Chimeric antigen receptor-expressing phagocytic cells and methods of making and using the same

Abstract

CAR phagocytic cells are provided as well as methods of making and administering the same to treat neurodegenerative diseases.

Description

CHIMERIC ANTIGEN RECEPTOR-EXPRESSING PHAGOCYTIC CELLS AND METHODS OF MAKING AND USING THE SAME BACKGROUND

[0001] Accumulation of toxic misfolded protein species and neuroinflammation are key features of neurodegenerative diseases (NDDs). Recent clinical evidence demonstrates clearance of A0 aggregates may have a disease-modifying impact. The central nervous system (CNS) is protected from pathogens through phagocytic immunity. There are three populations of cells that provide phagocytic immunity to the CNS. The first are microglia, which are the resident professional phagocytic cell of the CNS. Microglia are long-lived and begin to seed the CNS from the yolk sack early in embryonic development. The second are astrocytes, which are active in the elimination of synapses and neuronal debris in the developing or injured brain. The third are monocytes, which are derived from myeloid precursors in the bone marrow and either enter the inflamed CNS from the blood and differentiate into macrophages or act in perivascular spaces to affect aggregate clearance. CNS macrophages can be potent phagocytes. The fates of CNS-macrophages seem to be heterogeneous; some persist in the perivascular compartment per above (Barrier Associated Macrophages - BAM), some differentiate into long-lived cells that are phenotypically identical to microglia, and some remain macrophages and turn over.

[0002] A need exists for compositions and methods for increasing the phagocytic capacity and for modulating inflammatory tone in or near the CNS, including for the treatment of NDDs.BRIEF DESCRIPTION

[0003] In one aspect, compositions and methods are provided for the treatment of NDDs. In one aspect, a chimeric antigen receptor (CAR) construct or a construct encoding a CAR construct is provided, the CAR construct comprising: an antigen-binding domain (e.g., a single-chainvariable fragment (scFv)); optionally, a hinge; optionally, a linker; optionally, a transmembrane protein (e.g., CD28, CD8); and a phagocytosis-inducing intracellular signaling domain. In some aspects, the antigen-binding domain targets protein aggregates and / or the phagocytosis-inducing intracellular signaling domain induces phagocytosis upon oligomerization. In some aspects, the phagocyte expressing the protein aggregate-targeting, phagocytosis-inducing CAR is capable of phagocytosing the targeted protein aggregate.

[0004] In another aspect, a method is provided for clearing disease-causing or pathogenic protein aggregates (e.g., amyloid-P peptides) in a subject having pathogenic protein aggregates (e.g., Alzheimer’s disease (AD) or Cerebral Amyloid Angioapathy (CAA)), the method comprising expressing the CAR in a phagocyte or introducing the CAR-expressing phagocyte to a protein aggregate.

[0005] In another aspect, a method is provided for clearing amyloid-P peptides, the method comprising administering CAR-expressing peripheral immune cells to a subject having an Ap associated NDD.

[0006] In another aspect, a method is provided for clearing amyloid-P peptides, the method comprising administering CAR-expressing monocyte precursor cells or Hematopoietic Stem Cells (HSCs) to a subject having an Ap associated NDD. In some aspects, the CAR is a protein aggregate-targeting, phagocytosis-inducing CAR.

[0007] In another aspect, a method is provided for treating a neurodegenerative disease associated with accumulation of disease-causing or pathogenic proteins, the method comprising: administering antigen-targeted, phagocytosis inducing CAR-expressing peripheral immune cells to a subject having an NDD associated with accumulation of disease-causing or pathogenic proteins or a significant risk of contracting such a disease.

[0008] In another aspect, a method is provided for treating an NDD associated with accumulation of disease-causing or pathogenic proteins, the method comprising: administering a CAR-expressing monocyte precursor cells or Hematopoietic Stem Cells (HSCs) to a subject having an NDD associated with accumulation of disease-causing or pathogenic proteins or a significant risk of contracting such a disease.

[0009] In another aspect, a method is provided for phagocyte (e.g., microglia, astrocyte) replacement in a subject, the method comprising: depleting resident phagocytes (e.g., microglia), optionally with an MCSF-R inhibitor (e.g., such as PLX5622); and / or administering antigen-targeted, phagocytosis-inducing CAR transduced peripheral phagocytes (e.g., macrophage, monocyte, HSC) peripherally to the subject to repopulate the brain phagocytes.

[0010] In another aspect, a method is provided for generating phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells, the method comprising: providing phagocyte or phagocyte precursor cells; and / or transducing a phagocytosis-inducing, protein aggregatetargeting CAR via a viral vector or non-viral particle or molecule (e.g., a transposon) into the phagocyte cells or phagocyte precursor cells for an amount of time sufficient to virally transduce or non-virally transfect the phagocytosis-inducing, protein aggregate-targeting CAR into the phagocyte cells or phagocyte precursor, resulting in CAR-transduced or transfected phagocyte cells.

