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WO2025080949A1 - Methods and compositions for improving immunotherapy - Google Patents

Methods and compositions for improving immunotherapy Download PDF

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Publication number
WO2025080949A1
WO2025080949A1 PCT/US2024/050927 US2024050927W WO2025080949A1 WO 2025080949 A1 WO2025080949 A1 WO 2025080949A1 US 2024050927 W US2024050927 W US 2024050927W WO 2025080949 A1 WO2025080949 A1 WO 2025080949A1
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vps72
cells
cell
mice
inkt
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French (fr)
Inventor
Qing-Sheng MI
Li Zhou
Jie Wang
Kalpana SUBEDI
Nirmal PARAJULI
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Henry Ford Health System
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Henry Ford Health System
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the present disclosure relates to compositions and methods to treat immune related diseases such as autoimmune diseases and inflammation related diseases and conditions.
  • VPS72 vacuolar protein sorting-associated protein 72 homolog
  • YL1 vacuolar protein sorting-associated protein 72 homolog
  • VPS72 functions as a histone chaperone for H2A to H2A.Z exchange in chromatin remodeling and belongs to two multi-subunit chromatin-remodeling complexes: the Snf2-related CBP-activator protein chromatin remodeling (SRCAP) complex (FIG.l); and the TRRAP/TIP60 complex (FIG. 1).
  • SRCAP Snf2-related CBP-activator protein chromatin remodeling
  • TRRAP/TIP60 complex FIG. 1
  • VPS72-mediated H2A.Z exchange is required for nuclear reassembly after mitosis in HeLa cells, and VPS72-H2A.Z interaction with the help of Znhitl determine Lgr5+ stem cell fate (FIG. 1).
  • VPS72 is also reported to regulate the acetylation of non-histone proteins, including autophagy gene ATG8a in Drosophila (FIG. 1).
  • Chromatin remodeling is an important layer of epigenetic regulation and plays a key role in immune cell development, homeostasis, and function.
  • Nucleosomes are the basic unit of chromatin.
  • the histone octamer packages DNA into nucleosomes, maintains the nucleosome morphology, and serves as a regulatory 7 layer for gene expression.
  • This octamer consists of histone proteins such as H2A, H2B, H3, and H4.
  • H2A which comprises an H2A variant known as histone H2A.Z.
  • H2A.Z has two different isoforms, H2A.Z1 and H2A.Z2, which differ by only three amino acids and are encoded by tw o separate genes, H2AFZ and H2AFV, respectively.
  • H2A.Z has been linked to diverse biological processes such as memory, epithelial-to-mesenchymal transition, microglial development, and neuronal survival through promoting nuclear-encoded mitochondrial gene expression and organelle function.
  • H2A.Z is associated with a greater proliferative capacity in multiple cancers such as metastatic melanoma, colorectal, liver, and lung.
  • Exchanging H2A for variant histone H2A.Z through the VPS72/SRCAP/TIP60 complex (Fig. 1) has been shown to modulate local chromatin structure to activate or repress target genes that regulate cellular processes, such as cell cycle progression, autophagy regulation, metabolic processes, and mitochondrial function especially in HSC and embryonic stem cell development.
  • the functions of VPS72-mediated H2A.Z exchange in immune cells remain unknown.
  • VPS72 is a chaperone protein for H2A.Z and deposits the H2A.Z in the nucleosome exchange.
  • the present disclosure provides a method of altering an immune response, particularly in subjects having a malfunctioning immune activated state, for example, in autoimmune diseases and diseases where inflammation is a factor or cause.
  • the subj ect is treated by administering a therapeutically effective amount of VPS72 inhibitor which decreases the expression of VPS72 in the immune cells that contribute to the hyper immune response or decreases the activity of VPS72 by acting as a blocking peptide with its cognate binding molecule H2A.Z.
  • the present disclosure provides a VPS72 peptide inhibitor that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2.
  • the present disclosure provides a VPS72 inhibitor that is a nucleic acid, for example, a DNAi or an RNAi molecule that inhibits the transcription or translation of VPS72 in the immune cell that contributes to the hyper immune state which causes the autoimmune and/or inflammatory disease or condition.
  • a VPS72 inhibitor that is a nucleic acid, for example, a DNAi or an RNAi molecule that inhibits the transcription or translation of VPS72 in the immune cell that contributes to the hyper immune state which causes the autoimmune and/or inflammatory disease or condition.
  • the present disclosure provides a combination of a VPS72 inhibitor and at least one of an anti-inflammatory, and an immune suppressive agent for the treatment of an autoimmune disease and/or diseases w here inflammation is at least a symptom or cause of a disease or condition.
  • FIG. 1 depicts a schematic representation of the historic studies involving the role and function of VPS72 in chronological order.
  • FIG. 2 CD4Cre mediated VPS72 deletion.
  • Panels A-B Expression pattern of VPS72 in iNKT cells VPS72 expression from ImmGen RNA-seq(C57BL/6, 6wks old) (Panel A) and qPCR (C57BL/6, 8-12wks old) (Panel B) in sorted DP, CD4, CD8, and iNKT cells.
  • Panel C Cytometry histogram shows VPS72 expression in DP, DN, CD4 T, CD8 T and iNKT cells.
  • Panel D Generation of VPS72 floxed allele and conditionally deleted allele.
  • FIG. 4 VPS72 is required for iNKT cell homeostasis. Histogram and bar graph show Ki-67 expression in total iNKT cells and substages of iNKT. Flow cytometry and bar graph show iNKT cell early and later apoptosis in WT and VPS72cKO.
  • Panels C-D Flow cytometry of iNKT 1/2/17 in total iNKT cells (Panel C) and NK1.1- subset (Panel D) from VPS72cKO and WT mice.
  • Panel E bar graph depicting total iNKTl/2/17 frequency and absolute.
  • Panel F Heatmaps show the iNKTl/2/17 signature expression in thymic iNKT cells from WT and VPS72cKO mice. *P ⁇ 0.05, ***P ⁇ 0.001, and ****P ⁇ 0.0001.
  • FIG. 5 VPS72 is required for iNKT function in tamoxifen induced VPS72uKO mice.
  • Panel E cell apoptosis and cytokine production in conventional CD4 and CD8 T cells. *P ⁇ 0.05 (for many of the heatmap-style figures disclosed herein, the red or “hot” zone is indicated with a star).
  • FIG. 6 VPS72 deletion led to accumulation of cellular organelles in iNKT cells
  • Panel A Heatmap shows gene signatures related to autophagy assembly in iNKT cells.
  • Panel B Flow cytometry showing Mitotracker Deep Red and Mitotracker Orange dye in total iNKT cells, and subsets of iNKT cells.
  • FIG. 7 VPS72 deletion alters the gene expression program in thymic DP cells undergoing iNKT cell selection.
  • Panel A IL-2 production from DN32.D3 iNKT cells with DP thymocytes from WT and VPS72cKO mice in the presence of a-GalCer.
  • Panel B qPCR shows Val4-Jal8 rearrangement in thymic DP cells.
  • Panels C-D Heatmap of DP RNA-Seq shows key TCR signaling pathways related to iNKT selection (Panel C) and key mitochondrial function pathway in DP thymocytes (Panel D).
  • FIG. 8 Defective iNKT cells in H2A.ZdKO mice.
  • Panel F Pie chart of different genomic regions obtained H2A.Z CUT&RUN-seq analysis of thymic iNKT cells.
  • FIG. 9 VPS72 is required for MAIT cell development.
  • (Panel A) Flow cytometry plots shows MAIT cells in thymus and peripheral organs.
  • Panel B Frequency and absolute number of MAIT cells in indicated organs. Each dot on bar graph represents an individual mouse. *P ⁇ 0.05, **P ⁇ 0.0I.
  • (Panel C) Flow cytometry plots shows developmental stages of MAIT cells in thymus.
  • Panel D Frequencies and absolute numbers of indicated stages of MAIT cells. Each dot on bar graph represents an individual mouse. * P ⁇ 0.05, p** ⁇ 0 01, and ***P ⁇ 0.001.
  • FIG. 10 Deletion of VPS72 alters MAIT cell CD4 and CD8 expression.
  • FIG. 11 Generation ofVPS72 floxed allele and conditionally deleted allele.
  • Panel A Schematic diagram showing technical strategy to generate conditional knockout mice.
  • Panel B Western blot shows deletion of VPS72 in CD4+, CD8+ and CD4+CD25+ cells from VPS72 cKO spleen.
  • FIG. 13 Treg specific loss of VPS72 results in lethal, multi organ autoimmune disease.
  • Panel A Gross anatomical analysis showing runted growth in FOXP3cre VPS72 KO compared to WT and heterozygote (Het) mice.
  • FIG. 14 VPS72 deletion in FOXP3+ Tregs causes activation of conventional T cells.
  • lack of VPS72 in Tregs show expansion of (Panel A) CD4+CD2LloCD44hi and (Panel B) CD8+CD62LloCD44hi effector cells in spleen and lymph nodes, with corresponding representative plots (left) and bar diagram showing their frequency (up) and absolute number (dowTi).
