Alzheimer’s Disease (AD) necessitates (but may not be limited to) consideration of pathology, clinical trials, chimeric antigen receptors (CARs), and design of phagocytic CARs against amyloid beta. The significance of AD is evidenced by following: (i) AD is the most common cause of dementia, accounts for 60-80% of the cases, (ii) AD is estimated to affect more than 6.2 million people in the United States alone, (iii) the AD-associated annual national cost reached approximately $355 billion in 2021 , (iv) more than 11 million Americans provide unpaid care for people with AD, and (v) the AD-associated unpaid work is valued at more than $256 billion annually. Age is the greatest risk factor for AD (FIG. 27A). As the oldest baby boomers are now 75 years old, the projected number of people with AD in the U.S. has been steadily increasing (FIG. 27B and FIG. 27C). Despite intensive research, the cause of death due to AD continues to increase (FIG. 27D).

[0251] Regarding clinical presentation of AD, which is the neurodegenerative disorder responsible for 60-80% of dementia, it is believed that the disease starts up to 20 years before symptoms present. Cardinal symptoms include memory impairment, impaired executive function, and behavioral and psychologic symptoms. The amyloid hypothesis implicated amyloid-beta (A|3) aggregation as a potential therapeutic target (FIG. 28A and FIG. 28B) by inhibiting the generation of A|3 via [3-secretase and / or y-secretase inhibitors, as well as by decreasing A|3 levels via active immunization and / or passive immunization approaches.

[0252] Amyloid-targeted treatment to date in mice and humans has shown promise with various antibodies, however not without drawbacks (FIG. 29(A-F)). Treatments with previous antibodies may have failed because they were started too late. Even so, if antibodies are proven successful, these therapies face challenges of low brain bioavailability, sustainability, Docket No.: 019794 / US4 and cost. CARs have been developed for other diseases (e.g., DLBCL) that included a singlechain fragment variable (SCFV), hinge, transmembrane, and signal transduction regions. CAR development is time and resource intensive due to cloning and gene synthesis, virus production, cell line infection and purification, and / or structural and functional validation of designs.

[0253] Developing Chimeric Antigen Receptors (CARs) for the treatment of AD aims to prevent A|3 deposition, to prevent A[3-associated pathology, and to only require a single dose. In order to overcome the antibody limitations, ideal characteristics of aA[3 chimeric antigen receptor protein include the ability to bind A|3, to induce phagocytosis of A|3, and to be expressed permanently by a brain-resident phagocyte (FIG. 30A). To enable binding to A|3, AD-directed CARs include specialized design of the scFv region (FIG. 30B). CAR design embodiment considerations with Crenezumab are shown in FIG. 31 (A-D). To induce phagocytosis of A[3, AD-directed CARs include specialized design with Mertk, CD3 and Megfl O induce phagocytosis upon crosslinking (FIG. 32). To be expressed permanently by a brain-resident phagocyte, a first step includes identifying the cell type of interest followed by a second step of permanently expressing a CAR by utilizing microglia to control plaque burden in AD (FIG. 33). Microglia are brain-resident macrophages that phagocytose apoptotic cells, debris, and inactive synapses. Gene variants affecting microglia have been associated with up to 3x the risk of developing AD.

[0254] In exemplary embodiments, T-cells are transduced with CAR-coding retroviruses. The process may proceed via leukapheresis, lentivirus + lymphocyte lentiviral infection, and finally CAR T-cell infusion. Depending upon the embodiment, a lentiviral expression system may be based on the HIV genome wherein genomic content between the LTRs is integrated into the host genome. According the present disclosure, a proposed A|3 CAR design embodiment is shown in FIG. 34. Essential criteria for successful CAR function in A|3 treatment therapy include proper protein expression, folding, and targeting of the membrane (FIG. 35(A-H)), as well as successful scFv binding of A|3 (FIG. 36) and resulting phagocytosis (FIG. 37(A-E)).

[0255] In summary: (i) the designed scFv allows HEK cells to bind AB on the membrane, (ii) Mertk CARs (CAR 1 and 2) are not properly being targeted to the membrane as evidenced by the cytoplasmic distribution, (iii) MegflO CAR (CAR 3) folds appropriately and is Docket No.: 019794 / US4 transported to the membrane and promotes phagocytosis, and (iv) CD3 CAR (CAR 4) does not fold properly and accumulates in the endoplasmic reticulum. Additional in vitro testing included scFv from other a-A[3 antibodies (e.g., Bapineuzumab) and CAR expression and effects on primary astrocytes and microglia. Additional ex vivo testing included Ap-containing brain slice co-culture with CAR-expressing cells. Additional in vivo testing included AAV- Gfap-CAR3 infection of 5xFAD mice and microglia replacement for treating AD model mice. See FIG. 38(A-G).