[0011] In another aspect, a method is provided for generating phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells, the method comprising: providing phagocyte cells; and / or transfecting a phagocytosis-inducing, protein aggregate-targeting CAR via a non-viral 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 thephagocyte cells, resulting in CAR-transduced phagocyte cells. In some aspects, the viral vector comprising a CAR is a CAR lentivirus or a CAR adeno-associated viral (AAV) vector. In some aspects, the viral vector is a lentiviral vector (LVV).

[0012] In another aspect, a method is provided for administering phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells to a subject in need thereof, the method comprising: isolating phagocyte cells from a subject or a donor; generating phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells; and / or administering a therapeutically effective amount of phagocytosis-inducing, protein aggregate-targeting CAR phagocyte cells into the subject.

[0013] In some aspects, the CAR construct comprises an antigen binding domain, or the antigen-binding domain comprises an A|3 binding domain. In some aspects, the antigen-binding domain comprises an scFv. In some aspects, the scFv is derived from (e.g., a truncated version or variant thereof) one or more Amyloid binding antibodies (e.g., Bapineuzumab, Crenezumab, Lecanemab, among others). In some aspects, the scFv is derived from (e.g., a truncated version or variant thereof) one or more Amyloid 13 binding antibodies that bind to Amyloid 13 fibrils present in CAA (Soederberg et al, Nature, 2024). In some aspects, the scFv is fused to the hinge or transmembrane domains from stable or non-dimerizing proteins (e.g., CD8, CD28). In some aspects, the antigen-binding domain is an scFv derived from the antigenbinding fragment (Fab) region of Crenezumab, Bapineuzumab or Lecanemab. In some aspects, the A0 binding domain comprises at least a portion, a fragment, or a variant of an a- A [3 antibody. In some aspects, the scFv, the hinge (if present), the transmembrane protein (if present), and the phagocytosis-inducing intracellular signaling domain are operably linked.

[0014] In some aspects, the scFv comprises a heavy chain (HC) Ig domain and / or a light chain (LC) Ig domain and a linker peptide. In some aspects, the HC or LC is derived from at least a portion, fragment, or variant of Crenezumab, Bapineuzumab, or Lecanemab antigen-binding fragments (Fabs). In some aspects, at least a portion, fragment, or variant of Crenezumab comprises a portion that interacts with Ap. In some aspects, the portion or variant thereof of the Crenezumab comprises the HC 13-16 N-terminus. In some aspects, the Ig domain is truncated at the terminal P-sheet. In some aspects, the hinge and / or the transmembrane domain, if present, supplies stability constraint. In some aspects, the hinge and / or the transmembrane domain, if present, are from or derived from stable and / or non-dimerizing proteins (e.g., CD8, CD28).

[0015] In some aspects, the intracellular domain is fused or operably linked to a linker, an extracellular domain, or a hinge. In some aspects, the intracellular domain is a phagocytosisinducing protein. In some aspects, the intracellular signaling domain is Mertk, CD3(^, MegflO, Dectinl, or CD19 PI3K. In some aspects, the intracellular signaling domain is a phagocytosisinducing protein, such as AXL, TYRO3, ME GF 10, DECTIN 1, MER, CD3c) Fc, CD64, or an Fc receptor. In some aspects, the intracellular signaling domain is MegflO. In some aspects, the intracellular signaling domain is Dectinl. In some aspects, the intracellular signaling domain induces phagocytosis of Ap upon crosslinking (or binding to AP). In some aspects, the peripheral immune cell is a monocyte or macrophage. In some aspects, the protein aggregate is pathological or associated with an NDD.

[0016] In some aspects, the NDD is associated with accumulation of a pathological protein, such as Amyloid B (AP), hyperphosphorylated tau, alpha-synuclein, or TDP-43. In some aspects, the NDD is Alzheimer’s Disease (AD), Parkinson's Disease (PD), dementia with Lewy bodies (LBD), multiple system atrophy (MSA), or Amyotrophic Lateral Sclerosis (ALS). In someaspects, the NDD is CAA. In some aspects, the CAR is expressed in myeloid cells such as monocytes or macrophages. In some aspects, the CAR is expressed in precursors of peripheral immune cells, for example a Hematopoietic Stem Cell (HSC) or other myeloid or myeloid precursors.