  • (Panel C) CD4+Ki67+ and CD8+Ki67+ proliferating cells in spleen and LN by flow cytometry' (left) and bar graphs (right). Bar graph represents mean SEM (n 5-8/group).
  • CLP common lymphoid progenitors
  • GMP granulocyte-macrophage progenitor
  • MEP megakaryocyte-erythroid progenitor
  • CMP common myeloid progenitor.
  • WT Mxl cre 'VPS72 fl/fl
  • KO Mxl cre+ VPS72 fl/fl .
  • FIG. 17 VPS72 is required for the maintenance of body weight in adult mice.
  • Panel A Scatter dot plot showing the body weight in gram
  • Panel B Representation of mouse image from Csflricre+VPS72fl/fl (KO) and Csflricre-VPS72fl/fl (WT) mice. Each dot represents individual events as indicated and each bar showed mean ⁇ SEM from the measurement n> 3 independent samples. * Significant differences (p ⁇ 0.05) were compared between KO and WT mice.
  • FIG. 19 VPS72 is required for CD45 in organ specific manner from embryonic (E14.5) to adult mice after birth. Fate-mapping analysis for Csflr lcie+ VPS72 flfl expressing mice.
  • Panel A Single cell suspension of epidermis, caudal skin/dermis, brain, eye, lung, kidney, heart and liver starting E14.5 to 3 weeks of age were prepared from Csftr lcre +VPS72 fl/fl (KO) and Csflr lcre -VPS72 fl/fl (WT) mice.
  • FIG. 20 VPS72 required for post-natal maintenance of LCs and AMs.
  • Panels A-B Single cell suspension of epidermis and lung were prepared from CDl lc cre +VPS72 fl/fl (KO) and CD1 lc cre -VPS72 fl/fl (WT) mice.
  • FIG. 22 VPS72-mediated H2A.Z exchange regulates peripheral Treg cell stability and function, and Treg adaptation to tumor microenvironment (TME).
  • FIG. 23 VPS72-mediated H2A.Z epigenetic axis is involved in the biological processes.
  • FIG. 26 Increase VPS72 expression in tumor infiltrated Tregs from human liver cancers and melanoma compared to that in Tregs from PBMCs based on sc-RNA-Seq data. *p ⁇ 0.05, **p ⁇ 0.01 and ****p ⁇ 0.0001.
  • FIG. 31 VPS72 is required for iTreg differentiation from CD4+ naive T cells.
  • CD4 Naive T cells were sorted from spleen WT and VPS72-TregKO mice and stimulated with anti-CD3 coated plate with anti-CD28, IL-2, anti-IL4, anti-IFNy and TGF-(3 for 5 days. Then cells were isolated and expression of FoxP3 was analysis by flow cy tometry'.
  • FIG. 32 H2AZ is not required for T cell and Treg development in thymus but required for peripheral Treg stability.
  • (Panel C) Representative image for Tregs (left) and their frequencies (right) in thymus, spleen and LN from WT and H2AZ-TKO mice. (N 4-6). Student t test. *p ⁇ 0.05 and ****p ⁇ 0.0001.
  • FIG. 33 H2A.Z CUT&RUN-seq analysis of spleen Tregs cells.
  • Panel A Pie chart: genomic distribution of H2A.Z binding.
  • Panel B Gene Ontology (GO) enrichment analy sis.
  • Panel C KEGG enriched pathways.
  • Panel D HOMER de novo motif analysis of H2A.Z binding peaks related to Tregs development.
  • Panel E Genomic track of peaks in the FoxP3 and Tgfbl regions.
  • FIG. 34 Deletion of VPS72 in Treg cells enhances the antitumor response against B16 melanoma.
  • Tumor grow th Panel A
  • tumor w eight Panel B
  • Tumor infiltrated lymphocytes were analyzed in CD45+ cells by flow cytometry (Panel C).
  • the levels of GZMB Panel D
  • IFN-y Panel E
  • Panels F-G CD4+FoxP3+ Tregs in the infiltrated CD4 cells were analyzed.
  • FIG. 36 VPS72-mediated H2A.Z epigenetic axis directly (Panel A) or indirectly (Panel B) regulates FoxP3.
  • the term “and/or.” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed.
  • the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B. and C in combination.
  • treatment and “treating”, are used interchangeably herein, and refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underly ing disorder.
  • the term “treat”, in all its verb forms, is used herein to mean to relieve, alleviate, prevent, and/or manage at least one symptom of a disorder in a subject.
  • a “subject,” as used herein, can refer to any animal which is subject to a viral infection, e.g., a mammal, such as an experimental animal, a farm animal, pet, or the like. In some embodiments, the animal is a primate, preferably a human. As used herein, the terms “subject” and “patient” are used interchangeably.
  • subject and “patient” refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), specifically a “mammal” including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzee and a human), and more specifically a human.
  • the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit).
  • the subject is a “human’”.
  • the term “linked” refers to unifying two molecules having the same or different function or structure, and the methods of fusing may include any physical, chemical or biological method capable of binding the peptide to the protein, the smallmolecule drug, the nanoparticle or the liposome.
  • the fusion may be mediated by a linker peptide, and for example, the linker peptide may be fused to the C-terminus of a peptide.
  • the therapeutic treatment includes the amelioration of at least one measurable physical parameter.
  • the therapeutic treatment includes the inhibition of the progression of a disease or worsening symptom, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g.. stabilization of a physical parameter, or both.
  • an “effective amount” refers to an amount sufficient to elicit the desired biological response.
  • the desired biological response is the reduction or amelioration of disease severity, duration, progression, reoccurrence or delayed onset of disease or symptoms associated with the disease, or to enhance or improve the prophylactic or therapeutic effect(s) of another therapy used to treat the disease (e.g., cancer).
  • the precise amount of the therapeutic composition administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • an “effective amount” of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a polypeptide described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed.
  • compounds described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.
  • peptide is used herein to refer to a chain of amino acid residues.
  • oligonucleotide or nucleic acid are used interchangeably herein to refer to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • polymer is used interchangeably herein to refer to a chain of covalently linked amino acid or nucleotide (RNA or DNA) monomers.
  • the term “reduce” or other forms of the word, such as “reducing” or “reduction.” generally refers to the lowering of an event or characteristic (e.g., one or more symptoms, or the numbers of cells). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. In some embodiments, the term “reducing,” is used in the context of “reducing the functional response of an immune cell”.
  • the binding properties of selected polypeptides can be quantified using methods well known in the art.
  • One such method entails measuring the rates of antigenbinding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (K on ) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • K on the “on rate constant”
  • Koff the “off rate constant”
  • the ratio of Koff/Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant KD. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity' can be measured by well-established methods known in the art, including those described herein.
  • reduced binding refers to a decrease in affinity for the respective interaction.
  • increased binding refers to an increase in binding affinity for the respective interaction.
  • a recombinant peptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is ⁇ 1 pM.
  • a KD can be 10 5 M or less (e.g., 10 6 M or less, 10 7 M or less. 10 s M or less. 10 s M or less, 10 l0 M or less, 10 1 1 M or less, 10 l2 M or less, 10 15 M or less, 10 14 M or less, 1 13 M or less, or 10 16 M or less).
  • an "isolated'’ peptide or oligonucleotide as used herein is obtained directly from a synthetic or recombinant generated peptide or oligonucleotide sequence that is identical, substantially related to, complementary, or modified from a known protein or polynucleotide or a fragment thereof.
  • substantially related means that the protein or oligonucleotide may have been modified by chemical, physical or other means (e.g. sequence modification).
  • Carrier refers to a delivery system.
  • a carrier delivery system functions to increase circulation time in the subject, increase solubility to enhance bioavailability, prevent protease or nuclease degradation, and allow drug targeting to specific cell types.
  • Various types of carriers are described below, each with unique advantages.
  • an inhibitor of the present disclosure provides an inhibitor for use to reduce the expression of VPS72 in targeted or exposed cells, or for use in inhibiting the activity of VPS72 in immune cells.
  • an inhibitor of the present disclosure includes a peptide or protein that binds to VPS72 and inhibits its binding to its binding partner H2A to H2A.Z exchange in chromatin remodeling.
  • a peptide inhibitor comprises a peptide having at least 80%, or 85%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or at least 100% amino acid identity to a peptide of SEQ ID NO: 2 over the entire length of the peptide of SEQ ID NO: 2.
  • a peptide inhibitor of the present disclosure comprises one, two, three, four or five amino acid conservative amino acid substitutions in the sequence of SEQ ID NO: 2 ranging from amino acid position 13 to 55 of SEQ ID NO: 2. with the exception of amino acids F29, Y30, Y34. F37, D43, E35, and Y64 of SEQ ID NO: 2.
  • a peptide inhibitor of the present disclosure comprises a peptide having 23 to 100 amino acids, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 2 ranging from amino acid position 13 to 55 of SEQ ID NO: 2.
