Methods of treating amyloid pathology

Abstract

Methods and compositions are provided for treating Alzheimer's disease by administering interleukin-33 (IL-33) to a subject. Methods of gene therapy are also provided for expressing IL-33 in vivo in the brain of a subject in effective amounts sufficient to treat amyloid plaque pathology and reduce numbers of plaques in the brain.

Description

METHODS OF TREATING AMYLOID PATHOLOGYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(c) of provisional application 63 / 711,463, filed October 24, 2024, which application is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “UCSF-835WO” created on October 20, 2025, and having a size of 8,473 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entireties.INTRODUCTION

[0003] Microglia are essential players in neurodegenerative diseases including Alzheimer’s disease (AD), which is the most common neurodegenerative disorder and increasing in prevalence as the population ages. A major feature of Alzheimer’s disease is the development of amyloid plaques, which can build up in the brain for years before symptoms like impaired cognition and memory loss become apparent. Development of new therapies for Alzheimer’s disease is of tremendous clinical and public health interest. Recently, some therapies have come to market that target the amyloid plaques, but their benefits appear to be modest, and their mechanism of action is not entirely clear. Thus, there remains a need for better methods of treating Alzheimer’s disease.SUMMARY

[0004] Methods and compositions are provided for treating Alzheimer’s disease by administering interleukin-33 (IL-33) to a subject. Methods of gene therapy are also provided for expressing IL-33 in vivo in the brain of a subject in effective amounts sufficient to treat amyloid plaque pathology and reduce numbers of plaques in the brain.

[0005] In one aspect, a method of treating Alzheimer’s disease in a subject is provided, the method comprising administering a therapeutically effective amount of interleukin-33 (IL-33) to the subject, wherein the IL-33 lacks a functional nuclear localization sequence.

[0006] In certain embodiments, the IL-33 is administered prophylactically before amyloid plaques are detected in the subject.

[0007] In certain embodiments, the nuclear localization sequence is deleted.

[0008] In certain embodiments, the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO:5.

[0009] In certain embodiments, the IL-33 further comprises a deletion of a chromatinbinding motif comprising the sequence of SEQ ID NO:4.

[0010] In certain embodiments, the IL-33 comprises a deletion of amino acids at positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

[0011] In certain embodiments, the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO: 2.

[0012] In certain embodiments, the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least about 80-100% sequence identity to the amino acid sequence of SEQ ID NO:3, including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0013] In certain embodiments, the IL-33 is conjugated to a binding agent that specifically binds to amyloid beta. In some embodiments, the binding agent is an antibody or an antigenbinding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer. In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.

[0014] In certain embodiments, the IL-33 is provided by a recombinant nucleic acid or a vector comprising a coding sequence encoding the IL-33.

[0015] In certain embodiments, the recombinant nucleic acid is RNA or DNA. In some embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL- 33 in the subject.

[0016] In certain embodiments, the vector is a viral vector or a plasmid. Exemplary viral vectors include, without limitation, adeno-associated viral (AAV) vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors.

[0017] In certain embodiments, the viral vector is an AAV vector of an AAV serotype that can cross a blood brain barrier. In some embodiments, the AAV serotype is PHP.eB.

[0018] In certain embodiments, wherein the viral vector is an AAV vector, the AAV vector further comprises a 5’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5’ ITR and the 3’-ITR.

[0019] In certain embodiments, the method further comprises administering a helper virus vector to the subject. In some embodiments, the helper virus vector encodes Ela and Elb or E2a and E4.

[0020] In certain embodiments, the recombinant nucleic acid or vector is administered by retroorbital injection.

[0021] In certain embodiments, the recombinant nucleic acid or vector is administered locally to the brain.

[0022] In certain embodiments, the vector comprises a promoter operably linked to a coding sequence encoding the IL-33.

[0023] In certain embodiments, the promoter is an astrocyte-specific promoter. In some embodiments the astrocyte-specific promoter is a gfaABCID promoter.

[0024] In certain embodiments, the promoter is constitutive or inducible.

[0025] In certain embodiments, the coding sequence encoding the IL-33 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the IL-33 at the chromosomal locus.

[0026] In certain embodiments, the IL-33 is provided by a viral particle comprising a viral vector comprising the coding sequence encoding the IL-33.

[0027] In certain embodiments, the viral particle is administered locally to the brain of the subject.

[0028] In certain embodiments, the IL-33 is provided by genetically modifying the genome of an astrocyte in the brain of the subject to express the IL-33.

[0029] In certain embodiments, the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, the Cas nuclease is Cas9 or Casl2a.

[0030] In certain embodiments, the subject is a mammal. In some embodiments, the mammal is human.

[0031] In another aspect, a method of decreasing numbers of amyloid plaques in a brain of a subject is provided, the method comprising introducing into an astrocyte in the brain of the subject an effective amount of interleukin- 33 (IL-33), wherein the IL-33 lacks a functional nuclear localization sequence.

[0032] In another aspect, a composition comprising IL-33 or a recombinant nucleic acid or vector encoding IL-33 for use in a method of treating Alzheimer’s disease in a subject is provided, wherein the IL-33 lacks a functional nuclear localization sequence.

[0033] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0034] In another aspect, use of IL-33 or a recombinant nucleic acid or vector encoding IL-33 in the manufacture of a medicament or pharmaceutical composition for treating Alzheimer's disease, wherein the IL-33 lacks a functional nuclear localization domain, is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGS. 1A-1E. IL-33 signaling deficiency worsens amyloid pathology in a mouse model of amyloid deposition, a pathological hallmark of Alzheimer's Disease. FIG. 1A) Images and quantification of amyloid plaques (M0AB-2+, labeling all types of A[> forms) in the prefrontal cortex of 5xFAD and globally IL-33 deficient mice (Z / 53- / -:5xFAD). Welch’s t-test by sex. This shows that plaques are worse in the absence of IL-33. FIG. IB) Images and quantification of dense core plaques (Methoxy-X04+ compacted A0) in the dorsal prefrontal cortex of 5xFAD and / / 3J- / -: XI A mice. Welch’s t-test by sex. This data supports FIG. 1A and shows that compacted plaques are increase in the absence of IL-33. FIG. 1C) Images and quantification of LAMP1 + dystrophic neurites shows overlap with neurotoxic protofibrillar Ap (MOAB2+MXO4neg) in 5xFAD and / / 33- / -:5xFAD mice. Welch’s t-test by sex. In the correlation analysis between MOAB-2 and LAMP1, the difference between the slopes was analyzed through the simple linear regression test. This data shows that neuronal damage is worse in the absence of IL-33. FIG. ID) Images and quantification of M0AB-2+ amyloid plaques in the prefrontal cortex of 5xFAD vs 5xFAD mice that lack the IL-33 receptor on all cells ( / / 7r / 7- / -:5xFAD). Welch’s t-test by sex. This data shows that loss of receptor signaling phenocopies the effect in FIG. A, indicating that IL-33 is acting through its receptor, as would be expected in canonical signaling. FIG. IE) Images and quantification of plaque compaction, analyzed as the ratio of MOAB-2 (all types of A forms) and MX04 (dense core plaque) volume in 5xFAD and 7 / 53- / -:5xFAD mice. Welch’s t-test by sex. This data combines the information in A and B to shows that plaques are less compacted without IL-33, suggesting that neurotoxic protofibrillar Ap is increased. In FIGS. 1 A-1E, data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0036] FIGS. 2A-2D. IL-33 signaling promotes microglial encapsulation of amyloid plaques (a process known to be disrupted in mouse models lacking Alzheimer’s risk genes such as TREM2). FIG. 2A) Images showing microglia (IBA1+), 4G8 (all types of A ), and MX04 (dense core plaques). Inset (dotted box) shows the region where microglial processes contact and encapsulate the dense core plaques. Bottom row shows an enlargement of this inset to illustrate the surface contact of microglia with dense-core plaques, far right image shows a 3Dreconstruction used to calculate coverage of microglia on plaques. “Engulfed A[3” panel (top right) illustrates the images used to quantify phagocytosis of Ap by microglia (orange signal inside microglia) in 5xFAD and 1133 - / -:5xF AD mice. FIG. 2B) Quantification of the data represented in A shows percentage of microglial coverage of dense-core plaques and the correlation analysis between the percentage of surface area coverage and MX04+ dense-core plaque volume. This data shows that microglial coverage of plaques is reduced in the absence of IL-33, and that this effect is even more pronounced around small (early stage) plaques. FIG. 2C) Quantification of Ibal + plaque-associated microglia volume. Unpaired t-test. This data suggests that impaired contact with plaques is a result of defective microglial chemotaxis towards plaques, since there is a trend towards increased, not decreased microglial volume around plaques. FIG. 2D) Quantification of the percentage of engulfed A inside Ibal + microglia around plaques. This data shows that IL-33 may not act via changes in microglial phagocytosis. Unpaired t-test. In all panels data are mean ± SEM. Dots represent individual mice (black dot for male and white dot for female).

[0037] FIG. 3A-3B. Astrocytic IL-33 protein is decreased in human brains with late-stage Alzheimer’s Disease (AD). FIG. 3A) Images of human astrocytic IL-33 in the cortex of healthy controls and human AD brains (n = 4-5 / group). Unpaired t-test. FIG. 3B) Correlation assay between astrocytic IL-33 and the distance from amyloid plaques in human AD brains. This data shows that IL-33 is profoundly reduced in AD brains, and that this is modestly correlated with distance from plaques. This is relevant because it suggests that there may be a deficit of IL-33 signaling in human AD brains. All human AD brains at Braak stage V / VI. Data are mean ± SEM. Dots represent individual patients.

[0038] FIGS.4A-4E. Viral delivery of an active form of the cytokine Interleukin-33 into brain astrocytes reduces amyloid pathology in a mouse model and rescues short term memory.FIG.4A) Schematic of strategy to deliver an active form of IL-33 that lacks its nuclear localization signal (“ANLS”, amino acids 1-67) termed “IL-33 ANLS”, below are images of astrocytes labeled with a control virus expressing the control virus (AAV-gfaABClD-tdT, reporter only, left) and the IL-33ANLS delivery construct (AAV-PHP.eB-gfaABClD-IL-33ANLS-tdT, right). This shows that IL-33 is only in the nucleus in the control, but is localized in the cytoplasm of astrocytes after expression of the IL-33ANLS construct, which is likely to facilitate its release outside the cell. See Fig. 5 for more detail. FIG. 4B) Images and quantification of astrocytes labeled with reporter virus in the dorsal prefrontal cortex of wild-type and 5xFAD mice show effective delivery of viral construct into the brain and more robust expression induced by plaques in the 5xFAD model. Welch’s t-test by sex. This suggests that production of active IL-33 may undergo a selfamplifying effect near regions affected by plaque pathology, which would be anticipated tominimize off-target effects on bystander tissues. FIG. 4C) Experimental schematic of IL-33ANLS AAV-based gain-of-function strategy in 5xFAD model mice. Construct was delivered at six months, and behavior and plaque pathology analyzed at 8 months. FIG. 4D) Images and quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD mice injected with control (tdTomato) and IL-33ANLS virus. This shows that viral delivery of active IL-33 reduces plaque pathology. Analyzed by 2 way ANOVA. FIG.4E) Quantification of spatial working memory assessed by the Y-maze test (see schematic) comparing 5xFAD mice injected with control (tdTomato) and IL-33ANLS virus. This shows that active IL-33 rescues the impairment of working memory in 5xFAD mice. Analyzed by one way ANOVA. In all panels data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0039] FIGS. 5A-5B. Full plasmid maps. FIG. 5A) Map of “control virus” (AAV-gfaABClD-tdT) that expresses the TdTomato reporter (TdT) under control of an astrocyte promoter (gfaBCID) but does not express IL-33. FIG. 5B) map showing IL-33ANLS delivery construct (AAV-gfaABClD-IL-33ANLS-tdT) which expresses TdTomato and IL-33 ANLS, an active form of IL-33 that lacks its nuclear localization signal (“ANLS”, amino acids 1-67), which also has a small epitope tag for identification (HA). [Note that the image on the left has restriction enzyme sites labeled, whereas the right docs not, which is not relevant here.]