[0017] In some aspects, the CAR is transduced with a CAR-coding retrovirus (e.g., lentivirus) or an AAV vector or non-viral particle or transfected with a molecule (e.g., a transposon) that genetically codes for a CAR of any one of the preceding claims. In some aspects, the CAR is transfected with a CAR-coding nucleic acid using a nonviral particle or LNP. In some aspects, the CAR is transfected with a CAR-coding RNA using a nonviral nanoparticle or LNP. In some aspects, the CAR is transfected with a CAR-coding DNA using a nonviral nanoparticle or LNP. In some aspects, the expression of CAR is achieved by lentiviral transduction of brain resident phagocytes (e.g., macrophages, microglia, astrocytes). In some aspects, the CAR targets protein aggregates of Ap, hyperphosphorylated tau, alpha-synuclein, or TDP-43. In some aspects, the CAR prevents A0 deposition; prevents A0-associated pathology; only requires a single dose; binds AP; or induces phagocytosis of Ap. In some aspects, CAR-dependent clearance promotes phagocytosis through noninflammatory pathways.Antigen-Binding Domains

[0018] In some aspects, the CAR construct can comprise an antigen-binding domain or target aggregates such as Ap, hyperphosphorylated tau, alpha-synuclein, or TDP-43. In one aspect, an scFv can be used to bind antigens. Targeting antibody fragments or scFvs can be against any NDD-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 Ap, hyperphosphorylated tau, alpha-synuclein, or TDP-43. ScFvs are well known in the art to be usedas 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).Therapeutic Methods

[0019] According to the methods described herein, a therapeutically active CAR phagocyte can be generated ex vivo and re-introduced into a patient using adoptive cell transfer or generated in vivo by administering a delivery vehicle such as a nonviral particle or a LNP that can deliver the RNA or DNA construct to the desired phagocyte or phagocyte precursor. The methods described herein also include extracorporeal delivery of expression constructs, where blood cells are temporarily, and for a short time, removed from the patients, exposed to a delivery vehicle containing the CAR RNA or DNA, and reintroduced into the patient.Definitions

[0020] Use of the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0021] Terms such as “connected,” “attached,” “linked,” and “conjugated” are used interchangeably herein and encompass direct as well as indirect connection, attachment, linkage, or conjugation unless the context clearly dictates otherwise. Where a range of values is recited, each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither, or both limits are included is also encompassed. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, values thatare “about” (that is, within ±10%) the same quantity or amount as the recited value are also within the scope. Where a combination is disclosed, each sub-combination of the elements of that combination is also specifically disclosed. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element is disclosed as having a plurality of alternatives, examples in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0022] Unless defined otherwise herein, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the relevant art. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2ndEd., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY, 1991, provide one of skill with a general dictionary of many of the terms used herein. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The terms defined immediately below are more fully defined by reference to the specification as a whole.

[0023] The “configuration” of a polynucleotide means the functional sequence elements within the polynucleotide and the order and direction of those elements.

[0024] The terms “corresponding transposon” and “corresponding transposase” are used to indicate an activity relationship between a transposase and a transposon. A transposase transposases its corresponding transposon.

[0025] The terms “DNA sequence,” “RNA sequence,” or “polynucleotide sequence” refer to a contiguous nucleic acid sequence. The sequence can be an oligonucleotide of 2 to 20 nucleotides in length to a full-length genomic sequence of thousands or hundreds of thousands of base pairs.

[0026] The term “expression construct” means any polynucleotide designed to transcribe an RNA, such as, for example, a construct that contains at least one promoter that is or may be operably linked to a downstream gene, coding region, or polynucleotide sequence (for example, a cDNA or genomic DNA fragment that encodes a polypeptide or protein, or an RNA effector molecule, for example, an antisense RNA, triplex-forming RNA, ribozyme, an artificially selected high affinity RNA ligand (aptamer), a double-stranded RNA, for example, an RNA molecule comprising a stem-loop or hairpin dsRNA, or a bi-finger or multi-finger dsRNA or a microRNA, or any RNA). An “expression vector” is a polynucleotide comprising a promoter that can be operably linked to a second polynucleotide. Transfection or transformation of the expression construct into a recipient cell allows the cell to express an RNA effector molecule, polypeptide, or protein encoded by the expression construct. An expression construct may be a genetically engineered plasmid, virus, recombinant virus, or an artificial chromosome derived from, for example, a bacteriophage, adenovirus, adeno-associated virus, retrovirus, lentivirus, poxvirus, or herpesvirus. Such expression vectors can include sequences from bacteria, viruses, or phages. Such vectors include chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids, and phagemids. An expression construct can be replicated in a living cell, or it can be made synthetically. The terms “expression construct,” “expression vector,” “vector,” and “plasmid” are used interchangeably herein to demonstrate theapplication of the invention in a general, illustrative sense, and are not intended to limit the invention to a particular type of expression construct.

[0027] The term “expression polypeptide” means a polypeptide encoded by a gene on an expression construct.