  • VPS72 inhibitor peptide of SEQ ID NO: 2 comprises a region of VPS72 as underlined below
  • GDQSDTEDEVDSDFDIDEGDEP Partial amino acid sequence of VPS72 (homo sapiens) - SEQ ID NO: 3, which is a modified form of SEQ ID NO: 2.
  • an inhibitor peptide blocks VPS72 from binding to H2A.Z-H2B dimer by simulating the critical amino acids in the N- terminal binding domain of VPS72 that interacts with H2A.Z-H2B as shown underlined relative to the VPS72 amino acid sequence of SEQ ID NO: 3, which includes F29, Y30, Y34, F37, D43, E35. and Y64 of SEQ ID NO: 3.
  • an inhibitor peptide comprises an amino acid sequence that comprises at least a region of the VPS72 amino acid sequence comprising positions F29, Y30, Y34, F37, D43, E35, and Y64 of SEQ ID NO: 3.
  • the inhibitor peptide ranges from 20 amino acids to 60 amino acids, more preferably from 25 amino acids to 50 amino acids, most preferably from 30 amino acids to 40 amino acids in length, wherein the inhibitor peptide has amino acids F29, Y30, Y34, F37, D43, E35, and Y64 relative to the entire amino acid sequence of SEQ ID NO: 3.
  • an exemplary 7 inhibitor peptide of VPS72 comprises a peptide having at least 95%, or 96%, or 97%, or 98%, or 98%, or 99%, or at least 100% sequence identity to a peptide of SEQ ID NO: 2, along its entire length.
  • a VPS72 inhibitor of the present disclosure is a nucleic acid that inhibits the transcription or expression or translation of VPS72 DNA or VPS72 mRNA.
  • RNA interference also called post-transcriptional gene silencing (PTGS)
  • PTGS post-transcriptional gene silencing
  • dsRNA long double-stranded RNA
  • siRNAs small interfering RNAs
  • RNAi can involve the introduction of a small hairpin RNA (shRNA); shRNA is a nucleic acid that includes the sequence of the tw o desired siRNA strands, sense and antisense, on a single strand, connected by a “loop” or “spacer” nucleic acid.
  • shRNA small hairpin RNA
  • the two complementary portions anneal intra-molecularly to fonn a “hairpin,” which is recognized and processed by the endogenous machinery.
  • a RNAi nucleic acid molecule as described herein is complementary to at least a portion of a target mRNA (i.e., a VPS72 mRNA), and typically is referred to as an “antisense strand”.
  • the antisense strand includes at least 15 contiguous nucleotides of the DNA sequence (e.g., the VPS72 nucleic acid sequence shown in SEQ ID NO:4 (homo sapiens and/or mus musculus for SEQ ID NOS: 4-9)); it would be appreciated that the antisense strand has the “RNA equivalent” sequence of the DNA (e.g., uracils instead of thymines; ribose sugars instead of deoxyribose sugars).
  • a RNAi nucleic acid molecule can be, for example, 15 to 500 nucleotides in length (e.g., 15 to 50, 15 to 45, 15 to 30, 16 to 47, 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74. 17 to 162, 17 to 305, 18 to 60.
  • 15 to 500 nucleotides in length e.g., 15 to 50, 15 to 45, 15 to 30, 16 to 47, 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to
  • the ‘"antisense strand” (e.g., a first nucleic acid) can be accompanied by a “sense strand” (e.g., a second nucleic acid), which is complementary to the antisense strand.
  • each nucleic acid e.g., each of the sense and antisense strands
  • 20 to 40 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides in length).
  • the hairpin structure has two annealing RNA sequences, where one of the annealing RNA sequences of the hairpin RNA structure includes a sense sequence identical to at least 20 consecutive nucleotides of the VPS72 nucleotide sequence, and where the second of the annealing RNA sequences includes an antisense sequence that is identical to at least 20 consecutive nucleotides of the complement of the VPS72 nucleotide sequence.
  • the DNA region can include an intron (e.g., a functional intron).
  • the intron When present, the intron generally is located between the two annealing RNA sequences in sense orientation such that it is spliced out by the cellular machinery (e.g., the splicesome).
  • a construct can be introduced into one or more plant cells to reduce the phenotypic expression of a VPS72 nucleic acid (e.g., a nucleic acid sequence that is normally expressed in a plant cell).
  • a construct e.g., an expression construct
  • a single promoter can be used to drive expression of the inverted- duplication of the VPS72 gene segment, and that the inverted-duplication typically contains at least one copy of the portion of the VPS72 gene in the sense orientation.
  • Such a construct can be introduced into one or more plant cells to delay, inhibit or otherwise reduce the expression of a VPS72 gene in the plant cells.
  • the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty 7 : 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0; gap extension penalty’: 5.0: and weight transitions: yes.
  • word size 1; window size: 5; scoring method: percentage: number of top diagonals: 5; and gap penalty 7 : 3.
  • the Tm between a nucleic acid that is less than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Section 11.46.
  • Sambrook et al. additionally disclose Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54).
  • the Tm between a nucleic acid greater than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al.
  • Expression elements that can be used to drive expression of a RNAi nucleic acid molecule are known in the art and include, without limitation, constitutive promoters such as, without limitation, the cassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the actin promoter, or the glyceraldehyde-3-phosphate dehydrogenase promoter, or tissue-specific promoters such as, without limitation, root-specific promoters such as the putrescine N-methyl transferase (PMT) promoter or the quinolinate phosphosibosyltransferase (QPT) promoter.
  • constitutive promoters such as, without limitation, the cassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the actin promoter, or the glyceraldehyde-3-phosphate dehydrogenase promoter
  • tissue-specific promoters such as, without limitation, root
  • VPS72 functions as a histone chaperone for H2A to H2A.Z exchange in chromatin remodeling and belongs to two multi-subunit chromatin-remodeling complexes: (1) the Snf2 -related CBP-activator protein chromatin remodeling (SRCAP) complex; and (2) the TRRAP/TIP60 complex.
  • SRCAP Snf2 -related CBP-activator protein chromatin remodeling
  • TRRAP/TIP60 complex Exchanging H2A for variant histone H2A.Z through the VPS72/SRCAP/TIP60 complex has been show n to modulate chromatin structure to activate or repress target genes, modify the local chromatin structure, and regulate cellular processes and mitochondrial function.
  • VPS72 is critical for invariant natural killer T cells (iNKT) cells, mucosal associated invariant T cells (MAIT) cell development and activity, Tregs, hematopoietic stem cells (HSC), Langerhans cells (LC), macrophages, and dendritic cell (DC) development and function.
  • iNKT invariant natural killer T cells
  • MAIT mucosal associated invariant T cells
  • Tregs hematopoietic stem cells
  • LC Langerhans cells
  • macrophages macrophages
  • DC dendritic cell
  • inhibitors of VPS72 function, for example, the disclosed VPS72
  • autoimmune diseases for example, allergic contact dermatitis, lupus, scleroderma, inflammatory bowel disease colitis (e.g., ulcerative colitis) systemic lupus erythematosus (i.e., lupus) multiple sclerosis and arthritis, and symptoms
  • the present disclosure provides a method of altering an immune cell response in a subject in need thereof, said method comprising administering an agent that inhibits VPS72 activity and/or expression in an immune cell of the subject.
  • the methods of the present disclosure anticipate inhibiting the expression of VPS72 in immune cells and/or inhibiting the expression of VPS72 in the target immune cells, for example, iNKT cells and MAIT cells.
  • iNKT cells arise in the thymus from the CD4 + CD8 + double-positive (DP) stage and are selected by CD Id-expressing DP thymocytes. In contrast to the selection of conventional T cells by thymic epithelial cells, iNKT cell selection requires stronger TCR signal strength.
  • iNKT cells CD24 + CD44 lo NKl.r
  • subsequent events elicit proliferation and a progression of maturation from immature CD24" CD44 lo NKl. l (stage 1. STI) to semi-mature CD24 CD44 hl NKl. T(stage 2, ST2) to mature CD44 hl NKl. l + (stage 3, ST3) iNKT cells.
  • the MR1-MAIT cell axis is strikingly conserved across 150 million years of mammalian evolution, with -90% sequence homology for MR1 between mouse and human, implying a strong evolutionary 7 pressure maintaining the MAIT cell repertoire. Nonetheless identifying the critical functional role(s) played by these cells has not proved straightforward, perhaps because these cells perform not a single, but several distinct functions. Indeed, several new MAIT cell functions have recently been discovered, representing distinct transcriptional programs which can be triggered via distinct activation pathways. [0109] Conventional T cells are implicated as effectors in many organ-specific autoimmune diseases such as type-1 diabetes or multiple sclerosis, but strong HLA associations in a range of systemic autoimmune diseases imply a pathogenic role in these diseases as well.
  • reducing Treg functional response can be advantageous in the treatment of certain autoimmune diseases.