[0040] FIGS. 6A-6C. Early (prophylactic) administration of AAV-IL-33ANLS reduces amyloid deposition in the 5xFAD model. FIG.6A) Experimental schematic of IL-33 ANLS AAV-based gain-of-function strategy in early 5xFAD model mice. Construct was delivered at 2.5 months, and plaque pathology was analyzed at 5 months. FIG. 6B) Images and quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD mice injected with control (tdTomato) and IL-33ANLS virus. This data demonstrates that early viral delivery of active IL-33 reduces plaque pathology. FIG. 6C) Images and quantification of GFAP+ reactive astrocytes in 5xFAD mice injected with control and IL-33ANLS virus. Viral delivery of an active IL-33 does not induce astrogliosis. Analyzed by an unpaired t-test for each sex. All data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0041] FIGS. 7A-7B. Confirmation that the effects of AAV-IL-33ANLS act via the canonical IL-33 signaling pathway as they require expression of the IL-33 receptor IL1RL1 (a.k.a ST2). FIG. 7A) Experimental schematic of IL-33ANLS AAV-based gain-of-function strategy in IL-33 receptor-deficient 5xFAD model mice (5xFAD:ST2 / _). Construct was delivered at 5 months, and plaque pathology was analyzed at 7 months. FIG. 7B) Quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD:ST2 / _mice injected with control (tdTomato) and IL-33ANLS virus. This data demonstrates that viral delivery of active IL-33 aredependent on the IL-33 receptor in the brain. Analyzed by an unpaired t-test for each sex. All data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0042] FIGS. 8A-8B. Viral delivery of AAV-IL-33ANLS reduces amyloid pathology in an independent mouse model used to study Alzheimer’s disease (APP-SAA knockin, MGI: J:325368 / PMID: 35690868). Images (FIG. 8A) and quantification (FIG. 8B) of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of APP-SAA KI mice injected with control (tdTomato) and IL-33ANLS virus. Construct was delivered at 6 months, and plaque pathology was analyzed at 8 months. This data demonstrates that viral delivery of active IL-33 exerts similar effects in the other AD mouse model, APP-SAA KI mice. Analyzed by two-way ANOVA. All data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0043] FIGS. 9A-9H. No evidence of inflammatory lymphocyte infiltration or change in immune cell subsets after AAV-IL-33ANLS. This is relevant because it supports the idea that this therapeutic will not lead to unwanted inflammatory responses in the brain. Quantification of the number of CD45+ total immune cells or the frequency of lymphocytes in the brain. Analyzed by unpaired t-test. All data are mean ± SEM. Dots represent individual male mice. FIG. 9A) shows a schematic of the experimental protocol. FIGS. 9B-9H show quantification of FIG. 9B) CD45+ total immune cells, FIG. 9C) B cells, FIG.9D) natural killer (NK) cells and other group 1 innate lymphoid cells (ILC1), FIG.9E) CD8+ T cells, FIG. 9F) CD4+ T regulatory (Treg) cells, FIG.9G) CD4+ resting conventional T (Tconv) cells, and FIG. 9H) group 2 innate lymphoid cells (ILC2).

[0044] FIG. 10. Human microglia respond to IL-33. Representative images of Ibal + microglia in acute human brain slices treated with either saline or human recombinant DeltaNLS-IL-33. This data demonstrates that microglia in the adult human brain are capable of responding to Delta-NLS-IL-33, supporting its potential use as a human therapeutic.DETAILED DESCRIPTION

[0045] Methods and compositions are provided for treating Alzheimer’s disease by administering interleukin-33 (IL-33) to a subject. Methods of gene therapy are also provided for expressing IL-33 in vivo in the brain of a subject in effective amounts sufficient to treat amyloid plaque pathology and reduce numbers of plaques in the brain.

[0046] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein arc incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0049] It must be noted that as used herein and in the appended claims, the singular forms “a", “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins and reference to "the protein" includes reference to one or more proteins and equivalents thereof, e.g., polypeptides and peptides, known to those skilled in the art, and so forth.

[0050] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0051] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitionsof a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.

[0052] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.DEFINITIONS

[0053] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted as well as those in which prevention is desired.

[0054] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0055] The terms “individual”, “subject”, and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Mammals include human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development ofanimal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.

[0056] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0057] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0058] The term "survival" as used herein means the time from the start of treatment to the time of death.

[0059] By "therapeutically effective dose or amount" of IL-33 (e.g., IL-33 lacking a functional nuclear localization sequence (IL-33-ANLS) or a conjugate of IL-33 comprising a binding agent that binds to amyloid plaques) is intended an amount that, when administered, as described herein, brings about a positive therapeutic response with respect to treatment of an individual for amyloid plaque pathology such as occurs in Alzheimer’s disease or amyloidosis. A therapeutically effective dose or amount of IL-33 is an amount that decreases formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject. Additionally, a therapeutically effective dose or amount may improve short-term memory, locomotor ability, reasoning, and survival. In some embodiments, the IL-33 (e.g., IL-33-ANLS or conjugate) is provided by a recombinant polynucleotide (e.g., DNA or mRNA) or a vector encoding the IL-33, wherein the IL-33 is expressed in vivo in the brain of a subject in effective amounts sufficient for treatment of an individual for amyloid plaque pathology, including decreasing formation of amyloid plaques, the size of amyloid plaques, and / or numbers of amyloid plaques in the brain of a subject. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the type of condition and the severity of the condition being treated, the particular drug or drags employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may bedetermined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0060] The terms "protein", "peptide", and "polypeptide" refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and / or imino molecules. No particular size is implied by use of the terms "protein", "peptide", and "polypeptide", and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005: 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem Biol.7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for descriptions of peptoids). Non-limiting lengths of peptides suitable for use in the present invention includes peptides of 3 to 5 residues in length, 6 to 10 residues in length (or any integer therebetween), 11 to 20 residues in length (or any integer therebetween), 21 to 75 residues in length (or any integer therebetween), 75 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length. Typically, polypeptides useful in this invention can have a maximum length suitable for the intended application. Preferably, the polypeptide is between about 3 and 100 residues in length. Generally, one skilled in art can easily select the maximum length in view of the teachings herein. Further, peptides and polypeptides, as described herein, for example synthetic peptides, may include additional molecules such as labels or other chemical moieties.

[0061] Thus, references to polypeptides or peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequencefrom which they were derived. Such derivatives can include postexpression modifications of the polypeptide or peptide, for example, glycosylation, acetylation, phosphorylation, and the like.

[0062] Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., IL-33 biological activity such as ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: increasing IL-33 biological activity, increasing ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject, or facilitating purification, delivery, or cell processing. Proteins or biologically active fragments thereof can be made recombinantly, synthetically, or in tissue culture.

[0063] The term “interleukin-33” or “IL-33” as used herein encompasses all forms of IL-33, including isoforms 1-4, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (c.g., ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject; ability to induce secretion of T-helper type 2-associated cytokines).

[0064] An IL-33 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of IL-33 nucleic acid and protein sequences are known. A representative nucleotide sequence of a human mRNA encoding an IL-33 protein is presented in SEQ ID NO:1. A representative amino acid sequence of a human IL-33 protein is presented in SEQ ID NO:2. A representative amino acid sequence of a human IL-33 protein having an N-terminal deletion of amino acids 1-67 containing its NLS is presented in SEQ ID NO:3. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_00119964I, NM_001314044, NM_033439, NM_001353802, NM_001199640, NM_001314045, NM_001314048, NM_001314047, NM_001314046, NM_133775, NM_001127689, NM_001285978, NM_001131597, NP_001186569, NP_001186570, NP_001300973, NP_001300974, NP_001300975, NP_001347654, NP_001014188, NP_001272907, NP_001125069, and NP_001003l80; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any ofthese sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to produce a IL-33 protein or recombinant polynucleotide comprising a coding sequence encoding an IL-33 protein for use in the methods described herein.

[0065] In some embodiments, amino acids 46 to 67 are deleted to produce an IL-33 protein having its NLS deleted. In some embodiments, amino acids 1 to 67 are deleted to produce an IL-33 protein having its NLS deleted. Although the foregoing numbering is relative to the reference human IL-33 sequence of SEQ ID NO:2, it is to be understood that the corresponding positions in other IL-33 isoforms and IL-33 proteins obtained from other species are also intended to be encompassed by the present invention.

[0066] By "fragment" is intended a molecule consisting of only a part of the intact full-length sequence and structure. The fragment can include a C-terminal deletion, an N- terminal deletion, and / or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide will generally include at least about 50-100 contiguous amino acid residues of the full length molecule, but may include at least about 75-150 contiguous amino acid residues of the full length molecule, and can include at least about 100-200 or more contiguous amino acid residues of the full length molecule, or any integer between 50 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject).

[0067] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0068] By "isolated" is meant, when referring to a protein, polypeptide, or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as itexists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0069] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0070] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject, as described herein. In general, the terms “variant” and “analog” refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al., Chem Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same biological activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0071] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conscrvali vc replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or nonconservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp / Woods and Kyte-Doolittle plots, well known in the art.

[0072] By “derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0073] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0074] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482489, 1981 forpeptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin SequenceAnalysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0075] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0076] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra,' DNA Cloning, supra,' Nucleic Acid Hybridization, supra.

[0077] Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0078] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0079] 'Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0080] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0081] Typical " control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0082] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0083] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.

[0084] "Expression cassette" or "expression construct" refers to an assembly that is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassettegenerally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0085] 'Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0086] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0087] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non- viral vectors, particulate earners, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0088] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived frombacterial plasmid vectors, viral vectors, non- viral vectors, adeno-associated viruses, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0089] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.

[0090] By " recombinant virus" is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.

[0091] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV” includes any AAV serotype, such as, but not limited to, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc.

[0092] An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0093] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector contained within an rAAV particle.

[0094] "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of a viral particle.

[0095] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated vims. AAV rep and cap are referred to herein as AAV "packaging genes."

[0096] By "AAV rep coding region" is meant the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are required to replicate the viral genome and to insert the viral genome into a host genome during latent infection. The term also includes functional homologues thereof such as the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204, 304-311). For a further description of the AAV rep coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 7 3-801. The rep coding region, as used herein, can be derived from any viral serotype, such as those described above. The region need not include all of the wild-type genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the rep genes present provide for sufficient integration functions when expressed in a suitable recipient cell.

[0097] By "AAV cap coding region" is meant the art- recognized region of the AAV genome which encodes the coat proteins of the virus which are required for packaging the viral genome. For a further description of the cap coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 793-801. The AAV cap coding region, as used herein, can be derived from any AAV serotype, as described above. The region need not include all of the wild-type cap genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the genes provide for sufficient packaging functions when present in a host cell along with an AAV vector.

[0098] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2d ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" need not have the wild-type nucleotide sequence depicted in the previously cited references, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a hostcell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.

[0099] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild- type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV arc known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0100] "Helper virus function(s)" refers to function(s) encoded in a helper virus genome which allow viral AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.

[0101] An "infectious" virus or viral particle is one that comprises a polynucleotide component which is capable of delivering into a cell for which the viral species is tropic. The temi does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” vims or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the vims comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and / or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (EEISA). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle areknown in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0102] A "replication-competent" virus (e.g., a replication-competent AAV) refers to a phcnotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes.

[0103] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0104] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0105] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0106] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an exon or an intron of a gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0107] The Cas 9 protein naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)). Thus, the Cas9 associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression.

[0108] The term “CRISPR agent” as used herein encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and / or synthetic sequences, that can be usedin a Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).

[0109] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising asequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) .1. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0110] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0111] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0112] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be in an exon or an intron or a specific allele.

[0113] By "homology ami" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology ami that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology amis to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0114] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complcmcntary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0115] A “zinc-finger nuclease” or “ZFN” is an artificial DNA endonuclease generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October;40(10):759-65; Ekker et al, Zebrafish 2008 Summer; 5(2): 121 -3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91 ; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371 / journal.ponc.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA.2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZEN agent” encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0116] A “transcription activator-like effector nuclease” or “TALEN” is an artificial DNA endonuclease generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8) :731 -4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8): 695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[0117] "Administering" a nucleic acid, such as a vector encoding IL-33, a CRISPR system or vector encoding a CRISPR system, a guide RNA, a donor polynucleotide (e.g., for HDR), a vector encoding a ZFN, or a vector encoding a TALEN to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

[0118] A "ligand" or "binding agent" is any molecule that can be used to target IL-33 to an amyloid plaque, amyloid beta, or other target. In certain embodiments, the ligand is a molecule that selectively binds to a target analyte of interest (e.g., amyloid beta) with high binding affinity. By high binding affinity is meant a binding affinity of at least about IO-4M, usually at least about 10‘6M or higher, e.g., 10"9M or higher. The ligand may be any of a variety of different types of molecules, as long as it exhibits the requisite binding affinity for the target analyte when conjugated to IL-33. In certain embodiments, the ligand has medium or even low affinity for its target analyte, e.g., less than about IO"4M. As such, the ligand may be a small molecule or large molecule ligand. By small molecule ligand is meant a ligand having a size of less than 10,000 Daltons, usually ranging in size from about 50 to about 5,000 Daltons, and more usually fromabout 100 to about 1000 Daltons in molecular weight. By large molecule is meant a ligand having a size of more than 10,000 Daltons in molecular weight.