Claims

Docket No.: 019794 / US4CLAIMSWhat is claimed is:1 . A chimeric antigen receptor (CAR) construct comprising: an antigen-binding domain (e.g., an scFv); a hinge; a linker; a transmembrane protein (e.g., CD28, CD8); and a phagocytosis-inducing intracellular signaling domain, wherein the antigen-binding domain targets protein aggregates; and / or the phagocytosis-inducing intracellular signaling domain induce phagocytosis upon oligomerization.

2. An expression vector capable of expressing the CAR construct of claim 1 in a host cell.

3. A method of treating a neurodegenerative disease associated with accumulation of disease-causing or pathogenic proteins in a subject in need thereof, the method comprising: administering to the subject an antigen-targeted, phagocytosis inducing CAR-expressing brain-resident phagocyte, wherein the phagocyte expresses the CAR construct of claim 1 .

4. A method of inducing phagocytosis of a protein aggregate (e.g., amyloid-[3 peptides), the method comprising expressing a protein aggregate-targeting, phagocytosis-inducing CAR in a phagocyte, wherein the phagocyte expressing the protein aggregate-targeting, phagocytosis-inducing CAR is capable of phagocytosing the targeted protein aggregate, wherein the CAR comprises the CAR construct of claim 1.

5. A method of clearing disease-causing or pathogenic protein aggregates (e.g., amyloid-p peptides) in a subject in need thereof, the method comprising: expressing theDocket No.: 019794 / US4CAR construct of any one of the preceding claims in a phagocyte and introducing the CAR expressing phagocyte to a protein aggregate, wherein the subject has a neurodegenerative disease (e.g., Alzheimer’s disease (AD)) or the subject has diseasecausing pathogenic protein aggregates.

6. A method of clearing amyloid-[3 peptides in a subject in need thereof, the method comprising: administering to the subject a composition comprising CAR expressing brain-resident phagocytes, wherein the subject has an A|3 associated disease, disorder, or condition (e.g., AD), wherein the CAR is a protein aggregatetargeting, phagocytosis-inducing CAR, and wherein the CAR comprises the CAR construct of claim 1 .

7. A method of phagocyte (e.g., microglia, astrocyte) replacement in a subject, comprising: depleting resident phagocytes (e.g., microglia), optionally with a MCSF-R inhibitor (e.g., such as PLX5622); and / or administering an antigen-targeted, phagocytosis inducing CAR transduced brainresident phagocytes (e.g., microglia) to the brain of the subject to repopulate the brain phagocytes.

8. A method of generating phagocytosis-inducing, protein aggregate-targeting chimeric antigen receptor (CAR) phagocyte cells comprising: providing phagocyte cells; and transducing a phagocytosis-inducing, protein aggregate-targeting CAR via a viral vector into the phagocyte cells for an amount of time sufficient to virally transduce the phagocytosis-inducing, protein aggregate-targeting CAR into the phagocyte cells, resulting in CAR-transduced phagocyte cells.

9. The method of claim 8, wherein the viral vector comprises a chimeric antigen receptor (CAR) is a CAR lentivirus or CAR adeno-associated viral (AAV) vector.Docket No.: 019794 / US410. The method of claim 8, wherein the viral vector is a lentiviral vector.

11. A method of administering phagocytosis-inducing, protein aggregatetargeting CAR phagocyte cells to a subject in need thereof comprising: isolating phagocyte cells from a subject or a donor; generating phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells according to claim 8; and administering a therapeutically effective amount of phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells into the subject.

12. The construct or method of any one of the preceding claims, wherein the CAR construct comprises an antigen-binding domain and the antigen-binding domain comprises an A[3 binding domain.

13. The construct or method of any one of the preceding claims, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

14. The construct or method of any one of the preceding claims, wherein the scFv is derived from (e.g., a truncated version or variant thereof) one or more Amyloid B binding antibodies (e.g., Bapineuzumab, Crenezumab, among others).

15. The construct or method of any one of the preceding claims, wherein the scFv is fused to the hinge or transmembrane domains from stable and non-dimerizing proteins (e.g., CD8, CD28).

16. The construct or method of any one of the preceding claims, wherein the antigen-binding domain is an scFv derived from the antigen-binding fragment (Fab) region of Crenezumab or Bapineuzumab.Docket No.: 019794 / US417. The construct or method of any one of the preceding claims, wherein the A|3 binding domain comprises at least a portion, a fragment, or a variant of an a-Ap antibody.