  • the VPS72 inhibitors and compositions containing such inhibitors can be used to effectively treat or reduce chronic graft versus host disease (cGVHD) in a patient.
  • the patient has received a bone marrow transplant, a hematopoietic stem cell transplant, or a progenitor cell transplant, from a donor.
  • the patient has received a solid organ transplant (e.g., kidney, liver, heart, lung, etc.) from a donor.
  • the methods described herein can be used to treat an autoimmune or alloimmune disease (e.g., chronic alloimmune or autoimmune responses).
  • a pharmaceutically acceptable carrier may contain inert ingredients which do not unduly inhibit the biological activity of the poly peptides.
  • the pharmaceutically acceptable excipient should be biocompatible, e.g., non-toxic, non-infl ammatory, non- immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.
  • Some examples of materials which can serve as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the peptide or oligonucleotides, also described as polymers, of the present invention may exist as salts, such as with pharmaceutically acceptable acids.
  • the present invention includes such salts.
  • Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, propri onates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.
  • Formulations described herein as being useful for pulmonary delivery’ are useful for intranasal delivery of a pharmaceutical composition of the invention.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation for buccal administration.
  • Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0. 1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and. optionally, one or more of the additional ingredients described herein.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example. U.S. Pat. No. 5,075.109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty 7 acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings: compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active compound is contained in a form within a matrix such as those described in U.S. Pat. Nos.
  • Long-term sustained release are used herein, means that the implant is constructed and arranged to delivery 7 therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Longterm sustained release implants are known to those of ordinary skill in the art and include some of the release systems described above.
  • Parenteral administration includes, e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of additional therapies.
  • the therapeutic drugs can be administered alone or can be co-administered to the patient.
  • Co-administration is meant to include simultaneous or sequential administration of the components individually or in combination.
  • the preparations can also be combined, when desired, with other active substances.
  • sequential administration includes that the administration of two agents (e.g., the agents described herein) do not occur on a same day.
  • compositions include overlapping in duration at least in part.
  • concurrent administration includes overlapping in duration at least in part.
  • two compositions e.g., any of the compositions described herein
  • their administration occurs within a certain desired time.
  • the administration of the compositions may begin and end on the same day.
  • the administration of one composition can also precede the administration of a second composition by day(s) as long as both compositions are taken on the same day at least once.
  • the administration of one composition can extend beyond the administration of a second composition as long as both agents are taken on the same day at least once.
  • the composition do not have to be taken at the same time each day to include concurrent administration.
  • “intermittent administration” includes the administration of an agent for a period of time (which can be considered a “first period of administration”), followed by a time during which the composition is not taken or is taken at a lower maintenance dose (which can be considered “off-period”) followed by a period during which the composition is administered again (which can be considered a “second period of administration”).
  • first period of administration a period of time
  • second period of administration a period during which the composition is administered again
  • the dosage level of the agent will match that administered during the first period of administration but can be increased or decreased as medically necessary.
  • VPS72 inhibitors whether protein, peptide or nucleic acid based inhibitors and pharmaceutically acceptable compositions described above, can be administered to humans and other animals orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated.
  • Dosage forms for topical or transdermal administration of a polypeptide described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention.
  • the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a polypeptide to the body.
  • Such dosage forms can be made by dissolving or dispensing the polypeptide in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the polypeptide across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the polypeptide in a polymer matrix or gel.
  • Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS 72 inhibitors is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
  • compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors as disclosed herein can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation.
  • Formulations having compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors suitable for use with a sprayer typically include a polypeptide composition in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one polypeptide per ml of solution or mg/gm, or any range, value, or fraction therein.
  • the formulation can include agents, such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality 7 of VPS72 inhibitors formulation can also include a surfactant, which can reduce or prevent surface- induced aggregation of the polypeptide composition caused by atomization of the solution in forming an aerosol.
  • a surfactant which can reduce or prevent surface- induced aggregation of the polypeptide composition caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation.
  • Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80. polysorbate 20. or the like.
  • compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors, such as preservatives and protease and nuclease inhibitors, can also be included in the formulation.
  • a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
  • an ultrasonic nebulizer high-frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy 7 is transmitted to the formulation of the polypeptide composition either directly or through a coupling fluid, creating an aerosol including the polypeptide composition.
  • particles of the polypeptide composition delivered by a nebulizer have a particle size less than about 10 gm, in some embodiments, in the range of about 1 pm to about 5 pm, or from about 2 pm to about 3 pm.
  • Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
  • Formulations of at least one polypeptide for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one VPS72 inhibitor as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
  • the desired outcome is a decrease in the number and/or activity of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC).
  • the desired outcome is a decrease or elimination of symptoms associated with a condition characterized by increased number and/or activity of MAIT cells.
  • the exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like.
  • the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every' third day, every' week, every' two weeks, every three weeks, or every' four weeks.
  • the desired dosage can be delivered using multiple administrations (e.g.. two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • an effective amount of a VPS72 inhibitor or a preparation for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a VPS72 inhibitor per unit dosage form.
  • the invention contemplates in vitro and in vivo uses of the VPS72 inhibitor immuno-inhibitory molecules and preparations provided herein.
  • the VPS72 inhibitor and preparations may be formulated as pharmaceutical compositions (or preparations), intending that they are suitable for administration to a subject.
  • a pharmaceutical composition need not be therapeutic or prophylactic however (i.e. , it may not eradicate an existing condition or prevent a condition from ever occurring in a subject).
  • an aberrant immune response such as an increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) based immune response, or an increase in Treg activity, and thereby optionally modulate symptoms resulting from the underlying iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell based immune response.
  • an aberrant immune response such as an increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell based immune response.
  • Such in vivo uses may be in subjects being treated for a particular condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell activity with the intention of providing some therapeutic or prophylactic benefit.
  • Tregs T- regulatory T-cells
  • HSCs T-regulatory T-cells
  • LCs macrophages and dendritic cells
  • DC dendritic cells
  • VPS72 inhibitor and preparations may be used in vivo for research purposes, inter alia, typically in non-human subjects.
  • the VPS72 inhibitor or preparations may be used in a method that involves contacting the VPS72 inhibitor (s) or preparation with an antigen presenting cell, and contacting the "loaded" antigen presenting cell with an iNKT cell, MAIT cell, T-regulatory T-cell (herein referred to as “Tregs”), HSC, LC, macrophage and dendritic cell (DC).
  • the antigen presenting cells typically will express CD Id on their surface.
  • a "loaded" antigen presenting cell intends an antigen presenting cell that has an immunoinhibitory molecule of the invention bound to its CDld and is therefore able to present such molecule to an iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs. LCs, macrophages and dendritic cells (DC).
  • the contacting may occur in the presence of an agent that stimulates iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC)such as the VPS72 inhibitor peptide of SEQ ID NO: 2.
  • the contacting may occur in the absence of such an immunostimulatory agent, and instead the iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) may be activated iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC).
  • Tregs T-regulatory T-cells
  • DC dendritic cells
  • Exemplary autoimmune diseases include, but are not limited to, multiple sclerosis, inflammatory bowel diseases such as ulcerative colitis, Crohn's disease, and ileitis, glomerulonephritis, Goodpasture's disease or syndrome, Graves' disease, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid, arthritis, psoriatic arthritis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis.
  • multiple sclerosis inflammatory bowel diseases such as ulcerative colitis, Crohn's disease, and ileitis, glomerulonephritis, Goodpasture's disease or syndrome
  • Graves' disease necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematos
  • anti-phospholipid antibody syndrome scleroderma, perphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), urveitis, Sjogren's syndrome, Reiter's syndrome, ankylosing spondylitis, Lyme arthritis, GuillainBarre syndrome, Hashimoto's thyroiditis, and cardiomyopathy.
  • vasculitis e.g., Wegener's granulomatosis, microscopic polyangiitis
  • the subject may be less than 10 years of age, less than 5 years of age, less than 1 year of age, less than 6 months of age, or less than 1 month of age, or 4 to 12 years of age, 12-18 years of age or 18-80 years of age and any age within these ranges.
  • the invention further contemplates administration of older subjects such as adults, 18 to 80 years of age.
  • the subject may be a pregnant subject or a female subject of child-bearing age, either of which may optionally be at increased risk of developing a condition characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or activity’ (e.g., an autoimmune disease, asthma, and the like).
  • Tregs T-regulatory T-cells
  • HSCs T-regulatory T-cells
  • LCs T-regulatory T-cells
  • macrophages dendritic cells
  • DC dendritic cells
  • HSCs, LCs, macrophages and dendritic cells (DC)cell numbers and/or activity may be treated once, twice or more times, over a period of time. This period of time may be days, weeks, months, or years.
  • the agents may be administered daily or weekly in a subject experiencing symptoms associated with a condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as "Tregs").
  • Tregs T- regulatory T-cells
  • HSCs, LCs, macrophages and dendritic cells (DC) cell numbers or activity 7 until such symptoms are reduced or eliminated.