[0119] A small molecule ligand may be any molecule, as well as binding portion or fragment thereof, that is capable of binding with the requisite affinity to the target analyte of interest (e.g., cellular marker). Generally, the small molecule is a small organic molecule that is capable of binding to the target analyte of interest. The small molecule will include one or more functional groups necessary for structural interaction with the target analyte, e.g., groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions. Where the target analyte is a protein, the drug moiety will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The small molecule will also comprise a region that may be modified and / or participate in conjugation to an IL-33, without substantially adversely affecting the small molecule's ability to bind to its target analyte.

[0120] Small molecule ligands may comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule ligands may also include organic compounds comprising alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Small molecules may include structures found among biomolecules, including peptides, carbohydrates, fatty acids, vitamins, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0121] The small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0122] As such, the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule employed will have demonstrated some desirable affinity for the protein target in a convenient binding affinity assay. Combinatorial libraries, as well as methods for the production and screening, are known in the art and described in: U.S. Pat. Nos. 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference.

[0123] As pointed out, the ligand can also be a large molecule. Of particular interest as large molecule ligands are antibodies, as well as antigen-binding fragments and mimetics thereof. Also suitable for use as binding agents arc peptoids and aptamers. The ligand or binding agent may include a domain or moiety that can be covalently attached to IL-33 or other detectable label without substantially abolishing the binding affinity for its target analyte (e.g., amyloid-beta).

[0124] The term "antibody" encompasses monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. ( 1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); diabodies, tetrabodies, affibodies, camelid antibodies, humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep.1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.

[0125] "Fv" is an antibody fragment which contains an antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although often at a lower affinity than the entire binding site.

[0126] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see, for example, Pluckthun, A. in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0127] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains arc forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097: WO 93 / 11161: and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448.

[0128] The term "affibody molecule" refers to a molecule that consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. A monoclonal antibody, for comparison, is 150 kDa, and a single-domain antibody, the smallest type of antigen-binding antibody fragment, 12-15 kDa. See, for exemplary details of affibody structures and uses, Orlova, A; Magnusson, M; Eriksson, T L; Nilsson, M; Larsson, B; Hoiden-Guthenberg, I; Widstrom, C; Carlsson, J et al. (2006). "Tumor imaging using a picomolar affinity IIER2 binding affibody molecule", Cancer Res. 66 (8): 4339-48. Exemplary Affibody. Molecules are commercially available from Abeam Corp. Cambridge Mass.

[0129] The phrase "specifically (or selectively) binds" with reference to binding of an antibody or other binding agent to an antigen or analyte (e.g., amyloid beta) refers to a binding reaction that is determinative of the presence of the antigen or analyte in a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified antibodies or other binding agents bind to a particular antigen or analyte at at least two times the background and do not substantially bind in a significant amount to other molecules present in thesample. Specific binding to an antigen or analyte under such conditions may require an antibody or other binding agent that is selected for its specificity for a particular antigen or analyte. For example, antibodies raised to an antigen from specific species such as rat, mouse, or human can be selected to obtain only those antibodies that arc specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

[0130] The term "conjugated" refers to the joining by covalent or noncovalent means of two compounds or agents (e.g., binding agent specific for amyloid beta conjugated to IL-33).Methods of Treating Amyloid Pathology with IL-33

[0131] Applicant has shown that amyloid pathology can be treated by administering IL-33 lacking a functional nuclear localization sequence. Without being bound by theory, interleukin-33 is expressed in the brain but is not easily secreted from cells due to its nuclear localization sequence, which results in localization of IL-33 in the nucleus. Removing the nuclear localization sequence increases secretion of IL-33 and its bioactivity in the brain. Treatment of amyloid pathology with IL-33 lacking a functional nuclear localization sequence decreases formation of amyloid plaques, the size of amyloid plaques, and the numbers of amyloid plaques in the brain of a subject. IL-33 lacking a functional nuclear localization sequence can also be administered prophylactically (before amyloid plaques are detected) to prevent or reduce amyloid deposition and amyloid plaque formation.

[0132] The nuclear localization sequence of IL-33 may be inactivated or deleted such that the IL-33 no longer localizes to the nucleus. In certain embodiments, the IL-33 further comprises a deletion of its chromatin-binding motif, MXLRSG (SEQ ID NO:4). In an exemplary embodiment, the IL-33 comprises a deletion of the nuclear localization sequence at amino acid positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2. In another exemplary embodiment, the IL-33 comprises an N-terminaldeletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

[0133] In certain embodiments, the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least about 80-100% sequence identity to the amino acid sequence of SEQ ID NO:3, including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.Conjugation of IL-33 to a Binding Agent

[0134] In certain embodiments, the IL-33 is conjugated to a binding agent that localizes the IL-33 to amyloid plaques. For example, IL-33 may be conjugated to any agent that specifically binds to an amyloid-beta peptide. Amyloid beta (A0 or Abeta) peptides such as A 36-43 are components of amyloid plaques associated with Alzheimer’ s disease. In some embodiments, the binding agent binds specifically to one or more amyloid peptides selected from the group consisting of AP36, A|337, A^38, AP39, AfMO, AP41, A[342, and AP43. In some embodiments, the binding agent binds to an amyloid-beta peptide with high affinity. Examples of binding agents include, without limitation, antibodies, antibody fragments, antibody mimetics, and aptamers as well as small molecules, peptides, peptoids, or ligands that bind selectively to amyloid plaques. The conjugates used in the subject methods comprise IL-33 attached to one or more binding agents that specifically bind to amyloid plaques. In some embodiments, the IL-33 in the conjugate lacks a functional nuclear localization sequence.

[0135] In certain embodiments, the binding agent comprises an antibody that specifically binds to amyloid-beta. Any type of antibody may be used in IL-33 conjugates, including, without limitation, monoclonal antibodies, polyclonal antibodies, as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. Antibodies may include hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883): nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); diabodies, tetrabodies, affibodies, camelid antibodies, humanized antibody molecules (see, e.g., Riechmannet al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.

[0136] In other embodiments, the binding agent comprises an aptamer that specifically binds to amyloid-beta. Any type of aptamer may be used, including a DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that specifically binds to the tumor antigen. Such aptamers can be identified, for example, by screening a combinatorial library. Nucleic acid aptamers (e.g., DNA or RNA aptamers) that bind selectively to a target tumor antigen can be produced by carrying out repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). Peptide aptamers that bind to a marker of interest may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection. For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Nucleic Acid Aptamers: Selection, Characterization, and Application (Methods in Molecular Biology, G. Mayer cd., Humana Press, 2016), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan. X. Fang eds., Springer, 2015), Cox et al. (2001) Bioorg. Med. Chem. 9(10):2525-2531; Cox et al. (2002) Nucleic Acids Res. 30(20): el08, Kenan et al. (1999) Methods Mol. Biol.118:217-231; Platelia et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304-4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9): 1440-1452; herein incorporated by reference in their entireties.

[0137] In other embodiments, the binding agent comprises an antibody mimetic that specifically binds to amyloid-beta. Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS I. 275 (ll):2668-2676), affilins (Ebersbach et al. (2007) I. Mol. Biol. 372 (1): 172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5): 1058- 1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (ll):2677-2683), avimers (Silverman et al. (2005) Nat. Biotechnol. 23 (12): 1556-1561), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15- 16):695-701), fynomers (Grabulovski et al. (2007) J. Biol. Chem. 282 (5):3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).

[0138] In other embodiments, the binding agent comprises a small molecule ligand. Small molecule ligands encompass numerous chemical classes, e.g., small organic compounds having amolecular weight of less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. The small molecule will include one or more functional groups necessary for structural interaction with the target analyte, e.g., groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions. Where the target analyte is a protein (e.g., amyloidbeta), the ligand will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, or preferably at least two of the functional chemical groups. The small molecule may also comprise a region that may be modified and / or participate in conjugation to a IL-33, without substantially adversely affecting the small molecule's ability to bind to its target analyte.

[0139] Small molecule ligands can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule ligands may also include organic compounds comprising alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tctracylincs, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Small molecule ligands are also found among biomolecules including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. The small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0140] As such, the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means,i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule employed will have demonstrated some desirable affinity for the protein target in a convenient binding affinity assay. Combinatorial libraries, as well as methods for the production and screening, arc known in the art and described in: U.S. Pat. Nos. 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5.641,862; 5,639,603; 5.593,853; 5,574,656; 5,571.698; 5.565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference.

[0141] IL-33 may be conjugated to binding agents by any suitable method. In some instances, the IL-33 and binding agent may be directly linked, e.g., via a single bond, or indirectly linked e.g., through the use of a suitable linker, e.g., a polymer linker, a chemical linker, or one or more linking molecules or moieties. In some instances, attachment of the IL-33 and binding agent may be by way of one or more covalent interactions. In some instances, the IL-33 or binding agent may be functionalized, e.g., by addition or creation of a reactive functional group. Functionalized IL-33s or binding agents may be modified to contain any convenient reactive functional group for conjugation such as an amine functional group, a carboxylic functional group, a sulfhydryl group, a thiol functional group, and the like.

[0142] Any convenient method of bioconjugation may be used including, but not limited to, glutaraldehyde crosslinking, carbodiimide crosslinking, succinimide ester crosslinking, imidoester, crosslinking, maleimide crosslinking, iodoacetamide crosslinking, benzidine crosslinking, periodate crosslinking, isothiocyanate crosslinking, and the like. Such conjugation methods may optionally use a reactive sidechain group of an amino acid residue of the binding agent (e.g., a reactive side-chain group of a Lys, Cys, Ser, Thr, Tyr, His or Arg amino acid residue of the protein, i.e., a polypeptide linking group may be amino-reactive, thiol-reactive, hydroxylreactive, imidazolyl-reactive or guanidinyl-reactive). In some cases, a chemoselective reactive functional group may be utilized. Other conjugation reagents that can be used include, but are not limited to, e.g., homobi functional conjugation reagents (e.g., (bis(2- [succinimidooxycarbonyloxy]ethyl) sulfone, l,4-Di-(3'-[2'pyridyldithio]-propionamido) butane, disuccinimidyl suberate, disuccinimidyl tartrate, sulfodisuccinimidyl tartrate, dithiobis(succinimidyl propionate), 3,3’-dithiobis(sulfosuccinimidyl propionate), ethylene glycol bis(succinimidyl succinate), and the like), heterobifunctional conjugation reagents (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-y-maleimidobutyryloxysuccinimide ester, N-y-maleimidobutyryloxysulfosuccinimide ester, N-(8-maleimidocaproic acid) hydrazide, N-(e-maleimidocaproyloxy) succinimide ester, N-(8-maleimidocaproyloxy) sulfo succinimide ester, N-(p-maleimidophenyl) isocyanate, N-succinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, succinimidyl 4-(p-maleimidophenyl) butyrate, N-sulfosuccinimidyl(4-iodoacctyl)aminobcnzoatc, sulfosuccinimidyl 4-(N-malcimidomcthyl) cyclohexane-l-carboxylate, sulfo succinimidyl 4-(p-maleimidophenyl) butyrate, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, maleimide PEG N-hydroxysuccinimide ester, and the like), photoreactive conjugation reagents (e.g., p-azidobenzoyl hydrazide, N-5-azido-2-nitrobenzyloxysuccinimide, p-azidophenyl glyoxal monohydrate, N-(4-[p-azidosalicylamido]butyl)-3'-(2'-pyridyldithio) propionamide, bis(P-[4-azidosalicylamido]-ethyl) disulfide, N-hydroxysuccinimideyl-4-azidosalicyclic acid, N-hydroxysulfosuccinimidyl-4-azidobenzoate, sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l,3-dithiopropionate, azido phenyl 2-(m-azido-o-nitrobenzamido)-ethyl-l,3'-propionate, sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, sulfosuccinimidyl (4- azidophenyl dithiojpropionate, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-l,3-dithiopropionate, and the like).