[0028] The term “expression system” means any in vivo or in vitro biological system that is used to produce one or more gene product encoded by a polynucleotide.

[0029] A “gene transfer system” refers to a vector or gene transfer vector, i.e., a polynucleotide comprising the gene to be transferred which is cloned into a vector (a “gene transfer polynucleotide” or “gene transfer construct”). A gene transfer system may also comprise other features to facilitate the process of gene transfer. For example, a gene transfer system may comprise a vector and a lipid or viral packaging mix for enabling a first polynucleotide to enter a cell, or it may comprise a polynucleotide that includes a transposon and a second polynucleotide sequence encoding a corresponding transposase to enhance productive genomic integration of the transposon. The transposases and transposons of a gene transfer system may be on the same nucleic acid molecule or on different nucleic acid molecules. The transposase of a gene transfer system may be provided as a polynucleotide or as a polypeptide.

[0030] Two elements are “heterologous” to one another if not naturally associated. For example, a nucleic acid sequence encoding a protein linked to a heterologous promoter means a promoter other than that which naturally drives expression of the protein. A nucleic acid is heterologous to a cell if not naturally found in the cell or if naturally found in the cell but in a different location (e.g., episomal or different genomic location) than the location described.

[0031] The term “host” means any prokaryotic or eukaryotic organism that can be a recipient of a nucleic acid. A “host” includes prokaryotic or eukaryotic organisms that can be geneticallyengineered. For examples of such hosts, see Maniatis et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). As used herein, the terms “host,” “host cell,” “host system,” and “expression host” can be used interchangeably.

[0032] An “isolated” polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Polypeptides or polynucleotides may be purified, that is, essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities.

[0033] The terms “nucleoside” and “nucleotide” include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.

[0034] An “Open Reading Frame” or “ORF” means a portion of a polynucleotide that, when translated into amino acids, contains no stop codons. The genetic code reads DNA sequences in groups of three base pairs, which means that a double-stranded DNA molecule can read in any of six possible reading frames-three in the forward direction and three in the reverse. An ORF typically also includes an initiation codon at which translation may start.

[0035] The term “operably linked” refers to functional linkage between two sequences such that one sequence modifies the behavior of the other. For example, a first polynucleotide comprising a nucleic acid expression control sequence (such as a promoter, enhancer, or array oftranscription factor binding sites) and a second polynucleotide are operably linked if the first polynucleotide affects transcription and / or translation of the second polynucleotide. Similarly, a first amino acid sequence comprising a secretion signal, i.e., a subcellular localization signal, and a second amino acid sequence are operably linked if the first amino acid sequence causes the second amino acid sequence to be secreted or localized to a subcellular location.

[0036] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” “nucleic acid molecule,” and “gene” are used interchangeably to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. These terms refer only to the primary structure of the molecule. Thus, the terms include triple-, double-, and single-stranded DNA, as well as triple-, double-, and single-stranded RNA. The terms also encompass modified, for example by alkylation and / or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide that is an N-or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (for example, peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule,” and these terms are used interchangeably herein. These terms include, for example, 3’-deoxy-2’, 5’-DNA,oligodeoxyribonucleotide N3’ P5’ phosphoramidates, 2’-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof including for example hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, “caps,” substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, or the like) with negatively charged linkages (for example, phosphorothioates, phosphorodithioates, or the like), and with positively charged linkages (for example, aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (for example, nucleases), toxins, antibodies, signal peptides, poly-L-lysine, or the like), those with intercalators (for example, acridine, psoralen, or the like), those containing chelates (of, for example, metals, radioactive metals, boron, oxidative metals, or the like), those containing alkylators, those with modified linkages (for example, alpha anomeric nucleic acids, or the like), as well as unmodified forms of the polynucleotide or oligonucleotide.

[0037] A “promoter” means a nucleic acid sequence sufficient to direct transcription of an operably linked nucleic acid molecule. A promoter can be used together with other transcription control elements (for example, enhancers) that are sufficient to render promoter-dependent gene expression controllable in a cell type-specific, tissue-specific, or temporal-specific manner, or that are inducible by external signals or agents; such elements, may be within the 3’ region of a gene or within an intron. In one aspect, the promoter may be operably linked to a nucleic acid sequence, for example, a cDNA, a gene sequence, or an effector RNA coding sequence, in such a way as toenable expression of the nucleic acid sequence, or a promoter is provided in an expression cassette into which a selected nucleic acid sequence to be transcribed can be conveniently inserted.