  • the agents may be administered one or more times in the early years of life of a subject and then may be administered again after several years, as a "boost" to the original administration. This latter administration schedule could be similar to that used in more traditional vaccination schemes.
  • the condition is an inflammatory condition.
  • the condition is asthma.
  • the condition is an autoimmune disease.
  • the condition is inflammatory bowel disease.
  • the condition is colitis (e.g., ulcerative colitis).
  • the condition is systemic lupus erythematosus (i.e., lupus).
  • the condition is multiple sclerosis.
  • the condition is arthritis.
  • the subject is human. In some embodiments, the subject is less than 5 years of age, less than 1 year of age, less than 6 months of age, or less than 1 month of age. In some embodiments, the subject is a pregnant subject and optionally is at high risk of developing a condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or activity. In some embodiments, the subject is a female subject of child-bearing age (e.g., in humans, approximately 15-55 years of age), and optionally is at increased (i.e..
  • HSCs, LCs, macrophages and dendritic cells in the presence of an agent that stimulates iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) such as an VPS72 inhibitor such as for example a peptide having at least 95% sequence identity to a peptide of SEQ ID NO:2.
  • the immunoinhibilory VPS72 inhibitor of the invention are able to compete and/or interfere with the expression and/or activity of VPS72, such as an inhibitor peptide having at least 95% sequence identity 7 to a peptide of SEQ ID NO: 2, thereby preventing or reducing the degree of immunostimulation that would otherwise occur in the absence of an inhibitor peptide having at least 95% sequence identity to a peptide of SEQ ID NO:2 or a VPS72 expression inhibitor, for example, a nucleic acid that inhibits the transcription, or translation ofVPS72.
  • iNKT cells MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and activity levels are normal (e.g., the levels in a subject that does not have an inflammatory or autoimmune diseases, conditions or symptoms related thereto and/or is not at elevated risk of developing an inflammatory or autoimmune diseases, conditions or symptoms related thereto (as a result of heredity', for example)), then the immunoinhibilory molecules and preparations may manifest no immunoinhibitory effect essentially because there is no observable background iNKT and/or MAITcell based immune stimulation.
  • T-regulatory T-cells herein referred to as “Tregs”
  • HSCs HSCs
  • LCs macrophages and dendritic cells (DC) numbers
  • Assays for measuring iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) activity are also known in the art and are described in the Examples herein.
  • These assays include cytokine production assays such as IFN-gamma, IL-13 and IL- 1 -beta production assays.
  • LCs LCs, macrophages and dendritic cells (DC) and/or increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as ‘'Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) activity.
  • 'Tregs T-regulatory T-cells
  • HSCs HSCs
  • LCs macrophages and dendritic cells
  • DC dendritic cells
  • symptoms include the symptoms associated with inflammatory conditions and autoimmune diseases and conditions.
  • An exemplary' but not limiting inflammatory condition is asthma.
  • An exemplary but not limiting autoimmune disease is colitis.
  • Desoximetasone Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; FluoromethoIone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Fur
  • the additional active agent may be an asthma medicament, meaning a medicament that reduces the symptoms, inhibits the asthmatic reaction, or prevents the development of an asthmatic reaction.
  • asthma medicaments meaning a medicament that reduces the symptoms, inhibits the asthmatic reaction, or prevents the development of an asthmatic reaction.
  • Various ty pes of medicaments for the treatment of asthma are described in the Guidelines for The Diagnosis and Management of Asthma. Expert Panel Report 2. NIH Publication No. 97/4051. Jul. 19. 1997, the entire contents of which are incorporated herein by reference.
  • Asthma medicaments include, but are not limited, PDE-4 inhibitors, Bronchodilator/beta-2 agonists, K+ channel openers, VLA-4 antagonists, Neurokin antagonists, TXA2 synthesis inhibitors, Xanthanines, Arachidonic acid antagonists, 5 lipoxygenase inhibitors. Thromboxin A2 receptor antagonists, Thromboxane A2 antagonists, Inhibitor of 5-lipox activation proteins, and Protease inhibitors.
  • Corticosteroids include, but are not limited to, beclomethasome dipropionate, budesonide, flunisolide, fluticaosone, propionate, and triamcinoone acetonide.
  • Systemic corticosteroids include, but are not limited to, methylprednisolone, prednisolone and prednisone.
  • the polypeptides are administered sufficiently close in time to have the desired therapeutic effect.
  • the period of time between each administration which can result in the desired therapeutic effect can range from minutes to hours and can be determined taking into account the properties of each composition such as potency, solubility, bioavailability. plasma half-life and kinetic profile.
  • a polymer and the second therapeutic agent can be administered in any order within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other or within about 30 minutes of each other.
  • the method of co-administration of a first amount of the polymer composition and a second amount of an additional therapeutic agent can result in an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect that would result from separate administration of the first amount of the polymer composition and the second amount of the additional therapeutic agent.
  • a synergistic effect can result in improved efficacy of agents in the prevention, management or treatment of a disorder.
  • a synergistic effect of a combination of therapies e.g., a combination of prophylactic or therapeutic agents
  • the unit dosage form comprising an inhibitor of VPS72 should be suitable for pulmonary delivery for example by aerosol.
  • kits may include agents in sterile aqueous suspensions that may be used directly or may be diluted with normal saline for intravenous injection or use in a nebulizer, or dilution or combination with surfactant for intratracheal administration.
  • the kits may therefore also contain the diluent solution or agent, such as saline or surfactant.
  • the kit may also include a pulmonary delivery device such as a nebulizer or disposable components therefore such as the mouthpiece, nosepiece, or mask.
  • Chromatin remodeling is an important layer of epigenetic regulation and can play a key role in immune cell development, homeostasis, and function.
  • Nucleosomes are the basic unit of chromatin.
  • the histone octamer packages DNA into nucleosomes, maintains the nucleosome morphology, and serves as a regulatory layer for gene expression.
  • This octamer consists of histone proteins such as H2A, H2B, H3, and H4 (Fig 1).
  • H2A which comprises an H2A variant known as histone H2A.Z15.
  • mice were housed under pathogen free conditions, allowed ad libitum food and water and maintained on standard conditions (25°C, 14: 10-h light- dark cycle). All experimental procedures were performed according to institutional guidelines for animal care and use committee of Henry Ford Health Sy stem, which conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
  • the LlL2_Bact_P cassette was inserted at position 95117966 of Chromosome 3 upstream of the critical exons (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site.
  • lysis buffer 0.3% NH4C12 buffer
  • wash buffer 1% FBS and 0.5M EDTA in PBS
  • RNA-Seq was perfonned in sorted splenic Tregs cells from Treg deficient VPS72 mice or WT mice.
  • cDNA synthesis and preamplification were performed to achieve optimal library 7 quality' using SMART-seq v4 Ultra Low Input RNA Kit (Clontech, Mountain View, CA) following the manufacturer’s instruction. Briefly, pre-amplification of cDNA was done using a thermal cycler (Applied Biosystems) for 12-14 cycles and fragmented into 150- 400bp using a Bioruptor Pico Sonicator (Diagenode, Denville, NJ).
  • iNKT cells arise in the thymus from the CD4+CD8+ double-positive (DP) stage and are selected by CD Id-expressing DP thymocytes. In contrast to the selection of conventional T cells by thymic epithelial cells, iNKT cell selection requires stronger TCR signal strength.
  • iNKT cells CD24+CD441oNKl. l-
  • stage 0, STO subsequent events elicit proliferation and a progression of maturation from immature CD24- CD441oNKl. l -(stage 1, STI) to semi-mature CD24-CD44hiNKl .1 -(stage 2, ST2) to mature CD44hiNKl.
  • iNKTl cells are PLZFloCD44hiNKl.l+ (i.e., ST3), express T-bet, and mainly produce IFN-y;
  • iNKT2 cells are PLZFhiCD44hiNKl.l- (i.e., ST2), express GATA3, and produce abundant IL-4; and
  • iNKTl 7 cells resemble iNKT2 cells in being PLZFintCD44hiNKl.
  • VPS72 mRNA in thymic T cells including iNKT cells, measured by RT- PCR (Fig.2E) and protein in DP thymocytes measured by Western blot (Fig.2F) were dramatically reduced in VPS72cKO mice.
  • iNKT CD24+ precursors progress through CD441o and CD44hi stages and lastly acquire NK1.1 expression during their final maturation.44 Therefore, the CD24/CD44/NK1.1 profiles of CDld-tetramer+ thymocytes were analyzed to determine if iNKT cell development and maturation were defective in VPS72cKO mice.
  • the absolute numbers at STO-2 in KO mice were comparable to control mice, but they were dramatically reduced in ST3 compared to WT controls (Fig.3D-E).
  • iNKT cell development requires positive selection by DP thymocytes through
  • NK1 (STI and ST2) iNKT cells and found that T- bet+NKTl. l- NKT1 precursors were dramatically reduced, while PLZFhiT-bet- iNKT2 cells were increased (Fig.4D).