[0143] In some instances, attachment of a IL-33 to a binding agent of interest is mediated by one or more functional linkers. A functional linker, as used herein, refers to any suitable linker that has one or more functional groups for the attachment of one molecule to another. For example, in some instances the functional linker comprises an amino functional group, a thiol functional group, a hydroxyl functional group, an imidazolyl functional group, a guanidinyl functional group, an alkyne functional group, an azide functional group, or a strained alkyne functional group. Further exemplary functional groups and methods of crosslinking and conjugation are described in, e.g., Hermanson Bioconjugnte Techniques (Academic Press, 3rdedition, 2013), herein incorporated by reference in its entirety.Production of IL-33

[0144] IL-33 proteins (or biologically active fragments thereof) can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, labeled, lipidated, amidated, acetylated, PEGylated, etc.). The IL-33 proteins may include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing proteins are well understood in the art. Proteins are preferablyprepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).IL-33 nucleic acid and protein sequences may be derived from any source. A number of IL-33 nucleic acid and protein sequences arc known. A representative nucleotide sequences of a human mRNA encoding an IL-33 protein is presented in SEQ ID NO:1, and a representative amino acid sequence of a human IL-33 protein is presented in SEQ ID NO:2. A representative amino acid sequence of a human IL-33 protein having an N-terminal deletion of amino acids 1-67 is presented in SEQ ID NO:3. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM_001199641, NM_001314044, NM_033439, NM_001353802, NM_0() 1199640, NM_001314045, NMJ101314048, NM_001314047, NM_001314046, NM_133775, NM_001127689, NM_001285978, NM_001131597, NP_001186569, NP_001186570, NP_001300973, NP_001300974, NP_001300975, NP_001347654, NP_001014188, NP_001272907, NP_001125069, and NP_001003180; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, or a biologically active fragment thereof, wherein the variant or biologically active fragment thereof has IL-33 biological activity (e.g., ability to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject) can be used to produce a IL-33 protein or recombinant polynucleotide comprising a coding sequence encoding an IL-33 protein for use in the methods described herein.

[0145] In certain embodiments, the IL-33 lack a functional nuclear localization sequence. For example, the nuclear localization sequence may be inactivated or deleted such that the IL-33 no longer localizes to the nucleus. In certain embodiments, the IL-33 further comprises a deletion of the chromatin-binding motif, MXLRSG (SEQ ID NO:4). In an exemplary embodiment, the IL-33 comprises a deletion of the nuclear localization sequence at amino acid positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2. In another exemplary embodiment, the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO: 2.

[0146] In certain embodiments, the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least about 80-100% sequenceidentity to the amino acid sequence of SEQ ID N0:3, including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0147] In some embodiments, IL-33 is generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode the desired proteins using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0148] The sequences encoding proteins can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g.. Edge (1981) Nature 292:756: Nambair etal. (1984) Science 223:1299; Jay etal. (1984) J. Biol. Chem. 259:6311; Stemmer et al. (1995) Gene 164:49-53.

[0149] Recombinant techniques are readily used to clone sequences encoding proteins that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0150] Once coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent fromthe teachings herein, a wide variety of vectors encoding modified proteins can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding proteins having deletions or mutations therein.

[0151] Numerous cloning vectors arc known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage (E. coll), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV 1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 Bacillus), pIJ61 (Slreplomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra,' Sambrook et al., supra,' B. Perbal, supra.

[0152] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. J555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).

[0153] Plant expression systems can also be used to produce the IL-33 proteins. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139:1-22.

[0154] Viral systems, such as a vaccinia-based infection / transfection system, as described in Tomei et aL, J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)74:1103-1113. will also find use with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0155] The IL-33 gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. Thecoding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Such sequences include, but arc not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0156] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0157] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0158] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0159] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CIIO) cells, IleLa cells, baby hamster kidney (BIIK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyc.es fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0160] Depending on the expression system and host selected, the fusion proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0161] In one embodiment, the transformed cells secrete the IL-33 protein product into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TP A) leader sequence, an interferon (y or a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted IL-33 protein product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0162] Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides or polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0163] For example, methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze -thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pretreatment used.

[0164] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides or polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0165] For example, one method for obtaining the intracellular peptides or polypeptides of the present invention involves affinity purification, such as by immunoaffinity chromatographyusing antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the peptides or polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.

[0166] The IL-33 proteins can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and Peter D. White eds., Oxford University Press, 1stedition, 2000) ; N. Leo Benoiton, Chemistry’ of Peptide Synthesis (CRC Press; 1stedition, 2005); Peptide Synthesis and Applications (Methods in Molecular Biology, John Howl ed., Humana Press, 1sted„ 2005); and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor & Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1stedition, 1999); herein incorporated by reference.

[0167] In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final peptide or polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and .1. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, (Springer- Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., ThePeptides: Analysis, Synthesis, Biology, Vol. 1, for classical solution synthesis. These methods are typically used for relatively small polypeptides, i.e., up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0168] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t- amyloxy carbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like.

[0169] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and di vinylbenzene-benzhydrylaminopoly styrene copolymers .

[0170] The IL-33 proteins can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Patent No. 4,631,211.Recombinant Nucleic Acids and Vectors

[0171] Recombinant nucleic acids and vectors comprising coding sequences encoding IL-33 (e.g., IL-33 lacking a functional nuclear localization sequence) are provided. In certain embodiments, an RNA or DNA comprising a coding sequence encoding IL-33 is provided. In certain embodiments, a recombinant nucleic acid or a vector comprising a coding sequence encoding IL-33 is administered to a subject, wherein the IL-33 is expressed in vivo in the subject in an effective amount sufficient to decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject.

[0172] Methods of introducing a nucleic acid (e.g., DNA or RNA) such as a recombinant nucleic acid comprising a coding sequence encoding IL-33 or a recombinant expression vector comprising a coding sequence encoding IL-33 into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., nucleic acid delivery by encapsulation in lipid nanoparticles (LNPs), viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. Introducing a recombinant nucleic acid or expression vector into a cell or cells can occur in any culture mediaand under any culture conditions that promote the survival of the cells. Introducing the recombinant nucleic acid or expression vector into a target cell can be carried out in vivo, ex vivo or in vitro.

[0173] In some embodiments, an IL-33-cncoding nucleic acid can be provided as RNA, such as a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the IL-33). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., encapsulated in LNPs, microinjection, electroporation, transfection, etc.). Nucleic acids may be provided to the cells using well-developed transfection techniques: see, e.g., Angel and Yanik (2010) PLoS One 5(7): el 1756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Minis Bio LLC. See also Beumer et al. (2008) Proc. Natl. Acad. Sci. USA 105(50): 19821-19826. In some embodiments, nucleic acids are introduced into cells by encapsulation in LNPs. For a description of LNP transfection techniques, see, e.g., Wang et al. (2023) J. Mater. Chem. B. 11 (23):5083-5093, Pozzi et al. (2023) ACS Pharmacol Trans! Sci.6(11): 1561-1573, and del Pozo-Rodngucz et al. (2011) Recent Pat Drug Deliv Formal. 5(3):214-26. LNPs for nucleic acid delivery are commercially available, for example, from LipExoGen Biotech (Baltimore, MD), Avanti Polar Lipids, Inc. (Alabaster, AL), Lonza Biologies (Hayward, CA), and Exelead, Inc. (Indianapolis, IN).

[0174] A vector may be provided directly to a target host cell, for example, by contacting the host cell with the vector (e.g., a recombinant expression vector comprising a coding sequence encoding IL-33) such that the vector is taken up by the cells. Methods of transfecting cells are well known in the art, and include, without limitation, electroporation, calcium chloride transfection, microinjection, and lipofection. For viral vector delivery, cells can be contacted with viral particles comprising viral expression vectors.

[0175] Nucleic acids encoding IL-33 can be inserted into an expression vector to create an expression cassette capable of producing the IL-33 in a suitable host cell. The ability of constructs to produce the IL-33 can be empirically determined. Expression cassettes typically include control elements operably linked to a coding sequence, which allow for the expression of a gene in vivo in the subject species. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0176] Promoters can be used to drive expression by an RNA polymerase (e.g., pol I, pol II, pol III). Suitable promoters can be derived from viruses (i.e., viral promoters) or an organism, including prokaryotic or eukaryotic organisms. Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMV1E), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), and human Hl promoter (Hl), and the like.

[0177] The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state) or an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF” is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). In some cases, a promoter is a spatially restricted promoter (e.g., tissue-specific promoter or cell type-specific promoter controlled by a transcriptional control element, enhancer, etc.). In some cases, a promoter is a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[0178] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0179] In some cases, the promoter is a spatially restricted promoter (i.e., cell type-specific promoter, tissue-specific promoter, organ-specific, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may be regulated by enhancers, transcriptional control elements, control sequences, etc. Any convenientspatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell). In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in a brain cell such as an astrocyte or other targeted cell.

[0180] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. A suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88: 1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

[0181] For illustration purposes, examples of spatially restricted promoters include, but are not limited to, brain- specific promoters, astrocyte-specific promoters, and neuron-specific promoters, and the like.

[0182] In some embodiments, the promoter is an astrocyte-specific promoter. Examples of astrocyte-specific promoters include, but are not limited to, the gfaABCID promoter (Heffernan et al. (2022) J Neurosci Methods 372:109530), the human glial fibrillary acidic protein(GFAP) promoter, gfa2 (Lee et al. (2008) Glia 56(5) :481 -93), and the human ALDH1L1 promoter (Koh et al. (2017) Exp Neurobiol 26(6):350-361).

[0183] In some embodiments, the promoter is a neuron-specific promoter. Examples of neuron-specific promoters include, but arc not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. No. 6,649,811, U.S. Pat. No. 5,387,742); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1161); a serotonin receptor promoter (see, e.g., GenBank S62283): a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384(1987) and Neuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMV enhancer / platelet-derived growth factor-. beta, promoter (see, e.g., Liu et al. (2620) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee ct al. (2620) Development 131:3295-3306); an alpha subunit of Ca2+-calmodulin-dependent protein kinase II (CaMKII) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250), and a retinal ganglion cell Nefh promoter (see, e.g., Hanlon et al. (2017) Front Neurosci. 11:521). Other suitable promoters include elongation factor (EF) 1 and dopamine transporter (DAT) promoters, and the like.

[0184] In some embodiments, cell subtype-specific expression of IL-33 is achieved by using a recombination system, e.g., Cre-Lox recombination, Flp-FRT recombination, etc. Cell type-specific expression of genes using recombination has been described in, e.g., Fenno et al., Nat Methods, 2014 July; 11(7):763; Gompf et al., Front. Behav. Neurosci. 2015 Jul. 2;9:152, and McCarthy et al. (2012) Skelet. Muscle. 2(1):8; which are herein incorporated by reference.

[0185] Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator / polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0186] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4:761, the enhancer / promoter derived from the long terminal repeat (LTR)of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[0187] Additionally, 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997) 22 150-161. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis vims (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), the polio leader sequence, the hepatitis A vims leader, the hepatitis C vims IRES, human rhinovims type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), an IRES element from the foot and mouth disease vims (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavims IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J.363(Pt l):37-44). These elements are readily commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES stmetures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple protein products in combination.

[0188] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products from a single vector. 2A linker peptides are inserted between the coding sequences in the multicistronic constmct. The 2A peptide, which is selfcleaving, allows co-expressed proteins from the multicistronic constmct to be produced at equimolar levels. 2A peptides from various vimses may be used, including, but not limited to 2Apeptides derived from the foot-and-mouth disease virus, equine rhinitis A vims, Thosea asigna vims and porcine teschovirus-1. See, e.g., Kim et al. (2011) PLoS One 6(4):e 18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001) Gene Ther. 8(11):864-873; herein incorporated by reference in their entireties.

[0189] In certain embodiments, cells containing a construct encoding IL-33 is identified in vitro or in vivo by including a selection marker expression cassette in the construct. Selection markers confer an identifiable change to the cell permitting positive selection of cells having the construct. For example, fluorescent or bioluminescent markers (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein, blue fluorescent protein, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin, or luciferase), cell surface markers, expression of a reporter gene (e.g., GFP, dsRed, GUS, lacZ, CAT), drug selection markers such as genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol may be used to identify cells. Alternatively, enzymes such as herpes simplex vims thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Any selectable marker may be used as long as it is capable of being expressed in the cell to allow identification of cells containing the constmct. Further examples of selectable markers are well known to one of skill in the art.

[0190] In certain embodiments, the selection marker expression cassette encodes two or more selection markers. Selection markers may be used in combination, for example, a cell surface marker may be used with a fluorescent marker, or a drag resistance gene may be used with a suicide gene. In certain embodiments, the selection marker expression cassette is multicistronic to allow expression of multiple selection markers in combination. The multicistronic vector may include an IRES or viral 2A peptide to allow expression of more than one selection marker from a single vector.

[0191] In certain embodiments, a suicide marker is included as a negative selection marker to facilitate negative selection of cells. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drag to a toxic compound in genetically modified cells. Examples include suicide genes encoding thymidine kinases, cytosine deaminases, intracellular antibodies, telomerases, caspases, and DNases. In certain embodiments, a suicide gene is used in combination with one or more other selection markers, such as those described above for use in positive selection of cells. In addition, a suicide gene may be used in cells containing constructs expressing IL-33, for example, to improve their safety by allowing their destraction at will. See, e.g., lones et al. (2014) Front. Pharmocol. 5:254, Mitsui et al. (2017)Mol. Ther. Methods Clin. Dev. 5:51-58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference.