[0038] The term “selectable marker” means a polynucleotide segment that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, a peptide, or a protein, or these markers can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds, or compositions. Examples of selectable markers include, but are not limited to: (1) DNA segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) DNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) DNA segments that encode products that suppress the activity of a gene product; (4) DNA segments that encode products that can be readily identified (e.g., phenotypic markers such as beta-galactosidase, GFP, and cell surface proteins); (5) DNA segments that bind products that are otherwise detrimental to cell survival and / or function; (6) DNA segments that otherwise inhibit the activity of any of the DNA segments described in Nos. 1-5 above (e.g., antisense oligonucleotides); (7) DNA segments that bind products that modify a substrate (e.g. restriction endonucleases); (8) DNA segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) DNA segments that encode a specific nucleotide sequence that can be otherwise non-functional (e.g., for PCR amplification of subpopulations of molecules); and / or (10) DNA segments, which when absent, directly or indirectly confer sensitivity to particular compounds.

[0039] A “transposase” is a polypeptide that catalyzes the excision of a corresponding transposon from a donor polynucleotide, for example a vector, and (providing the transposase is not integration-deficient) the subsequent integration of the transposon into a target nucleic acid.Non-limiting, suitable transposases are disclosed in U.S. Patent Nos. 10,233,454 and 11,060,098, each of which is incorporated herein by reference in its entirety.

[0040] The term “transposition” refers to the action of a transposase in excising a transposon from one polynucleotide and then integrating it, either into a different site in the same polynucleotide, or into a second polynucleotide.

[0041] The term “transposon” means a polynucleotide that can be excised from a first polynucleotide, for instance, a vector, and be integrated into a second position in the same polynucleotide, or into a second polynucleotide, for instance, the genomic or extrachromosomal DNA of a cell, by the action of a corresponding trans-acting transposase. A transposon comprises a first transposon end and a second transposon end, which are polynucleotide sequences recognized by and transposed by a transposase. A transposon usually further comprises a first polynucleotide sequence between the two transposon ends, such that the first polynucleotide sequence is transposed along with the two transposon ends by the action of the transposase. Natural transposons frequently comprise DNA encoding a transposase that acts on the transposon. Transposons as claimed herein are “synthetic transposons,” comprising a heterologous polynucleotide sequence that is transposable by virtue of its juxtaposition between two transposon ends.

[0042] The term “transposon end” means the cis-acting nucleotide sequences that are sufficient for recognition by and transposition by a corresponding transposase. Transposon ends of piggyBac-like transposons comprise perfect or imperfect repeats such that the respective repeats in the two transposon ends are reverse complements of each other. These are referred to as ITRs or terminal inverted repeats (“TIR”s). A transposon end may or may not include an additional sequence proximal to the ITR that promotes or augments transposition. Non-limiting, suitabletransposon ends are disclosed in U.S. Patent Nos. 10,233,454 and 11,060,098.

[0043] The term “vector,” “DNA vector,” or “gene transfer vector” refers to a polynucleotide that is used to perform a “carrying” function for another polynucleotide. For example, vectors are often used to allow a polynucleotide to be propagated within a living cell, to allow a polynucleotide to be packaged for delivery into a cell, or to allow a polynucleotide to be integrated into the genomic DNA of a cell. A vector may further comprise additional functional elements, such as, for example, a transposon.

[0044] The disclosure refers to several genes and proteins for which it provides an example “SEQ ID NO:.” Unless otherwise apparent from the context, reference to a gene or protein should be understood as including the specific SEQ ID NO, as well as allelic, species, and induced variants thereof having at least 90, 95, or 99% identity thereto.Prophetic ExamplesExample 1: In vivo reprogramming of monocytes via LNPs

[0045] Deploying anti-Ab CAR monocyte therapy to clear A0-aggregates in cerebral blood vessels could improve clearance of protein aggregates in the brain by restoring the natural clearance mechanisms of protein and cell debris and reducing vascular inflammation. LNPs are formulated using microfluidic mixing of a lipid-containing ethanol phase and a mRNA-containing aqueous phase via a microfluidic device. The ethanol phase is prepared by combining C 14-02 ionizable cationic lipid, l,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DOPE, Avanti Polar Lipids), cholesterol (Sigma), and a lipid anchored polyethylene glycol (C14-PEG 2000, Avanti Polar Lipids) at amol% of 35:16:46.5:2.5, respectively. The aqueous phase is prepared by diluting the anti-Ab-CAR mRNA to a concentration of .075 mg mL1in 10 mM citrate buffer (pH 3). Ethanol and aqueous phases are mixed at a 1:3 ratio using a single-channel staggered herringbonemicrofluidic mixing device. LNPs are collected in a 20 kDa MWCO dialysis cassette, dialyzed against lx PBS for 2 h, and sterile filtered using a .22 gm syringe filter (Thermo). The LNP-CAR particles are injected intravenously into a patient at 1 mg mRNA kg'1.Example 2: In vivo reprogramming of CD34+HSCs via LNPs