  • RNA-seq FACS-sorted thymic iNKT cells from VPS72cKO and WT mice.
  • VPS72 is required for well-developed iNKT cell IFN-y secretion, but not for conventional T cells.
  • VPS72 regulates iNKT cell autophagy.
  • RNA-seq analysis we found that autophagy assembly pathway-related genes were significantly reduced in VPS72cKO iNKT cells (Fig. 6A). It has been shown that defective autophagycauses accumulation of cellular organelles, ultimately leading to T cell apoptosis.
  • VPS72 Although the role of VPS72 interact with H2A.Z in SCARP complex has been elucidated in yeast and HeLa cells, it has not been defined in immune cells. We therefore performed Co-IP using two different H2A.Z antibodies in mouse iNKT cell line DN32.D3 against VPS72 antibody. As shown in Fig. 8A, we confirmed that VPS72 indeed interacts with H2A.Z in iNKT cells. To further confirm the functional specificity’ of VPS72 as a histone chaperone that recognizes and exchanges H2A.Z, we crossed
  • H2A.ZdKO mice CD4cre.H2A.Zl.H2A.Z2 double KO mice
  • H2A.ZdKO CD4cre.H2A.Zl.H2A.Z2 double KO mice
  • H2A.Z binding peaks were highly enriched in the GO terms of mitochondrial metabolism (transport, organization, protein complex, outer membrane, and nuclear envelope), transcription coactivator, translation regulator, and DNA-binding transcription factor (Fig.8G).
  • KEGG pathway analysis also indicates that the enriched peaks were found mostly in autophagy /mitophagy and TCR signaling pathway gene regions (Fig.8H).
  • iNKT cells within motifs for transcription factors in mitochondrial function, such as FOXO3, STAT3, and NRF-1, and in TCR signaling, such as NUR77 (Fig.81).
  • Thymic MAIT cells complete their functional differentiation in the thymus. We found that the frequency and absolute number of T-bet+MAITl cells and RORyt+ MAIT17 cells were reduced in VPS72 deficient thymus, liver, and spleen compared to WT (Fig 9E). Additionally. CD4CD8 double negative (DN) MAIT cells population were almost absent in VPS72 deletion MAIT cells.
  • MAIT cells function in cancer
  • Treg cells differentiate in the periphery from conventional T (Tconv) cells under certain conditions (peripherally derived Treg or pTreg cells) or by antigen stimulation in the presence of TGF-P and IL -2 (induced Treg or iTreg cells). Both pTreg and tTreg cells appear to be highly stable in the expression of FoxP3 and other Treg signature genes to mediate stable immune-suppressive function.
  • Tregs are unstable and plastic, and their phenotype and function change with the specific environment.
  • a third loxP site is inserted downstream of the targeted exon(s) at position 95119493.
  • the critical exon(s) is/are thus flanked by loxP sites.
  • a "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele.
  • VPS72 ft/fl were crossed with transgenic mice expressing Cre recombinase driven by the Cd4 promoter, resulting in T cell specific deletion of the VPS72 gene starting at the DP thymocyte stage.
  • the knockout efficiency was confirmed through the examination of VPS72 protein level in splenic CD4+CD25+ Treg cells (Fig. 11 B) [0301]
  • VPS72 is required for Treg homeostasis in periphery.
  • genes that are involved in regulating cytokine also found to increase the production of proinfl ammatory cytokines such as IL-10, IL-16 and IL-15ra, and chemokines such as CCL1, CXC13, CXCL10, and chemokine receptors and adhesion molecules (Slprl, Slpr4 and CCr4) in VPS72-deficient Tregs (Table 1 B), suggesting that Tregs lose suppressive function and failed to control against severe inflammation.
  • proinfl ammatory cytokines such as IL-10, IL-16 and IL-15ra
  • chemokines such as CCL1, CXC13, CXCL10, and chemokine receptors and adhesion molecules (Slprl, Slpr4 and CCr4) in VPS72-deficient Tregs (Table 1 B), suggesting that Tregs lose suppressive function and failed to control against severe inflammation.
  • Cancer is a leading cause of death worldwide accounting for nearly 10 million deaths in 2020. With this global burden, prevention of cancer is one of the most significant public health challenges these days. Cancer cells can evade the immune response and establish a very' complex balance for driving tumor progression, metastasis and resistance to therapy. Among skin cancers, melanoma is a highly aggressive cancer and metastasize to different organs, making it difficult to target via classical cancer therapies. Immunotherapy represents a novel emerging strategy 7 for targeting aggressive cancers, however a major hurdle remains with the immunosuppressive environment within the melanoma tumor mediated by regulatory T cells (Tregs).
  • Tregs regulatory T cells
  • VPS72 Role of histone demethylase Lsdl and lysine acetyltransferase Tip60 of HAT complex has been studied in hematopoietic stem cell maintenance. Novel gain-of-function screening approach identified VPS72 having a role for maintaining hematopoietic stem cell activity. However, the role of VPS72 in hematopoiesis in largely known. Given that the role VPS72, we hypothesize that VPS72 may serve as a therapeutic target to maintain HSC functions in different blood related diseases by regulating chromatin and gene expression.
  • VPS72fl/fl mice were bred with MxlCre mouse, which allows inducible deletion of VPS72 throughout all hematopoietic lineages and hematopoietic stem cells.
  • Injection of Mxlcre-vps72fl/fl and Mxlcre+vps72fl/fl mice with dsRNA poly (I: C) (Fig. 16 A) resulted in efficient VPS72 inactivation as shown by western blot, qPCR and flow cytometry (Fig.
  • TRM produces a wide range of growth factors for tissue development and achieve endocytic as well as phagocytic activity’ for cellular/tissue specific defense. Additionally, study showed that TRMs is necessary' embryogenesis and organogenesis in numerous organs including lung, kidney. Following these diversified roles of TRM, macrophage mediated immunotherapies may become a potential tool against derailed tissue specific physiological and compromised pathological conditions. Therefore, any differentiated TRM cells or any important regulatory’ protein of TRMs can be utilized as a possible treatment regime for various diseases including cancer. Hence, our aim is to provide current experimental evidence on the role of VPS72 protein on TRMs during embryonic development and adult mice after birth.
  • a third loxP site is inserted downstream of the targeted exon(s) at position 95119493.
  • the critical exon(s) is/are thus flanked by loxP sites.
  • a "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele.
  • Vps72 fl/fl mice were crossed with transgenic mice expressing Cre recombinase driven by the CD11c promoter, resulting in dendritic cell specific deletion of the Vps72 gene.
  • CD45hiMHCIIhi in adult CD45hiMHCIIhi in adult
  • dermal macrophage DM
  • CD1 lbintF4/80hi brain and eye microglia
  • CD1 lbintF4/80hi at PO CD1 lbintCD39hi in adult
  • lung AMs CD1 lbintF4/80hi at PO, CD1 IchiSiglecFhi in adult
  • kidney, pancreas heart TRMs (CDllbintF4/80hi at PO and in adult) and liver KCs (CDllbintF4/80hior CD1 lbintTIM4hi) in CKO mice compared to WT littennate.
  • the frequencies of all TRMs in epidermis, dermis, liver, lung, and kidney were dramatic decreased in CKO mice at some or all specific time compared to WT littermate after birth (Fig. 18 A, B, Table 2).
  • VPS72 is required for the post-natal maintenance of LCS and AMs.
  • CD11c ere dendritic cell specific (CD11c ere) VPS72 deletion mouse model and examine the TRM in both epidennis and lung in adult CD1 lccreVPS72fl/fl (CKO) mice by comparing to wild type (WT) littermates.
  • WT wild type
  • ACD Allergic contact dermatitis
  • ACD is the most common occupational disease that might irritate the skin or trigger an allergic reaction.
  • ACD is hypersensitivity response by an individual's immune system with direct skin contact with chemicals and appears as a red rash on irritant exposed skin site.
  • LCs there is reduced number of LCs in ACD patients.
  • any therapy modulates reestablishing LCs this may become an important tool to treat this disease.
  • our experiment data suggest that VPS72 is necessary’ for the development and maintenance of LCs (Fig. 18). We believe, if any intervention either pharmacological or genetic based topical activation of VPS72 is carried out, we can treat this allergic contact dermatitis.
  • Skin cancer is the most common type of cancer mostly consists of squamous cell carcinoma, basal cell carcinoma, and melanoma. Among them, melanoma is uncommon cancer with high mortality rate and is highly malignant to other tissue areas. During having melanoma, various studies indicated that there is a decline in LCs in the epidermis of skin, suggesting association of LC in advancing melanoma cancer. Likewise, the basal cell carcinoma has increased number of epidermal LCs in the periphery and decreases frequency of LC in tumor indicating the essential role of LCs to limit tumor growth. Moreover, if any therapy modulates reestablishing this derailed LCs population, this may become an important tool to treat this disease.
  • TRM tumor associated macrophages
  • a fully functioning immune system is essential for maintaining good health.