18. The construct or method of any one of the preceding claims, wherein the scFv, the hinge (if present), the transmembrane protein (if present), and the phagocytosis inducing intracellular signaling domain are operably linked.

19. The construct or method of any one of the preceding claims, wherein the scFv comprises a heavy (H) chain Ig domain, a light (L) chain Ig domain, and a linker peptide.

20. The construct or method of any one of the preceding claims, wherein the H or L chain is derived from at least a portion, fragment, or variant of Crenezumab or Bapineuzumab antigen-binding fragments (Fabs).21 . The construct or method of any one of the preceding claims, wherein at least a portion, fragment, or variant of Crenezumab comprises a portion that interacts with A .

22. The construct or method of any one of the preceding claims, wherein the portion or variant thereof of the Crenezumab comprises the heavy chain 13-16 N- terminus.

23. The construct or method of any one of the preceding claims, wherein the Ig domain is truncated at the terminal p-sheet.

24. The construct or method of any one of the preceding claims, wherein the hinge and / or the transmembrane domain, if present, supplies stability constraint.Docket No.: 019794 / US425. The construct or method of any one of the preceding claims, wherein the hinge and / or the transmembrane domain, if present, are from or derived from stable and non-dimerizing proteins (e.g., CD8, CD28).

26. The construct or method of any one of the preceding claims, wherein the intracellular domain is fused or operably linked to a linker, an extracellular domain, or a hinge.

27. The construct or method of any one of the preceding claims, wherein the intracellular domain is a phagocytosis-inducing protein.

28. The construct or method of any one of the preceding claims, wherein the intracellular signaling domain is Mertk, CD3 , MegflO, Dectinl , or CD19 PI3K.

29. The construct or method of any one of the preceding claims, wherein the intracellular signaling domain is phagocytosis-inducing protein, such as AXL, TYR03, MEGF10, DECTIN1 , MER, CD3 , Fc, CD64, or Fc receptor.

30. The construct or method of any one of the preceding claims, wherein the intracellular signaling domain is Megfl O.31 . The construct or method of any one of the preceding claims, wherein the intracellular signaling domain is Dectinl .

32. The construct or method of any one of the preceding claims, wherein the brain-resident phagocyte is a macrophage, microglial cell, or astrocyte.

33. The construct or method of any one of the preceding claims, wherein the brain-resident phagocyte is an autologous microglial cell.Docket No.: 019794 / US434. The construct or method of any one of the preceding claims, wherein the brain-resident phagocyte is a natural phagocyte, a native phagocyte, or a donor phagocyte.

35. The construct or method of any one of the preceding claims, wherein the protein aggregate is pathological or associated with a neurodegenerative disease.

36. The construct or method of any one of the preceding claims, wherein the neurodegenerative disease, disorder, or condition is associated with accumulation of a pathological protein, such as Amyloid B (A|3), hyperphosphorylated Tau, alpha- synuclein, or TDP-43.

37. The construct or method of any one of the preceding claims, wherein the neurodegenerative disease is AD, Parkinson's Disease, dementia with Lewy bodies and multiple systems atrophies, or Amyotrophic Lateral Sclerosis (ALS).

38. The construct or method of any one of the preceding claims, wherein the CAR is expressed in a brain-resident phagocyte (e.g., macrophages, astrocytes, microglia).

39. The construct or method of any one of the preceding claims, wherein the CAR is transduced with a CAR-coding retrovirus (e.g., lentivirus) or Adeno-associated viral (AAV) vector that genetically codes for a CAR of any one of the preceding claims.

40. The construct or method of any one of the preceding claims, wherein the expression of CAR is achieved by lentiviral transduction of brain-resident phagocytes (e.g., macrophages, microglia, astrocytes).41 . The construct or method of any one of the preceding claims, wherein the CAR targets protein aggregates Amyloid B (A|3), hyperphosphorylated Tau, alpha- synuclein, or TDP-43.Docket No.: 019794 / US442. The construct or method of any one of the preceding claims, wherein the CAR prevents A|3 deposition; prevents A[3-associated pathology; only requires a single dose; binds A|3; induces phagocytosis of A|3; and / or is expressed permanently by a brainresident phagocyte.

43. The construct or method of any one of the preceding claims, wherein CAR dependent clearance promotes phagocytosis through non-inflammatory pathways.

44. The construct or method of any one of the preceding claims, wherein the CAR comprises an scFv derived from at least a portion, fragment, or variant of Crenezumab and the intracellular signaling domain is selected from Megfl 0 and Dectinl .

45. The construct or method of claim 44, wherein the CAR further comprises a CD8 hinge and transmembrane domain.

Images