[0192] Once complete, the construct encoding IL-33 can be administered to a subject using standard gene delivery protocols. Methods for gene delivery arc known in the art. Sec, c.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.

[0193] A number of viral based systems have been developed for gene transfer into mammalian cells. Suitable vectors include viral vectors based on adeno-associated virus (AAV) (see, e.g., Li et al. (2020) Nat Rev Genet. 21(4):255-272, Balakrishnan et al. (2014) Curr. Gene Ther. 14(2):86-100, Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sei 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:38223828; Mendelson et al., Virol. (1988) 166:154165; and Flotte et al., PNAS (1993) 90:1061310617); rabies virus (e.g., Wickersham et al. (2007) Nat Methods. 4(l):47-49, Wickersham et al. (2007) Neuron. 53(5):639-647, Hagendorf et al. (2015) Cold Spring Harb Protoc.2015(12):pdb.prot089417, Suzuki et al. (2020) Front Neural Circuits 13:77, Osakada et al. (2013) Nat. Protoc. 8(8):1583-601); adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); SV40; herpes simplex virus (e.g., Artusi et al. (2018) Diseases 14;6(3):74, Lachmann (2004) Int. J. Exp. Pathol. 85(4): 177-90); human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); vaccinia vims (e.g., Xie et al. (2022) Vaccine 40(49):7022-7031, Yang et al. (2018) J Cancer Res Clin Oncol. 144(12):2433-2440, Mackett et al. (1986) J Gen Virol. 67 ( Pt 10):2067-82); poliovirus (e.g., Girard et al. (193) Biologicals 21(4):371-7); anellovirus (Prince et al. (2024) biorxiv.org / content / 10.1101 / 2024.03.27.586964vl); and retroviruses, including lentivirus (e.g., Cockrell et al. (2007) Mol. Biotechnol. 36(3):184-204, Wang et al. (2021) Sci China Life Sci. 64(11): 1842- 1857, Lever et al. (1999) Biochem Soc Trans.27(6):841-7), / -retrovirus such as murine leukemia vims and feline leukemia vims, an avian retrovims such as spleen necrosis vims, and vectors derived from retrovimses such as Rous Sarcoma Vims, Harvey Sarcoma Vims, avian leukosis vims, human immunodeficiency vims, myeloproliferative sarcoma vims, and mammary tumor vims; and the like. See also, e.g., Warnocket al. (2011) Methods Mol. Biol. 737:1-25: Walther et al. (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol. 21 (3): 117- 122; herein incorporated by reference.

[0194] In some embodiments, an adeno-associated virus (AAV) vector is used for delivery of a nucleic acid encoding IL-33. The components of the AAV DNA genome consists of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). The Rep and Cap regions are translated to produce multiple distinct proteins including Rep78, Rep68, Rep52, and Rep40 and the capsid proteins VP1, VP2, and VP3 required for production of rAAV virions. In addition, AAV requires a helper plasmid containing genes from a helper vims such as adenovirus, including El a, Elb, E4, E2a, and VA genes for AAV replication. An expression cassette (e.g., encoding IL-33 lacking a functional nuclear localization sequence) can be inserted between the 5’-ITR and 3’-ITR. The structural (cap) and packaging (rep) proteins can be delivered in trans. Various AAV vector systems are available, and AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol, and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; Zhou et al., J. Exp. Med. (1994) 179:1867-1875; Li et al. (2020) Nat Rev Genet. 21(4):255-272; and Balakrishnan et al. (2014) Curr. Gene Ther. 14(2):86-100; herein incorporated by reference.

[0195] Retroviruses provide another convenient platform for gene delivery. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant vims can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No.5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop.3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentivimses are a class of retrovimses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Vimses 3(2): 132- 159; herein incorporated by reference).

[0196] Commonly used retroviral vectors are “defective”, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid arc packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art (see, e.g., Kafri et al. (2004) Methods Mol Biol. 246:367-390, herein incorporated by reference).

[0197] Another vector system useful for delivering nucleic acids encoding IL-33 is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

[0198] Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the IL-33 include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the IL-33 can be constructed as follows. The DNA encoding the IL-33 coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5 -bromodeoxy uridine and picking viral plaques resistant thereto.

[0199] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with.respect to the production of vaccinia viruses. See, e.g., WO 91 / 12882; WO 89 / 03429; and WO 92 / 03545.

[0200] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael ct al., J. Biol. Chcm. (1993) 268:6866-6869 and Wagner ct al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0201] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis vims (SIN), Semliki Forest vims (SFV), and Venezuelan Equine Encephalitis vims (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-vims derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95 / 07995, WO 96 / 17072; as well as Dubensky, Jr., T. W„ et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr„ T. W„ U.S. Patent No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavims vectors comprised of sequences derived from Sindbis vims and Venezuelan equine encephalitis vims. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772; herein incorporated by reference in their entireties.

[0202] A vaccinia-based infcction / transfcction system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, an IL-33 expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia vims recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia vims recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747: Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0203] As an alternative approach to infection with vaccinia or avipox vims recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translationof the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94 / 26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

[0204] In addition, anelloviral vectors can be used to deliver genes. An anellovector, based on a virus of the Betatorquevirus genus, has been developed (see, e.g., Prince et al. (2024) (biorxiv.org / content / 10.1101 / 2024.03.27.586964vl). The vector comprises a self-amplifying trans-complementation of a universal recombinant anellovector (SATURN) system, which relies on a self-replicating plasmid to provide viral proteins in trans that drive replication and capsiddependentpackaging of vector genomes. The SATURN system uses Cre-lox-based recombination to generate single unit-sized circular genomes inside a MOUT-4 production cell line. Capsid protein-dependent particles that cncapsidatc single stranded DNA vector genomes can be produced using the SATURN system.

[0205] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991.) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.

[0206] Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., .1. Biol. Chem. (1990) 265:10189-10192), in functional form.

[0207] Cationic liposomes are readily available. For example, N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under thetrademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. Sec, c.g., Szoka ct al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198: PCT Publication No. WO 90 / 11092 for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0208] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0209] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in Methods of Immunology (1983), Vol. 101, pp. 512-527; Szoka ct al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76: 145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0210] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

[0211] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J. P., et al., J MicroencapsuL 14(2): 197-210, 1997; O’Hagan D. T., et al., Vaccine 11(2):149-54, 1993.

[0212] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L„ Advanced Drug Delivery Reviews (1990) 5: 163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No. 5,831,005, issuedNov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[0213] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding IL-33. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needleless injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0214] Recombinant vectors carrying a synthetic expression cassette encoding IL-33 are formulated into compositions for delivery to a subject. These compositions may either be prophylactic or therapeutic. The compositions will comprise a "therapeutically effective amount" of the nucleic acid of interest such that an amount of the IL-33 protein (or a biologically active fragment thereof) can be produced in vivo in the individual to which it is administered that brings about a positive therapeutic response with respect to treatment of the individual for amyloid pathology such as in the brain of an individual having Alzheimer’s disease or amyloidosis. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the degree of protection desired; the type of condition and severity of the condition being treated; the particular IL-33 protein produced and its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.

[0215] The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may be present in such vehicles. Certain facilitatorsof nucleic acid uptake and / or expression can also be included in the compositions or coadministered.

[0216] Once formulated, the compositions can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[0217] Direct delivery of synthetic expression cassette compositions in vivo will generally be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe, needless devices such as Bioject™ or a gene gun, such as the Accell™ gene delivery system (PowderMed Ltd, Oxford, England).Production of Recombinant Virions

[0218] The present disclosure further provides host cells comprising the vectors described herein. A subject host cell can be an isolated cell, e.g., a cell in in vitro culture or a cell in an organism, organ, or tissue. A subject host cell is useful for producing recombinant virions. If a subject host cell is used to produce recombinant virions, it is referred to as a “packaging cell.” In some cases, a subject host cell is stably genetically modified with a vector. In other cases, a subject host cell is transiently genetically modified with a vector. The vectors described herein can be used in a variety of host cells for recombinant virion production.

[0219] In some embodiments, a vector system is provided, comprising a helper virus vector in addition to the vector for production of IL-33 (e.g., IL-33 lacking a functional nuclear localization sequence), as described herein. For example, for production of AAV virions, a helper virus vector encoding El a and Elb or E2a and E4 may be included in the vector system. In addition, the vector system may comprise expression cassettes encoding the AAV Rep proteins and the capsid proteins, VP1, VP2, and VP3. For production of lentivirus virions, one or more vectors encoding Gag, Pol, Rev, and VSV-G may be included in the vector system.

[0220] Suitable host cells that are transfected with a vector system are rendered capable of producing recombinant virions. Vectors of a vector system can be introduced into a host cell, either simultaneously or serially, using established transfection techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, and the like. In some embodiments, vectors forproducing recombinant virions are introduced into a host cell, and a vector comprising an expressible sequence encoding a gene product of interest is introduced later when production of the gene product of interest is desired.Genetically Modifying Cells

[0221] The genome of a cell may be genetically modified to express or increase expression of IL-33 or delete the nuclear localization sequence from IL-33. Various gene editing approaches can be used for this purpose, including, without limitation, the use of genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties. These gene editing techniques involve creating a double-strand break (DSB) in the DNA at a target site of the intended gene edit. In some embodiments, the DSB is repaired by homology-directed repair (HDR) using a donor DNA template that is inserted into the genome at the target locus using homologous recombination to replace a portion of the genomic sequence with a modified sequence.

[0222] In some embodiments, the donor polynucleotide comprises a nucleotide sequence encoding IL-33, which is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to a genomic locus (e.g., intron or exon) where the coding sequence encoding the IL-33 is integrated into the genome. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3’ genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology anus, which relates to the relative position of the homology arms to the nucleotide sequence encoding the IL-33 within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[0223] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity thereto, wherein the nucleotide sequence encoding the IL-33 is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0224] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and / or a specific site for introducing the nucleotide sequence encoding the IL-33. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0225] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0226] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a sequence of the genomic target locus to target the nuclease-gRNA complex to a target site.

[0227] In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease.Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5c (CasD), Cas6, Cas6c, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0228] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coll (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10)16091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0229] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double- stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer- adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[0230] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the intron sequence of the TCR gene targeted by a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.

[0231] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0232] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl) also referred to as CRISPR associated protein 12a(Casl2a) may be used. Casl2ais another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Casl2a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cast 2a for targeting than Cas9. Casl2a is capable of cleaving either DNA or RNA. The PAM sites recognized by Casl2a have the sequences 5'-YTN-3’ (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Casl2a cleavageof DNA produces double-stranded breaks with sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Casl2a, see, e.g., Ledford et al. (2015) Nature. 526 (7571):17-17 , Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang ct al. (2017) Front. Plant Sei. 8:177, Fernandes ct al. (2016) Postcpy Biochcm. 62(3) :315-326; herein incorporated by reference.

[0233] C2clis another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2cl, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[0234] In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6) :569-576; herein incorporated by reference.

[0235] The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell, organoid, or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a non-human primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the gRNA and / or RNA-guided nuclease is introduced into cells, the gRNA and / or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell. Recombinant nucleic acids encoding the gRNA, RNA-guided nuclease, and / or donor polynucleotide can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection. Alternatively, a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.

[0236] In some embodiments, the CRISPR system is introduced into cells with a viral vector that encodes the RNA-guided nuclease and guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells withvarious viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:25611, Ranet al. (2015) Nature 520:186-191, Swiechet al. (2015) Nat Biotechnol. 33:102-106; herein incorporated by reference.

[0237] Alternatively, a gRNA and a messenger RNA encoding the RNA-guidcd nuclease can be introduced into cells, wherein the RNA-guided nuclease is produced by translation of the mRNA in the cytoplasm. The gRNA and RNA-guided nuclease then form a complex in the cytoplasm and enter the nucleus. RNA transfection of cells can be performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(l):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12): 1152-1161, Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incoiporated by reference.

[0238] Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos.4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotricstcr method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al.. Meth. Enzymol. (1979) 68:109. In view of the short lengths of gRNAs (typically about 20 nucleotides in length) and donor polynucleotides (typically about 100-150 nucleotides), gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.