  • the immune system undergoes deterioration with advancing age. and contributes towards increased susceptibility against infection, autoimmunity, and cancer.
  • macrophage immune cells can be an important player for maintaining tissue homeostasis and protecting tissue against infection.
  • those macrophages mediated function are derailed with age.
  • VPS72 in metabolic syndromes Dysregulation of TRM function can have multiple consequences in many diseases, including cardiovascular and metabolic condition, obesity, cancer, amyloidosis, and infections. Reestablishment of functional macrophages may become an opportunity for treating those metabolic disease. Accumulating evidence suggest that post-natal microglial activation may become chronic inflammatory source to drive progressive neurodegeneration and induce diabetic retinopathy. Further, Kupffer cells play important roles in iron metabolism, cholesterol metabolism, and immune surveillance. In case, if liver KC become dysfunctional or diminished, this may lead to pathogenic situation by increased lipogenesis, released inflammatory cytokines, and activated stellate cells to generate fibrosis condition like non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • VPS72 is a specific H2A.Z deposition chaperone, later is a widely conserved histone variant; the crystal structure of human VPS72-H2A.Z- H2B complex have identified the specific binding sites of VPS72
  • Previous study identified critical binding sits of VPS72
  • VPS72 vacuolar protein sorting-associated protein 72 homolog
  • Aim 1 Define the roles of VPS72 and H2A.Z in peripheral Treg cell stability and function.
  • Aim 3 Identify TME factors that upregulate VPS72 expression for Treg adaptation. It can be determined whether TME cytokines (such as TGF-(31), metabolites (low glucose & amino acids), and hypoxia induce VPS72-mediated FI2A.Z exchange in Tregs and dissect the underlying molecular mechanisms. [0362] In view of these aims, new insights can be gleaned into the role of VPS 72- mediated H2A.Z exchange in Treg stability and function, which will not only provide new insight into the biology of Tregs, but also facilitate the development of Treg-based intervention strategies for cancer.
  • TME cytokines such as TGF-(31)
  • metabolites low glucose & amino acids
  • hypoxia induce VPS72-mediated FI2A.Z exchange in Tregs and dissect the underlying molecular mechanisms.
  • VPS72 overexpression has been identified in different types of cancers, such as hepatocarcinoma and melanoma (TCGA GDAC team) and is recognized as an oncogene.
  • VPS72 is on the top 20 list of highly expressed genes in skin cutaneous melanoma (TCGA- GDAC database).
  • TCGA- GDAC database high H2A.Z expression w as positively correlated with high expression of VPS72 in different types of the aforementioned cancers (TCGA database), and high expression of VPS72 in hepatocarcinoma was associated with poor survival.
  • VPS72 expression measured by mean fluorescence intensity (MFI) was comparable in CD4+ and CD8+ T cells from spleen and tumors of those same mice (Fig. 25 D). Consistent with our preliminary data in mice, analysis of published single-cell data revealed a dramatic increase in VPS72 expression in tumor infiltrating Tregs from human melanoma and liver cancers relative to Tregs from human peripheral blood mononuclear cells (PBMC) (Fig. 26). Therefore, our preliminary discoveries imply that TME-specific factors induce both VPS72 and FoxP3 upregulation and may control Treg stability and adaptation.
  • MFI mean fluorescence intensity
  • VPS72-mediated H2A.Z chromatin remodeling is required for Treg stability and immune suppressive function.
  • a T cell-specific VPS72 knockout (KO) mouse model (VPS72-TKO) was generated (see Fig. 11 A and Fig. 27 A and B).
  • Cell-specific VPS72 deletion did not affect thymic conventional T cell and Treg cell development, but it did result in dramatically reduced peripheral Tregs (Fig. 28 A-C).
  • Fig. 29 T cell-specific VPS72 knockout mice model
  • VPS72-specific VPS72 KO mice were generated. Loss of VPS72 did not affect thymic Treg development but resulted in significantly reduced peripheral Treg cell frequency and FoxP3 expression (Fig. 30 A-B), with Fig. 30C showing a representative image of Teff cells with their respective absolute numbers.
  • the VPS72-TregKO mice spontaneously developed a severe and lethal multi-organ autoimmune disorder starting at 3-4 weeks of age and died very quickly (Fig. 13).
  • Fig. 31 A- C illustrates data showing that VPS72 is required for TGF-0-induced Treg (iTreg differentiation and FoxP3 expression).
  • VPS72 and H2A.Z regulate peripheral Treg stability and function in tumor immunity.
  • VPS72-H2A.Z has a role in Treg and Treg immunosuppressive function in the TME, and anti-tumor immune therapies that target VPS72 can be developed.
  • Novel mouse models with spatial/temporal-specific VPS72 and H2A.Z KO can be generated to dissect VPS72/H2A.Z function in Tregs; and multi-omics analysis can be employed, including scRNA-seq/scATAC-seq/CUT&RUN-seq and imaging mass cytometry (IMC).
  • mice can be matched for biological variables including sex, age, and weight in all experiments. Both male and female mice can be used. All findings can be repeated to confimi reproducibility and rigor. Transparency can be maintained in reporting methodological details and experimental results so that other investigators may reproduce and extend our findings. Rigorous statistical analyses can be applied to each Aim. Whenever possible, altered genotypes can be confirmed by measuring protein and mRNA expression. Where appropriate, examples of primary (typically FACS) data can be shown with the gating scheme and quantified across mice/experiments. For quantification, statistical analysis can be performed with GraphPad Prism 9, and analyses can be stated in the figure legends and methods. Comparisons can be based on at least 3 biological replicates. For animal studies, a sample size of 8-10 mice per group has >85% power to detect a significant difference of P ⁇ 0.05 with an effect size of 1.7.
  • VPS72 and H2A.Z are upregulated in mouse tumor Tregs. Given that VPS72 and H2A.Z are upregulated in tumor tissue, Treg cells expressing VPS72 and H2A.Z were analyzed. It was found that the VPS 72 expression in tumor Tregs was significantly higher than that in Tregs from spleens of the same tumor-bearing mice (Fig. 25 A). Interestingly and unexpectedly, H2A.Z1 and H2A.Z2 mRNA expression was also upregulated (Fig. 25 B). Furthermore, VPS72 protein levels by FACS analysis showed higher expression in tumor Tregs than in Tregs from spleen of tumor-bearing or wild ty pe skin (Fig. 25 C). However, VPS72 expression in CD4+ and CD8+ T cells was unaltered (Fig. 25 D). These data suggest that TME-specific factors may simultaneously upregulate FoxP3, VPS72, and H2A.Z expression in tumor Tregs.
  • Treg-specific VPS72 KO mice develop a lethal, multi-organ autoimmune disease.
  • Treg-specific VPS72 KO (VPS72-TregKO) mice were generated by breeding VPS721oxp mice with FoxP3-Cre-YFP knock-in mice (Jax: 016959) (Fig. 11 A).
  • VPS72 protein deletion in splenic CD4+CD25+ cells (Fig. 27 B) from VPS72-TregKO mice was confirmed by Western blot. Consistently, VPS72-TregKO mice developed normally in the first two postnatal weeks. Starting at 3-4 weeks of age, VPS72-TregKO mice had a runted size (Fig.
  • VPS72 is required for TGF-P-induced Treg (iTreg) differentiation and FoxP3 expression.
  • VPS72 is required for generating iTreg in vitro.
  • CD4 naive cells were sorted from VPS72-TregKO mice to generate iTreg in vitro and found significantly lower Treg differentiation (Fig. 31 A-B) with dramatically decreased expression of FoxP3 (Fig 31 C).
  • VPS72 is also required for in vitro iTreg differentiation and FoxP3 expression.
  • VPS72 suppression in Treg cells enhances the anti-tumor response against pre- established B 16 melanoma.
  • VPS72-TregKO mice die at an early age, an inducible deletion Cre-ERT2 mouse model was used. FoxP3eGFP-Cre-ERT2 mice w ere crossed to VPS72-floxed mice to allows for inducible deletion of VPS72 in Tregs (VPS72-TregiKO) following TAM treatment.
  • WT and age and sex-matched inducible VPS72-KO mice were subcutaneously injected with B16 melanoma cells (0.3M/mice) and monitored for tumor progression.
  • VPS72 and H2A.Z results in reduced peripheral Treg frequency and FoxP3 expression in Tregs with elevated autoimmunity, indicating an essential role for VPS72-H2A.Z-mediated chromatin remodeling in Treg maintenance and immunosuppressive function.
  • the present disclosure serves delineate the cellular mechanisms underlying the effect of VPS72-H2A.Z deficiency in Treg suppressive functions in vitro and in mice and then define the role of H2A.Z1 and H2A.Z2 in maintaining Treg stability and function. Finally, the mechanisms by which VPS72 and H2A.Z regulate FoxP3 expression can be identified.