[0046] LNP (Kim et al, Nature Biotechnology, 2024 and US63 / 632,354, incorporated herein by reference in its entirety) is prepared by combining ionizable cationic lipid, 1,2-distearoyl- w-glycero-3-phospho-ethanolamine (DOPE, Avanti Polar Lipids), cholesterol (Sigma), and a lipid anchored polyethylene glycol (C14-PEG 2000, Avanti Polar Lipids). The aqueous phase is prepared by diluting the anti-Ab-CAR mRNA in 10 mM citrate buffer (pH 3). Ethanol and aqueous phases are mixed at a 1 :3 ratio using NanoAssembly Spark (Precision Nanosystems). The Ab-CAR-LNP composition is sterile-filtered with a .22 pm filter and injected intravenously at 0.25 mg kg'1.Example 3: Autologous adaptive transfer of transfected monocytes

[0047] Peripheral blood mononuclear cells (PBMCs) are isolated from human blood using Ficoll-Paque (GE Healthcare, Chicago, IL) or Leucosep (Greiner Bio-One, Monroe, NC) tubes and density gradient centrifugation. CD14+monocytes are isolated using CD14 MicroBeads (Milteny Biotec, San Diego, CA) and an autoMAC column (Milteny Biotec, San Diego). A gene construct encoding for the CAR construct is placed under the control of a CD1 lb promotor and inserted between the inverted terminal repeat sequences of a transposon (using, e.g., the Leap-In® transposase technology provided by DNA TWOPOINTO, Inc. dba ATUM). Insulator sequences derived from human D4Z4 locus and chicken b-globin are included at or near the 5’ and 3’ ends of the gene construct, respectively, to reduce or eliminate position effects within the transposon. The transposon vector is transfected into human monocytes together with in vitro transcribedmRNA encoding for Leapin® transposase (ATUM) using ThermoFisher Neon instrumentation according to the manufacturer’s instructions. Macrophages are derived by culturing CD 14+ monocytes in DenriMACS GMP medium supplemented with 120 ng / ml m-CSF for 6 days. Between 1 and 10xl06cells / kg are infused into the patient once a month for a period of 6 months. Cognition is assessed 3 months and 6 months following the first cell infusion using ADCOMS and ADAS-cog scores. Inclusion criteria for a patient include diagnosis of mild AD as assessed by Mini Mental Status exam (score 19-24) and confirmed presence of A0 aggregates as measured by either Seed Amplification Assay (SAA), e.g., SAAmplify® SAA from Amprion, Inc. or PrecivityAD®. Possible exclusion criterium is the presence of alpha-synuclein aggregates in the CSF as measured by SAA. CAR-MACs will be delivered by i.v infusion.Example 4: Autologous adaptive transfer of transfected monocytes derived from iPSCs

[0048] Human iPSCs (hiPSCs) are maintained in mTeSR-E8 to reach 80% confluence. On day 1, hiPSCs are dissociated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) for 5 min at room temperature to obtain small cell clumps. The cells are seeded into MatrigeL coated plates (75 mg / mL) at a density of 10,000 cell / cm2(1:30 split ratio). Cells are cultured in TeSR-E8 for 24 h and switched to IF9S medium, supplemented with 50 ng / mL BMP4 (R&D Systems), 15 ng / mL ACTIVIN A (Miltenyi Biotec), and 1.5 mM CHIR99021 (Axon Medchem) for the first 2 days (day 0 to day 2). On day 2, cells are refreshed with IF9S supplemented with 50 ng / mL VEGF (R&D Systems), 50 ng / mL bFGF (PeproTech), 50 ng / mL SCF (Miltenyi Biotec), and 10 mM SB431542 (Tocris Bioscience). On day 5 and day 7, cells are refreshed with IF9S supplemented with 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL SCF, 50 ng / mL IL-6 (Miltenyi Bio- tec), 50 ng / mL TPO (Miltenyi Biotec), and 10 ng / mL IL-3 (Miltenyi Biotec). On day 9, floating cells are collected, and adherent cells are dissociated with TrypLE (Life Technologies) for10 min at 37 °C. Floating and adherent cells are combined and resuspended in IF9S medium supplemented with 50 ng / mL IL-6, 10 ng / mL IL-3, and 80 ng / mL M-CSF (Miltenyi Biotec). Cells collected from one 12-well plate are plated into one 24-well ultra-low attachment plate (Corning Life Sciences). Medium is refreshed on day 13 and day 15 with IF9S medium containing 50 ng / mL IL-6, 10 ng / mL IL-3, and 80 ng / mL M-CSF. Cells are cultured at 37 °C, 5% CO2, under normoxia conditions throughout the differentiation. On day 15 of differentiation, all cells in suspension are collected and washed once with FACS buffer (PBS, 0.5% BSA, 2 mM EDTA). CD14+cells are isolated using CD14 MicroBeads (Miltenyi Biotec) following the manufacturer’s instructions; 60 mL of MicroBeads are used for 1 x 107total cells. Isolated CD14+cells are cryopreserved in CryoStor CS10 medium (STEMCELL Technologies) or further differentiated into macrophages. Monocyte phenotype is verified by antibody staining and flow cytometry (CD14+, CDllb+, CD18+, ICAM-1+, CD31+, CD105+, E-selectin+, VCAM-1+, VE-cadherin+). A CAR construct is introduced to the monocyte using either a LNP (as, by example, described in Example 1), transposon (as described in Example 3), or any other viral and transposon-based method known in the art. To generate macrophages, hiPSC-derived CD14+cells are plated on FCS-coated tissue culture plates at a density of 40,000 cells / cm2in IF9S medium supplemented with 80 ng / mL M-CSF. After 4 days of culture, all monocytes differentiate into macrophages (M0) with more than 90% confluency. M0 macrophages are polarized to Ml or M2 macrophages for 48 h in IF9S medium supplemented with different stimuli: 100 ng / mL LPS (Sigma) and 20 ng / mL IFN-g (Miltenyi Biotec) for Ml; 20 ng / m IL-4 (Miltenyi Biotec) for M2.