[0239] Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10):759-65; Ekker et al, Zebrafish 2008 Summer; 5(2): 121 -3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371 / journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA.2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0240] Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell’s genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.Pharmaceutical Compositions

[0241] Any of the compositions described herein, including IL-33 (e.g., IL-33 lacking a functional nuclear localization sequence), conjugates of IL-33 with binding agents that bind to amyloid plaques, recombinant nucleic acids or vectors capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents (e.g., ZNFs, TALENS) capable of expressing or increasing expression of IL-33, or deleting or inactivating its NLS, can be formulated into pharmaceutical compositions, optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodiumchloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0242] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0243] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the IL-33, conjugates of IL-33 with binding agents that bind to amyloid plaques, vectors capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, or other components of the preparation. Suitable antioxidants for use include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0244] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0245] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0246] The amount of the IL-33, conjugates of IL-33 with binding agents that bind to amyloid plaques, vectors capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., avial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[0247] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0248] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional compositions include those for oral, topical, transcutaneous, transdermal intratympanic, ocular, or localized delivery. Formulations suitable for topical, transcutaneous, transdermal, intratympanic, or ocular administration may be prepared through use of appropriate suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Such formulations may be utilized as liquid drops or with a means to provide continuous administration, for example, incorporation into slow-release pellets or controlled-release patches.

[0249] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.Preferably, the compositions comprising the IL-33 are in unit dosage form, meaning an amount of a composition appropriate for a single dose, in a premeasured or pre-packaged form.

[0250] The compositions herein may optionally include one or more additional agents, such as other drugs for treating Alzheimer’s disease or amyloidosis such as, but not limited to, donepezil, rivastigmine, and galantamine, memantine, lecanemab, remtemetug, bortezomib, daratumumab, cyclophosphamide, dexamethasone, lenalidomide, pomalidomide, carfilzomib, ixazomib, isatuximab, prednisone, and vutrisiran, and / or other medications used to treat a subject for a disease. Compounded preparations may include the IL-33, a conjugate of IL-33 with a binding agent that binds to amyloid plaques, a recombinant nucleic acid or vector capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, and one or more other agents for treating Alzheimer’s disease or amyloidosis. Alternatively, such agents can be contained in a separate composition from the composition comprising the IL-33, vectors capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents, and co-administered concurrently, before, or after the composition comprising the IL-33, vectors capable of expressing IL-33, viral particles comprising such vectors, or CRISPR systems or other genome modifying agents.Administration

[0251] At least one therapeutically effective dose of IL-33 (e.g., IL-33 lacking a functional nuclear localization sequence or a conjugate of IL-33 comprising a binding agent that binds to amyloid plaques) is administered. By "therapeutically effective dose or amount" of IL-33 is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from Alzheimer’s disease or amyloidosis. In particular, a "therapeutically effective dose or amount" of IL-33 may decrease formation of amyloid plaques, the size of amyloid plaques, and / or the numbers of amyloid plaques in the brain of a subject. Additionally, a therapeutically effective dose or amount may improve short-term memory, locomotor ability, reasoning, and survival.

[0252] In some embodiments, the IL-33 is provided by a recombinant polynucleotide (e.g., DNA or mRNA) or a vector encoding the IL-33, wherein the IL-33 is expressed in vivo in the brain of a subject in effective amounts sufficient for treatment of an individual for amyloid plaque pathology, including decreasing formation of amyloid plaques, the size of amyloid plaques, and / or numbers of amyloid plaques in the brain of a subject. In some embodiments, a CRISPR system or other genome modifying agent (e.g., ZNFs, TALENS) capable of expressing or increasing expression of IL-33 and / or inactivating or deleting its NLS is used to provide IL-33 in effectiveamounts sufficient for treatment of an individual for amyloid plaque pathology such as occurs in Alzheimer’s disease or amyloidosis, including decreasing formation of amyloid plaques, the size of amyloid plaques, and / or numbers of amyloid plaques in the brain of a subject. In some embodiments, a genome modifying agent is used to delete the nuclear localization sequence from the IL-33 in a brain cell (e.g., astrocyte).

[0253] In certain embodiments, multiple therapeutically effective doses of IL-33 will be administered according to a daily dosing regimen, or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, and so forth. For example, in some embodiments, the IL-33 will be administered twice-weekly or thrice-weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of IL-33 are administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses arc administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy (i.e., twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. In certain embodiments, the IL-33 is administered no more frequently than biweekly. In certain embodiments, the IL-33 is administered for at least one month, at least two months, or at least three months. The IL-33 can be administered by any acceptable route of administration as noted herein below.

[0254] In other embodiments, the phamraceutical composition comprising the IL-33 is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0255] II .-33 (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents for treatingAlzheimer’s disease or amyloidosis such as, but not limited to, donepezil, rivastigmine, and galantamine, memantine, lecanemab, remtemetug, bortezomib, daratumumab, cyclophosphamide, dexamethasone, lenalidomide, pomalidomide, carfilzomib, ixazomib, isatuximab, prednisone, and vutrisiran; and the like, and other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[0256] The IL-33 can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, IL-33 can be provided in the same or in a different composition. Thus, IL-33 and one or more other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising IL-33 and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating ocular fibrosis, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, IL-33 and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[0257] Toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and / or defining a therapeutic dosage range and / or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0258] The pharmaceutical compositions comprising the IL-33 and optionally other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art. Suitable routes of administration include parenteral administration, such as intraocular, subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous (IV), or infusion, and oral, pulmonary, topical, and transdemial. In some embodiments, the pharmaceutical composition comprising the IL-33 is administered by retroorbital injection or stereotactic injection into the brain. In some embodiments, the IL-33 is administered locally to sites of amyloid lesions.

[0259] Factors influencing the respective amount of the various compositions to be administered include, but are not limited to, the mode of administration, the frequency of administration (i.e., daily, or intermittent administration, such as twice- or thrice-weekly), the severity of the Alzheimer’s disease or amyloidosis, whether the individual is undergoing concurrent therapy with another therapeutic agent, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Generally, a higher dosage of an agent is preferred with increasing weight of the subject undergoing therapy.Kits

[0260] Also provided are kits for treating a patient with IL-33, a conjugate of IL-33 comprising a binding agent that binds to amyloid plaques, or a recombinant nucleic acid (DNA or mRNA), vector system, viral particles, or CRISPR system or other genome modifying agent (e.g., ZNFs, TALENS) capable of producing IL-33, as described herein. The IL-33, recombinant nucleic acid, vector system, viral particles, or genome modifying agent, and optionally other therapeutic agents may be contained in separate compositions or in the same composition. Kits may include unit doses of the formulations comprising the IL-33 (or recombinant nucleic acid, vector system, viral particles, or genome modifying agents capable of producing IL-33) suitable for use in the treatment methods described herein, e.g., in tablets or injectable dose(s). Transfection agents may also be included in the kit such as lipid nanoparticles (LNPs), calcium phosphate, polyethyleneimine (PEI), DEAE-dextran, liposomes, and the like.

[0261] Formulations suitable for retroorbital injection are of particular interest, and in such embodiments the kit may further include a syringe or other device to accomplish such administration, which syringe or device may be pre-filled with a composition comprising the IL-33 or conjugate, or recombinant nucleic acid, vector system, viral particles, or genome modifying agents capable of producing IL-33. The instructions can be printed on a label affixed to the container or can be a package insert that accompanies the container.

[0262] In certain embodiments, the kit comprises an IL-33 lacking a functional nuclear localization sequence or a recombinant nucleic acid (e.g., DNA or mRNA) or vector encoding an IL-33 lacking a functional nuclear localization sequence. For example, the nuclear localization sequence may be inactivated or deleted such that the IL-33 no longer localizes to the nucleus. In certain embodiments, the IL-33 further comprises a deletion of the chromatin-binding motif, MXLRSG (SEQ ID NO:4). In an exemplary embodiment, the kit comprises an IL-33 comprising a deletion of the nuclear localization sequence at amino acid positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2. In another exemplary embodiment, the kit comprises an IL-33 comprising an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2. In certain embodiments, the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least about 80-100% sequence identity to the amino acid sequence of SEQ ID NO:3, including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain embodiments, the IL-33 is conjugated to a binding agent that specifically binds to amyloid-beta.

[0263] Kits may comprise one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre-filled with a solution comprising a unit dose of the IL-33 or conjugate, or a vector system or viral particles or genome modifying agent capable of producing the IL-33, and a pharmaceutically acceptable excipient; and instructions to administer a unit dose according to a desired regimen or exemplary regimen dependent upon a patient’s age, weight, gender, and the like.

[0264] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging ofthe kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Examples of Non-Limiting Aspects of the Disclosure

[0265] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -90 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method of treating Alzheimer's disease in a subject, the method comprising administering a therapeutically effective amount of interleukin- 33 (IL-33) to the subject, wherein the IL-33 lacks a functional nuclear localization sequence.2. The method of aspect 1, wherein the nuclear localization sequence is deleted.3. The method of aspect 1 or 2, wherein the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO: 5.4. The method of any one of aspects 1-3, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the amino acid sequence of SEQ ID NO:4.5. The method of any one of aspects 1-4, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67 or an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.6. The method of aspect 1, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID N0:3.7. The method of any one of aspects 1-6, wherein the IL-33 is administered prophylactically before amyloid plaques are detected in the subject.8. The method of any one of aspects 1-7, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.9. The method of aspect 8, wherein the amyloid peptide is selected from the group consisting of A036, Ap37, A 38, A039, A 4O, A041, Ap42, and A 43.10. The method of aspect 8 or 9, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.11. The method of aspect 10, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.12. The method of any one of aspects 1-11, wherein the IL-33 is provided by a recombinant nucleic acid or a vector comprising a coding sequence encoding the IL-33.13. The method of aspect 12, wherein the recombinant nucleic acid is RNA or DNA.14. The method of aspect 12, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the TL-33 in the subject.15. The method of aspect 12, wherein the vector is a viral vector or a plasmid.16. The method of aspect 15, wherein the viral vector is an adeno- associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.17. The method of aspect 16, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.18. The method of aspect 17, wherein the AAV serotype is PHP.cB.19. The method of any one of aspects 16-18, wherein the AAV vector further comprises a 5 ’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5’ ITR and the 3'-ITR.20. The method of any one of aspects 15-19, further comprising administering a helper virus vector to the subject.21. The method of aspect 20, wherein the helper virus vector encodes El a and Elb or E2a and E4.22. The method of any one of aspects 12-21, wherein the recombinant nucleic acid or vector is administered by rctroorbital injection.23. The method of any one of aspects 12-21, wherein the recombinant nucleic acid or vector is administered locally to the brain.24. The method of any one of aspects 12-23, wherein the vector comprises a promoter operably linked to a coding sequence encoding the IL-33.25. The method of aspect 24, wherein the promoter is an astrocyte-specific promoter.26. The method of aspect 24 or 25, wherein the promoter is constitutive or inducible.27. The method of any one of aspects 12-23, wherein the coding sequence encoding the IL-33 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the IL-33 at the chromosomal locus.28. The method of any one of aspects 15-27, wherein the IL-33 is provided by a viral particle comprising the viral vector comprising the coding sequence encoding the IL-33.29. The method of aspect 28, wherein the viral particle is administered locally to the brain of the subject.30. The method of any one of aspects 1-7, wherein the IL-33 is provided by genetically modifying the genome of an astrocyte of the subject to express the IL-33.31. The method of aspect 30, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).32. The method of aspect 31, wherein the Cas nuclease is Cas9 or Cas 12a.33. The method of any one of aspects 1-32, wherein the subject is a mammal.34. The method of aspect 33, wherein the mammal is human.35. A method of decreasing numbers of amyloid plaques in a brain of a subject, the method comprising introducing into an astrocyte in the brain of the subject an effective amount of interleukin-33 (IL-33), wherein the IL-33 lacks a functional nuclear localization sequence.36. The method of aspect 35, wherein the nuclear localization sequence is deleted.37. The method of aspect 35 or 36, wherein the nuclear localization sequence comprises the sequence of SEQ ID NO:5.38. The method of any one of aspects 35-37, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the sequence of SEQ ID NO:4.39. The method of aspect 35, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.40. The method of aspect 35, wherein the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.41. The method of aspect 35, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3.42. The method of any one of aspects 35-41, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.43. The method of aspect 42, wherein the amyloid peptide is selected from the group consisting of Ap36, Ap37, Ap38, Ap39, Ap40, A 41, Ap42, and Ap43.44. The method of aspect 42 or 43, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.45. The method of aspect 44, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.46. The method of any one of aspects 35-45, wherein the IL-33 is provided by a recombinant nucleic acid or a vector comprising a coding sequence encoding the IL-33.47. The method of aspect 46, wherein the recombinant nucleic acid is RNA or DNA.48. The method of aspect 47, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject.49. The method of aspect 46, wherein the vector is a viral vector or a plasmid.50. The method of aspect 49, wherein the vector is an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.51. The method of aspect 50, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.52. The method of aspect 51, wherein the AAV serotype is PHP.eB.53. The method of any one of aspects 50-52, wherein the AAV vector further comprises a 5 ’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5’-ITR and the 3’-ITR.54. The method of any one of aspects 50-52, further comprising administering a helper virus vector to the subject.55. The method of aspect 54, wherein the helper virus vector encodes Ela and Elb or E2a and E4.56. The method of any one of aspects 46-55, wherein the recombinant nucleic acid or vector is administered by retroorbital injection.57. The method of any one of aspects 46-55, wherein the recombinant nucleic acid or vector is administered locally to the brain.58. The method of any one of aspects 46-57, wherein the vector comprises a promoter operably linked to a coding sequence encoding the IL-33.59. The method of aspect 58, wherein the promoter is an astrocyte-specific promoter.60. The method of aspect 58 or 59, wherein the promoter is constitutive or inducible.61. The method of any one of aspects 46-57, wherein the coding sequence encoding the IL-33 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the IL-33 at the chromosomal locus.62. The method of any one of aspects 46-61, wherein the IL-33 is provided by a viral particle comprising the vector comprising the coding sequence encoding the IL-33.63. The method of aspect 62, wherein the viral particle is administered locally to the brain of the subject.64. The method of any one of aspects 35-45, wherein the IL-33 is provided by genetically modifying the genome of an astrocyte of the subject to express the IL-33.65. The method of aspect 64, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).66. The method of aspect 65, wherein the Cas nuclease is Cas9 or Cas 12a.67. The method of any one of aspects 35-66, wherein the subject is a mammal.68. The method of aspect 67, wherein the mammal is human.69. A composition comprising IL-33 or a recombinant nucleic acid or vector encoding IL-33 for use in treating Alzheimer’s disease in a subject, wherein the IL-33 lacks a functional nuclear localization sequence.70. The composition of aspect 69, further comprising a pharmaceutically acceptable excipient.71. The composition of aspect 69 or 70, wherein the nuclear localization sequence is deleted.72. The composition of any one of aspects 69-71, wherein the nuclear localization sequence comprises the sequence of SEQ ID NO:5.73. The composition of any one of aspects 69-72, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the sequence of SEQ ID NO:4.74. The composition of aspect 69, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO: 2.75. The composition of aspect 69, wherein the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.76. The composition of aspect 69, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3.77. The composition of any one of aspects 69-76, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.78. The composition of aspect 77, wherein the amyloid peptide is selected from the group consisting of Ap36, AP37, Ap38, A[339, AP40, AP41, AP42, and AP43.79. The composition of aspect 77 or 78, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.80. The composition of aspect 79, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.81. The composition of any one of aspects 69-80, wherein the recombinant nucleic acid is RNA or DNA.82. The composition of aspect 81, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject.83. The composition of aspect 69, wherein the vector is a viral vector or a plasmid.84. The composition of aspect 83, wherein the vector is an adcno-associatcd viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.85. The composition of aspect 84, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.86. The composition of aspect 85, wherein the AAV serotype is PHP.eB.87. The composition of aspect 86, wherein the AAV vector further comprises a 5’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5 ’-ITR and the 3 ’-ITR.88. The composition of any one of aspects 85-87, further comprising a helper virus vector.89. The composition of aspect 88, wherein the helper virus vector encodes El a and Elb or E2a and E4.90. Use of IL-33 or a recombinant nucleic acid or vector encoding IL-33 in the manufacture of a medicament or pharmaceutical composition for treating Alzheimer’s disease, wherein the IL-33 lacks a functional nuclear localization domain.EXAMPLES