Claims

CLAIMS1. A method for generating phagocytosis-inducing, protein aggregate-targeting chimeric antigen receptor (CAR) phagocyte cells, the method comprising:a. providing phagocyte cells or phagocyte precursor cells;b. transducing or transfecting a phagocytosis-inducing, protein aggregate-targeting CAR via a viral vector or non-viral particle or non-viral molecule into the phagocyte cells or phagocyte precursor cells; andc. allowing an amount of time sufficient to virally transduce or tranfect the CAR into the phagocyte cells or phagocyte precursors, resulting in CAR-transduced or transfected phagocyte cells.

2. The method of claim 1, wherein the vector encoding for the CAR comprises a CAR lentivirus, a CAR adeno-associated viral (AAV) vector, non-viral lipid nanoparticle (LNP), or a transposon.

3. The method of claim 1, wherein the vector is a viral vector and comprises a lentiviral vector (LVV) or an AAV vector.

4. The method claim 1, wherein the vector is a non-viral vector and comprising a non-viral lipid nanoparticle (LNP) or a transposon.

5. The method of claim 1, wherein the CAR construct comprises an antigen binding domain.

6. The method of claim 5, wherein the antigen binding domain comprises an Ap binding domain.

7. The method of claim 6, wherein the A binding domain comprises at least a portion, a fragment, or a variant of an a-Ap antibody.

8. The method of claim 1, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

9. The method of claim 8, wherein the scFv is fused to the hinge or transmembrane domains from stable or non-dimerizing proteins.

10. The method of claim 8, wherein the scFv is derived from one or more Amyloid p binding antibodies.

11. The method of claim 8, wherein the scFv is derived from one or more Amyloid 3 binding antibodies that bind to Amyloid P fibril present in Cerebral Amyloid Angioapathy (CAA).

12. The method of claim 8, wherein the antigen-binding domain is an scFv derived from the antigen binding fragment (Fab) region of Crenezumab, Bapineuzumab, or Lecanemab.

13. The method of claim 9, wherein the scFv, the hinge, the transmembrane protein, and the phagocytosis-inducing intracellular signaling domain are operably linked.

14. The method of claim 13, wherein the hinge and / or the transmembrane domain, supply stability constraints.

15. The method of claim 13, wherein the hinge and / or the transmembrane domain are from or derived from stable and / or non-dimerizing proteins.

16. The method of claim 13, wherein the intracellular domain is fused or operably linked to a linker, an extracellular domain, or a hinge.

17. The method of claim 13, wherein the intracellular domain is a phagocytosis-inducing protein.

18. The method of claim 13, wherein the intracellular signaling domain is Mertk, CD3(^, Megfl 0, Dectinl, or CD19 PI3K.

19. The method of claim 13, wherein the intracellular signaling domain encodes for a phagocytosis-inducing protein, such as AXL, TYR03, MEGF10, DECTIN1, MER, CD3(^, Fc, CD64, or Fc receptor.

20. The method of claim 19, wherein the intracellular signaling domain is MEGF10.

21. The method of claim 19, wherein the intracellular signaling domain is DECTIN1.

22. The method of claim 13, wherein the intracellular signaling domain induces phagocytosis of Ap upon crosslinking to A .

23. The method of claim 8, wherein the scFv comprises a heavy chain (HC) Ig domain and / or a light chain Ig (LC) domain and a linker peptide.

24. The method of claim 23, wherein the HC or LC is derived from at least a portion, fragment, or variant of a Crenezumab, Bapineuzumab, or Lecanemab Fab.