[0266] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations shouldbe accounted for. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLE 1: VIRAL DELIVERY OF INTERLEUKIN-33 INTO BRAIN ASTROCYTES AS METHOD TO REDUCE AMYLOID PATHOLOGY AS OCCURS IN ALZHEIMER’S DISEASE

[0267] The cytokine interleukin-33 is highly expressed in the brain but is not easily secreted from living cells due to a nuclear localization domain in the protein, which sequesters it in the nucleus. Our group has performed many studies in mouse models showing that deficiency of IL-33 in the brain can lead to impaired brain development, increased epileptiform activity (seizure like events), and impaired learning. We have shown that the mechanism for this relates to IL-33’s activation of microglia, the brain’s resident immune cells, which are necessary for many healthy brain activities but can also become inflammatory and contribute to disease.

[0268] Microglia are essential players in neurodegenerative diseases including Alzheimer’s Disease (AD), which is the most common neurodegenerative disorder and increasing in prevalence as the population ages. A major feature of Alzheimer’s Disease is the development of amyloid plaques, which can build up in the brain for years before symptoms like impaired cognition and memory loss become apparent. Development of new therapies for Alzheimer’s Disease is of tremendous clinical and public health interest. Recently, some therapies have come to market that target the Amyloid plaques but their benefits appear to be modest and their mechanism of action is not entirely clear.

[0269] Here we have developed a method to increase IL-33 levels in the brain, and found that this strategy can reduce amyloid plaques and even rescue behavioral deficits in a mouse model of plaque pathology (5xFAD). To do this, we used an existing adeno associated virus (AAV) backbone and a previously described promoter (gfaABCID) that targets gene expression to astrocytes, a structural support cell in the brain. We then inserted a bioactive portion of Interleukin-33 that lacks a nuclear localization domain that normally would sequester the protein in the nucleus and prevent its bioactivity (“delta NLS-IL33”). Finally, we expressed this AAV construct in the PHP.eB serotype of the virus (a serotype that can cross the blood brain barrier, previously developed by the Gradinaru lab (Caltech).

[0270] Using this active IL-33 expression approach, we delivered this AAV-PHP.eB-gfaABClDdeltaNLS-IL33 virus via retroorbital injection into mice that develop plaque pathology by six months of age. We observed a 30-50% reduction in plaque pathology and a complete rescue in a behavioral test of short-term memory (FIG. 1, attached). We also observe that IL-33 expression is markedly reduced in the astrocytes of patients that died with AD. Viral delivery ofactive IL-33 to astrocytes is an attractive therapeutic strategy because it modulates the function of microglia, which are thought to be important to the pathogenesis of AD. Furthermore, using a virus that restricts its expression to astrocytes is innovative because these cells are only present in the brain, thereby limiting potential off target effects of IL-33 delivery to other tissues.

[0271] For treatment of AD in humans, an AAV serotype that can deliver a recombinant polynucleotide comprising a coding sequence encoding the human IL-33 protein to the human brain is used. AAV mediated delivery of a human DeltaNLS-IL-33 to astrocytes is performed similarly as described above.Results

[0272] As shown in FIGS. 1A-1E, IL-33 signaling deficiency worsens amyloid pathology in a mouse model of amyloid deposition, a pathological hallmark of Alzheimer’s Disease. FIG.1A shows images and quantification of amyloid plaques (M0AB-2+, labeling all types of Ap forms) in the prefrontal cortex of 5xFAD and globally IL-33 deficient mice ( / / 33- / -:5xFAD). Welch’s t-test by sex. This shows that plaques are worse in the absence of IL-33. FIG. IB shows images and quantification of dense core plaques (Methoxy-X04+ compacted A ) in the dorsal prefrontal cortex of 5xFAD and Z / 35- / -:5xFAD mice. Welch’s t-test by sex. This data supports FIG. 1 A and shows that compacted plaques are increased in the absence of IL-33. FIG. 1C shows images and quantification of LAMP1+ dystrophic neurites shows overlap with neurotoxic protofibrillar Ap (MOAB2+MXO4ne8) in 5xFAD and Z / 55- / -:5xFAD mice. Welch’s t-test by sex. In the correlation analysis between MOAB-2 and LAMP1, the difference between the slopes was analyzed through the simple linear regression test. This data shows that neuronal damage is worse in the absence of IL-33. FIG. ID shows images and quantification of M0AB-2+ amyloid plaques in the prefrontal cortex of 5xFAD vs 5xFAD mice that lack the IL-33 receptor on all cells (IHrll- / -:5xFAD). Welch’s t-test by sex. This data shows that loss of receptor signaling phenocopies the effect in FIG. A, indicating that IL-33 is acting through its receptor, as would be expected in canonical signaling. FIG. IE shows images and quantification of plaque compaction, analyzed as the ratio of MOAB-2 (all types of Ap forms) and MX04 (dense core plaque) volume in 5xFAD and / G5- / -:5xFAD mice. Welch’s t-test by sex. This data combines the information in A and B to shows that plaques are less compacted without IL-33, suggesting that neurotoxic protofibrillar Ap is increased. In FIGS. 1A-1E, data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0273] As shown in FIGS. 2A-2D, IL-33 signaling promotes microglial encapsulation of amyloid plaques (a process known to be disrupted in mouse models lacking Alzheimer’s riskgenes such as TREM2). FIG. 2A shows images showing microglia (IBA1+), 4G8 (all types of AP), and MX04 (dense core plaques). Inset (dotted box) shows the region where microglial processes contact and encapsulate the dense core plaques. Bottom row shows an enlargement of this inset to illustrate the surface contact of microglia with dense-core plaques, far right image shows a 3D reconstruction used to calculate coverage of microglia on plaques. “Engulfed A ” panel (top right) illustrates the images used to quantify phagocytosis of AP by microglia (orange signal inside microglia) in 5xFAD and Z / 35- / -:5xFAD mice. FIG. 2B shows quantification of the data represented in A shows percentage of microglial coverage of dense-core plaques and the correlation analysis between the percentage of surface area coverage and MX04+ dense-core plaque volume. This data shows that microglial coverage of plaques is reduced in the absence of IL-33, and that this effect is even more pronounced around small (early stage) plaques. FIG. 2C shows quantification of Ibal-r- plaque-associated microglia volume. Unpaired t-test. This data suggests that impaired contact with plaques is a result of defective microglial chemotaxis towards plaques, since there is a trend towards increased, not decreased microglial volume around plaques.FIG. 2D shows quantification of the percentage of engulfed AP inside Iba 1+ microglia around plaques. This data shows that IL-33 may not act via changes in microglial phagocytosis. Unpaired t-tcst. In all panels data arc mean ± SEM. Dots represent individual mice (black dot for male and white dot for female).

[0274] As shown in FIG. 3A-3B, astrocytic IL-33 protein is decreased in human brains with late-stage Alzheimer’s Disease (AD). FIG. 3A shows images of human astrocytic IL-33 in the cortex of healthy controls and human AD brains (ra = 4-5 / group). Unpaired t-test. FIG. 3B show a correlation assay between astrocytic IL-33 and the distance from amyloid plaques in human AD brains. This data shows that IL-33 is profoundly reduced in AD brains, and that this is modestly correlated with distance from plaques. This is relevant because it suggests that there may be a deficit of IL-33 signaling in human AD brains. All human AD brains at Braak stage V / VI. Data are mean ± SEM. Dots represent individual patients.

[0275] As shown in FIGS. 4A-4E, viral delivery of an active form of the cytokine Interleukin- 33 into brain astrocytes reduces amyloid pathology in a mouse model and rescues short term memory. FIG.4A shows a schematic of the strategy to deliver an active form of IL-33 that lacks its nuclear localization signal (“ANLS”, amino acids 1-67) termed “IL-33 NLS”, below are images of astrocytes labeled with a control virus expressing the control virus (AAV-gfaABClD-tdT, reporter only, left) and the IL-33ANLS delivery construct (AAV-PHP.eB-gfaABClD-IL-33ANLS-tdT, right). This shows that IL-33 is only in the nucleus in the control, but is localized in the cytoplasm of astrocytes after expression of the IL-33ANLS construct, whichis likely to facilitate its release outside the cell. See FIG. 5 for more detail. FIG.4B shows images and quantification of astrocytes labeled with reporter virus in the dorsal prefrontal cortex of wildtype and 5xFAD mice show effective delivery of viral construct into the brain and more robust expression induced by plaques in the 5xFAD model. Welch’s t-test by sex. This suggests that production of active IL-33 may undergo a self-amplifying effect near regions affected by plaque pathology, which would be anticipated to minimize off-target effects on bystander tissues. FIG.4C shows an experimental schematic of IL-33ANLS AAV-based gain-of-function strategy in 5xFAD model mice. Construct was delivered at six months, and behavior and plaque pathology analyzed at 8 months. FIG. 4D shows images and quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD mice injected with control (tdTomato) and IL-33ANLS virus. This shows that viral delivery of active IL-33 reduces plaque pathology. Analyzed by 2 way ANOVA. FIG. 4E shows quantification of spatial working memory assessed by the Y-maze test (see schematic) comparing 5xFAD mice injected with control (tdTomato) and IL-33ANLS vims. This shows that active IL-33 rescues the impairment of working memory in 5xFAD mice. Analyzed by one way ANOVA. In all panels data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0276] FIGS. 5A-5B show full plasmid maps. FIG. 5A shows a map of a “control vims” (AAV-gfaABClD-tdT) that expresses the TdTomato reporter (TdT) under control of an astrocyte promoter (gfaBCID) but does not express IL-33. FIG.5B shows a map of an IL-33ANLS delivery constmct (AAV-gfaABClD-IL-33ANLS-tdT), which expresses TdTomato and IL-33ANLS, an active form of IL-33 that lacks its nuclear localization signal (“ANLS”, amino acids 1-67), which also has a small epitope tag for identification (HA).

[0277] FIGS.6A-6C shows that early (prophylactic) administration of AAV-IL-33ANLS reduces amyloid deposition in the 5xFAD model. FIG. 6A shows an experimental schematic of IL-33ANLS AAV-based gain-of-function strategy in early 5xFAD model mice. The constmct was delivered at 2.5 months, and plaque pathology was analyzed at 5 months. FIG. 6B shows images and quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD mice injected with control (tdTomato) and IL-33ANLS vims. This data demonstrates that early viral delivery of active IL-33 reduces plaque pathology. FIG. 6C shows images and quantification of GFAP+ reactive astrocytes in 5xFAD mice injected with control and IL-33ANLS vims. Viral delivery of an active IL-33 does not induce astrogliosis. The data was analyzed by an unpaired t-test for each sex.

[0278] As shown in FIGS.7A-7B, AAV-IL-33 ANLS act via the canonical IL-33 signaling pathway as they require expression of the IL-33 receptor IL1RL1 (also known as ST2). FIG. 7Ashows an experimental schematic of IL-33ANLS AAV-based gain-of-function strategy in IL-33 receptor-deficient 5xFAD model mice (5xFAD:ST2_ / ). Construct was delivered at 5 months, and plaque pathology was analyzed at 7 months. FIG.7B shows quantification of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of 5xFAD:ST2 / _mice injected with control (tdTomato) and IL-33ANLS virus. This data demonstrates that viral delivery of active IL-33 are dependent on the IL-33 receptor in the brain. Analyzed by an unpaired t-test for each sex. All data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0279] FIGS. 8A-8B show that viral delivery of AAV-IL-33ANLS reduces amyloid pathology in an independent mouse model used to study Alzheimer's disease (APP-SAA knockin, MGI: J:325368 / PMID: 35690868). Images (FIG.8A) and quantification (FIG.8B) of M0AB-2+ amyloid plaques in the dorsal prefrontal cortex of APP-SAA KI mice injected with control (tdTomato) and IL-33ANLS virus are shown. The construct was delivered at 6 months, and plaque pathology was analyzed at 8 months. This data demonstrates that viral delivery of active IL-33 exerts similar effects in the other AD mouse model, APP-SAA KI mice. Analyzed by two-way ANOVA. All data are mean ± SEM. Dots represent individual mice (square for male and triangle for female).

[0280] As shown in FIGS. 9A-9H, there is no evidence of inflammatory lymphocyte infiltration or change in immune cell subsets after administration of AAV-IL-33ANLS. The number of CD45+ total immune cells or the frequency of lymphocytes in the brain were quantitated and. analyzed by unpaired t-test. All data are mean ± SEM. FIG.9A shows a schematic of the experimental protocol. FIGS.9B-9H show quantification of FIG.9B) CD45+ total immune cells, FIG. 9C) B cells, FIG. 9D) natural killer (NK) cells and other group 1 innate lymphoid cells (ILC1), FIG.9E) CD8+ T cells, FIG.9F) CD4+ T regulatory (Treg) cells, FIG.9G) CD4+ resting conventional T (Tconv) cells, and FIG. 9H) group 2 innate lymphoid cells (ILC2).

[0281] FIG. 10 shows representative images of Ibal + microglia in acute human brain slices treated with either saline or IL-33. This data demonstrates that microglia in the adult human brain are capable of responding to IL-33.

[0282] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. It is also to be understood that the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0283] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWHAT is CLAIMED IS:

1. A method of treating Alzheimer's disease in a subject, the method comprising administering a therapeutically effective amount of interleukin- 33 (IL-33) to the subject, wherein the IL-33 lacks a functional nuclear localization sequence.

2. The method of claim 1, wherein the nuclear localization sequence is deleted.

3. The method of claim 1 or 2, wherein the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO:5.

4. The method of any one of claims 1-3, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the amino acid sequence of SEQ ID NO:4.

5. The method of any one of claims 1-4, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67 or an N-tcrminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

6. The method of claim 1, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3.

7. The method of any one of claims 1-6, wherein the IL-33 is administered prophylactically before amyloid plaques are detected in the subject.

8. The method of any one of claims 1-7, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.

9. The method of claim 8, wherein the amyloid peptide is selected from the group consisting of A036, A 37, A038, Ap39, A 4O, A041, A042, and A043.

10. The method of claim 8 or 9, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.

11. The method of claim 10, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.

12. The method of any one of claims 1-11, wherein the IL-33 is provided by a recombinant nucleic acid or a vector comprising a coding sequence encoding the IL-33.

13. The method of claim 12, wherein the recombinant nucleic acid is RNA or DNA.

14. The method of claim 12, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject.

15. The method of claim 12, wherein the vector is a viral vector or a plasmid.

16. The method of claim 15, wherein the viral vector is an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

17. The method of claim 16, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.

18. The method of claim 17, wherein the AAV serotype is PHP.eB.

19. The method of any one of claims 16-18, wherein the AAV vector further comprises a 5 ’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5’-ITR and the 3’-ITR.

20. The method of any one of claims 15-19, further comprising administering a helper virus vector to the subject.

21. The method of claim 20, wherein the helper virus vector encodes Ela and Elb or E2a and E4.

22. The method of any one of claims 12-21, wherein the recombinant nucleic acid or vector is administered by retroorbital injection.

23. The method of any one of claims 12-21, wherein the recombinant nucleic acid or vector is administered locally to the brain.

24. The method of any one of claims 12-23, wherein the vector comprises a promoter operably linked to a coding sequence encoding the IL-33.

25. The method of claim 24, wherein the promoter is an astrocyte-specific promoter.

26. The method of claim 24 or 25, wherein the promoter is constitutive or inducible.

27. The method of any one of claims 12-23, wherein the coding sequence encoding the IL-33 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the IL-33 at the chromosomal locus.

28. The method of any one of claims 15-27, wherein the IL-33 is provided by a viral particle comprising the viral vector comprising the coding sequence encoding the IL-33.

29. The method of claim 28, wherein the viral particle is administered locally to the brain of the subject.

30. The method of any one of claims 1-7, wherein the IL-33 is provided by genetically modifying the genome of an astrocyte of the subject to express the IL-33.

31. The method of claim 30, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).

32. The method of claim 31, wherein the Cas nuclease is Cas9 or Casl2a.

33. The method of any one of claims 1-32, wherein the subject is a mammal.

34. The method of claim 33, wherein the mammal is human.

35. A method of decreasing numbers of amyloid plaques in a brain of a subject, the method comprising introducing into an astrocyte in the brain of the subject an effective amount of interleukin-33 (IL-33), wherein the IL-33 lacks a functional nuclear localization sequence.

36. The method of claim 35, wherein the nuclear localization sequence is deleted.

37. The method of claim 35 or 36, wherein the nuclear localization sequence comprises the sequence of SEQ ID NO:5.

38. The method of any one of claims 35-37, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the sequence of SEQ ID NO:4.

39. The method of claim 35, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

40. The method of claim 35, wherein the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

41. The method of claim 35, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3.

42. The method of any one of claims 35-41, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.

43. The method of claim 42, wherein the amyloid peptide is selected from the group consisting of A036, A037, Ap38, A039, A 4O, A041, A042, and Ap43.

44. The method of claim 42 or 43, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.

45. The method of claim 44, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.

46. The method of any one of claims 35-45, wherein the IL-33 is provided by a recombinant nucleic acid or a vector comprising a coding sequence encoding the IL-33.

47. The method of claim 46, wherein the recombinant nucleic acid is RNA or DNA.

48. The method of claim 47, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject.

49. The method of claim 46, wherein the vector is a viral vector or a plasmid.

50. The method of claim 49, wherein the vector is an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

51. The method of claim 50, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.

52. The method of claim 51 , wherein the AAV serotype is PHP.eB.

53. The method of any one of claims 50-52, wherein the AAV vector further comprises a 5 ’-inverted terminal repeat (ITR) and a 3’-ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5’-ITR and the 3’-ITR.

54. The method of any one of claims 50-52, further comprising administering a helper virus vector to the subject.

55. The method of claim 54, wherein the helper virus vector encodes Ela and Elb or E2a and E4.

56. The method of any one of claims 46-55, wherein the recombinant nucleic acid or vector is administered by retroorbital injection.

57. The method of any one of claims 46-55, wherein the recombinant nucleic acid or vector is administered locally to the brain.

58. The method of any one of claims 46-57, wherein the vector comprises a promoter operably linked to a coding sequence encoding the IL-33.

59. The method of claim 58, wherein the promoter is an astrocyte-specific promoter.

60. The method of claim 58 or 59, wherein the promoter is constitutive or inducible.

61. The method of any one of claims 46-57, wherein the coding sequence encoding the IL-33 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the IL-33 at the chromosomal locus.

62. The method of any one of claims 46-61, wherein the IL-33 is provided by a viral particle comprising the vector comprising the coding sequence encoding the IL-33.

63. The method of claim 62, wherein the viral particle is administered locally to the brain of the subject.

64. The method of any one of claims 35-45, wherein the IL-33 is provided by genetically modifying the genome of an astrocyte of the subject to express the IL-33.

65. The method of claim 64, wherein the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRLSPR)-associated(Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

66. The method of claim 65, wherein the Cas nuclease is Cas9 or Cas 12a.

67. The method of any one of claims 35-66, wherein the subject is a mammal.

68. The method of claim 67, wherein the mammal is human.

69. A composition comprising IL-33 or a recombinant nucleic acid or vector encoding IL-33 for use in treating Alzheimer’s disease in a subject, wherein the IL-33 lacks a functional nuclear localization sequence.

70. The composition of claim 69, further comprising a pharmaceutically acceptable excipient.

71. The composition of claim 69 or 70, wherein the nuclear localization sequence is deleted.

72. The composition of any one of claims 69-71 , wherein the nuclear localization sequence comprises the sequence of SEQ ID NO:5.

73. The composition of any one of claims 69-72, wherein the IL-33 further comprises a deletion of a chromatin-binding motif comprising the sequence of SEQ ID NO:4.

74. The composition of claim 69, wherein the IL-33 comprises a deletion of amino acids at positions 46 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

75. The composition of claim 69, wherein the IL-33 comprises an N-terminal deletion of amino acids at positions 1 to 67, wherein positions of the amino acids are numbered relative to the reference sequence of SEQ ID NO:2.

76. The composition of claim 69, wherein the IL-33 comprises or consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3.

77. The composition of any one of claims 69-76, wherein the IL-33 is conjugated to a binding agent that specifically binds to an amyloid beta peptide.

78. The composition of claim 77, wherein the amyloid peptide is selected from the group consisting of A 36, Ap37, Ap38, Ap39, Ap40, Ap41, Ap42, and Ap43.

79. The composition of claim 77 or 78, wherein the binding agent is an antibody or an antigen-binding fragment thereof, an antibody mimetic, a peptide, a peptoid, or an aptamer.

80. The composition of claim 79, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a nanobody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fvfragment, and a scFv fragment.

81. The composition of any one of claims 69-80, wherein the recombinant nucleic acid is RNA or DNA.

82. The composition of claim 81, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the IL-33 in the subject.

83. The composition of claim 69, wherein the vector is a viral vector or a plasmid.

84. The composition of claim 83, wherein the vector is an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

85. The composition of claim 84, wherein the AAV vector is an AAV serotype that can cross a blood brain barrier.

86. The composition of claim 85, wherein the AAV serotype is PHP.eB.

87. The composition of claim 86, wherein the AAV vector further comprises a 5’-inverted terminal repeat (ITR) and a 3’ -ITR, wherein the coding sequence encoding the IL-33 is positioned between the 5 ’-ITR and the 3 ’-ITR.

88. The composition of any one of claims 85-87, further comprising a helper virus vector.

89. The composition of claim 88, wherein the helper vims vector encodes El a and Elb or E2a and E4.

90. Use of IL-33 or a recombinant nucleic acid or vector encoding IL-33 in the manufacture of a medicament or pharmaceutical composition for treating Alzheimer’s disease, wherein the IL-33 lacks a functional nuclear localization sequence.

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