25. The method of claim 24, wherein at least a portion, fragment, or variant of Crenezumab comprises a portion that interacts with Ap.

26. The method of claim 25, wherein the portion or variant thereof of the Crenezumab comprises the HC 13-16 N-terminus.

27. The method of claim 23, wherein the Ig domain is truncated at the terminal P-sheet.

28. A phagocytic cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises:a. an antigen-binding domain;b. optionally, a hinge;c. optionally, a linker;d. optionally, a transmembrane protein; ande. a phagocytosis-inducing intracellular signaling domain.

29. The phagocytic cell of claim 28, wherein the CAR comprises a protein aggregate-targeting, phagocytosis-inducing CAR.

30. The phagocytic cell of claim 28, wherein the CAR is expressed in myeloid cells such as monocytes or macrophages.

31. The phagocytic cell of claim 28, wherein the CAR is expressed in precursors of peripheral immune cells.

32. The phagocytic cell of claim 28, wherein the CAR is transduced with a CAR-coding retrovirus (e.g., lentivirus) or an adeno-associated viral (AAV) vector or transfected with a non-viral particle or molecule.

33. The phagocytic cell of claim 32, wherein the CAR is transfected with a CAR-coding nucleic acid using a nonviral particle or Lipid Nanoparticle (LNP) or transposon.

34. The phagocytic cell of claim 32, wherein the CAR is transfected with a CAR-coding RNA using a nonviral particle or Lipid Nanoparticle (LNP).

35. The phagocytic cell of claim 32, wherein the CAR is transfected with a CAR-coding DNA using a nonviral particle or Lipid Nanoparticle (LNP) or transposon.

36. The phagocytic cell of claim 28, wherein the expression of CAR is achieved by lentiviral transduction of brain resident phagocytes.

37. The phagocytic cell of claim 28, wherein the CAR targets protein aggregates Amyloid B (A0), hyperphosphorylated Tau, alpha-synuclein, or TDP-43.

38. The phagocytic cell of claim 28, wherein the CAR:a. prevents A0 deposition;b. prevents Ap-associated pathology;c. only requires a single dose;d. binds AP; ande. induces phagocytosis of A .

39. The phagocytic cell of claim 38, wherein the CAR promotes phagocytosis through noninflammatory pathways.

40. The phagocytic cell of claim 28, wherein:a. the antigen-binding domain of the CAR targets protein aggregates; orb. the phagocytosis-inducing intracellular signaling domain induces phagocytosis upon oligomerization; orc. the antigen-binding domain of the CAR targets protein aggregates and the phagocytosis-inducing intracellular signaling domain induces phagocytosis upon oligomerization.

41. The phagocytic cell of claim 28, wherein the phagocyte expressing the CAR is capable of phagocytosing the targeted protein aggregate.

42. A method for administering the phagocytic cells of claim 28 to a subject in need thereof, the method comprising:a. isolating phagocyte cells from a subject or a donor;b. generating the CAR phagocyte cells of claim 28; andc. administering a therapeutically effective amount of the CAR phagocyte cells into a subject.

43. The method of claim 42 wherein administration of a therapeutically effective amount of the CAR phagocyte cells into a subject clears the disease-causing or pathogenic protein aggregates in a subject having a neurodegenerative disease or pathogenic protein aggregates.

44. The method of claim 43, wherein , the pathogenic protein aggregate is pathological or associated with a neurodegenerative disease.

45. The method of claim 43, wherein the CAR phagocyte cells are monocytes, macrophages, Hematopoietic Stem Cells (HSC), or other myeloid or myeloid precursors.

46. A method for treating a neurodegenerative disease associated with the accumulation of disease-causing or pathogenic proteins, the method comprising administering the phagocytic cells of claim 28 to a subject having a neurodegenerative disease associated with accumulation of disease-causing or pathogenic proteins or having a significant risk of contracting such a disease.

47. The method of claim 46, wherein the neurodegenerative disease, disorder, or condition is associated with accumulation of a pathological protein, such as Amyloid B (A0), hyperphosphorylated Tau, alpha-synuclein, or TDP-43.

48. The method of claim 46, wherein the neurodegenerative disease is Alzheimer’s Disease, Parkinson's Disease, dementia with Lewy bodies, and / or multiple systems atrophies, or Amyotrophic Lateral Sclerosis (ALS).

49. The method of claim 46, wherein the neurodegenerative disease is Cerebral Amyloid Angioapathy (CAA).

50. A method of phagocyte replacement in a subject, comprising:a. depleting resident phagocytes, optionally with an MCSF-R inhibitor; and b. administering antigen-targeted, phagocytosis inducing CAR transduced peripheral phagocytes peripherally to the subject to repopulate the brain phagocytes.