  • Treg cells attain their immunosuppressive ability through the expression of inhibitory cell surface receptors (e.g., CTLA-4, PD-1, and G1TR) and the generation of regulatory cytokines such as IL-10. Therefore, to determine how VPS72 maintains Treg function, we will analyze the surface markers on Tregs related to Treg immunosuppressive function, such as CD25, ICOS, CTLA-4, Helios, PD-1, CD44, and Nrpl, as well as IL-10 production in Tregs from the spleen and lymph nodes in VPS72-TregiKO and WT mice.
  • inhibitory cell surface receptors e.g., CTLA-4, PD-1, and G1TR
  • regulatory cytokines such as IL-10. Therefore, to determine how VPS72 maintains Treg function, we will analyze the surface markers on Tregs related to Treg immunosuppressive function, such as CD25, ICOS, CTLA-4, Helios, PD-1, CD44, and Nrpl,
  • CD4+CD25+YFP+ Tregs sorted from VPS72-TregKO and WT mice will be co-cultured with sorted naive CD4+CD25-CD441oCD62Lhi T cells labelled with carboxyfluorescein succinimidyl ester (CFSE) in different ratios in 96-well U-bottom plates for 3 days.
  • Immunosuppressive Treg function will be analyzed by the proliferation of activated CD4+ T cells as determined by CFSE dilution.
  • apoptosis and proliferation in Tregs from spleen and lymph nodes can be assessed by annexin V and Ki67 staining, respectively.
  • FoxP3 is a lineage-specific TF required for Treg stability’, particularly in inflammatory conditions. Tregs can lose FoxP3 expression and convert into pathogenic effector T cells under certain inflammatory conditions, suggesting that inflammatory environmental factors may destabilize FoxP3 expression.
  • WT and VPS72-deficient Tregs can be co-cultured with different cytokines known to depolarize Tregs (IL-6, IFN-y, IL-4 and TNF-a.
  • VPS72-deficient Tregs produce Thl, Th2, or Thl7 cytokines can be analyzed and their FoxP3 expression can be examined by intracellular staining. We expect that loss of VPS72 will lead to Treg depolarization and production of Thl, Th2, or Th 17 cytokines, which is further enhanced by co-cultivation with inflammatory’ cytokines.
  • H2A.Z-TdKO mice T cell-specific CD4Cre.H2A.Zl/H2A.Z2 double KO mice and observed comparable levels of T cells and Tregs in the thymus and significantly reduced levels of peripheral Treg and T cells, suggesting that H2A.Z is not required for thymic T cell and Treg development but is required for peripheral Treg and T cell homeostasis (Fig. 32), which phenotypically resembles VPS72-TKO mice (Fig. 28).
  • Treg-specific H2A.Zl-TregKO, H2A.Z2-TregKO, and H2A.Z1.Z2- TregdKO mice can be generated and then dissected if H2A.Z1, H2A.Z2, or both are required for Treg cell development and function, evaluating (1) the frequency and number of Treg cells in the thymus and peripheral organs; (2) Treg immunosuppressive function and reprogramming; and (3) Treg apoptosis and proliferation. This will uncover independent and/or redundant roles for both H2A.Z isoforms in VPS72-mediated Treg stability and function. Because VPS72 acts as a chaperone for H2AZ, similar Treg phenotypes are likely to exist in VPS72.TregKO mice.
  • RNA-Seq can be performed to identify' the genes regulated by the VPS72-mediated H2A.Z chromatin remodeling in Tregs from VPS72-TregKO, H2A.Z-TregKO, and WT mice.
  • VPS72 is an H2A.Z chaperone that does not directly bind to DNA.
  • H2A.Z CUT&RUN-seq can be used to analyze DNA-protein interactions and has many advantages over ChlP-seq, including generating fewer false-positive binding sites, requiring fewer input cells, and having higher signal-to- noise ratios and higher resolution.
  • H2A.Z CUT&RUN can be performed using 50,000 splenic CD3+CD4+CD25+ Tregs from C57BL/6 mice and identified over 1400 genes and TF binding motifs, including genes related to apoptosis, MAPK, and Tgf- pathways (Fig. 33). Unexpectedly, H2A.Z binding peaks were mapped to the FoxP3 and Tgf-pi regions.
  • binding motifs for two FoxP3 TFs, RUNX1 and NF AT were significantly enriched within the FoxP3 promoter and CNS2 regions, while the binding motif for MYB, a Tgf-01 TF, was significantly enriched in the Tgf-pi promoter region.
  • VPS72-H2A.Z mediated chromatin remodeling may regulate FoxP3 by directly interacting with RUNX1 and NF AT or indirectly controlling FoxP3 expression by targeting TGF-pi through MYB.
  • the peaks can be annotated with the priority order (promoter > exon > intron > intergenic) using ChlPseeker (version 1.26.2) when a single peak spans more than two genomic features.
  • RPKM values can be calculated using a 5 kb sliding window ⁇ to compare genome-wide ChlP-seq enrichment among samples or promoter regions (TSS ⁇ 1 kb) can be used to compare promoter ChlP-seq enrichment among samples.
  • TSS ⁇ 1 kb promoter regions
  • Hierarchical clustering will be performed in R by stats package and hclust function ith RPKM values via Pearson's correlations.
  • We will perform peak calling using model-based analysis of ChlP-seq (MACS) and define the decreasing and increasing number of H2A.Z peaks.
  • the accumulation of H2A.Z peaks in promoter regions may be associated with genes required for Treg function.
  • VPS72 Loss or decrease of VPS72 can likely decrease Treg expression of surface markers related to immunosuppressive Treg function, such as ICOS, CTLA-4, PD-1, and CD44 and lead to reduced IL- 10 production and increased Treg immune suppressive function in vitro and in vivo.
  • surface markers related to immunosuppressive Treg function such as ICOS, CTLA-4, PD-1, and CD44
  • in vitro overexpression of FoxP3 in VPS72-deficient Tregs may partially rescue immunosuppressive function.
  • VPS72 regulates chromatin structure through H2A.Z exchange specific H2A.Z1 or H2A.Z2 deletion in Tregs can impair Treg immunosuppressive function.
  • VPS72/H2AZ suppression it is possible for targeted VPS72/H2AZ suppression to result in an elevated antitumor immune response and that VPS72/H2AZ-TregiKO mice can have better tumor rejection than WT mice.
  • FoxP3 MFI and levels of suppressive markers PD-1, CTLA-4, GITR, and CD 103 can be measured by flow cytometiy in CD4+CD25+FoxP3+ Tregs.
  • MDSCs myeloid-derived suppressive cells
  • the skin graft can be treated by topical application of 4-hydroxy -tamoxifen (TMX).
  • TMX 4-hydroxy -tamoxifen
  • Melanoma development in the graft can be monitored weekly for the first 5 weeks and daily thereafter.
  • recipient mice can be grouped and euthanized during early (day 30), middle (day 45), and late phase (day 60 or after) tumor grow th.
  • VPS72 depletion which decreases Treg suppressive function in VPS72-TregiKO mice
  • anti-PD-1 treatment which enhances Teff cell function
  • 8- to 10-week-old VPS72- TregiKO and WT mice can be injected with B16 melanoma cancer cells. Tumor size can be measured, and anti-PD-1 treatment can begin when tumors reach 100 mm3 (usually 7-10 days after tumor cell injection).
  • VPS72-mediated H2A.Z remodeling likely regulates Treg stability and function through targeting FoxP3 expression, and it follows that TME-specific factors induce VPS72 expression, which controls Treg stability and adaptation through targeting FoxP3.
  • TME-specific factors can be advantageous for reprogramming the immune response during tumor immune escape.
  • Intratumoral Tregs have the capability to adapt to the harsh elements promoted by the fast-growing malignant tumor cells within the TME.
  • Tregs cultured in low glucose medium had significantly enhanced VPS72 expression (Fig. 35). indicating that tumor-infiltrating Tregs can maintain their immunosuppressive function through VPS72 upregulation in response to low glucose.
  • TME factors may affect Treg VPS72 expression.
  • the molecular mechanisms underlying how these TME factors potentiate Treg immunosuppressive function by inducing VPS72 can be further elucidated.
  • TGF-P produced by tumor cells induces VPS72 expression in Tregs.
  • Tumor cells and infdtrating immune cells produce a variety of cytokines (e.g., TGF- pi) that promote immune evasion by inducing Treg activity and maintaining Treg stability.
  • TGF- suppression alters tumor-conditioned media-induced USP21 and USP22 expression, which can be analyzed using a TGF-P inhibitor (TGFpi).
  • TGFpi TGF-P inhibitor
  • Two further experiments can be performed: (1) Tregs cultured in tumor-conditioned medium with or without TGFpi LY3200882 at 25 pg/ml; and (2) Tregs cultured in regular medium with or without 20ng/mL TGF-P or with TGFpi for 8 hours.
  • VPS72 expression can be determined by qPCR and FACS analysis. This can indicate how the TME cytokine TGF- P induces VPS72 expression in Tregs. TGF- P induces gene transcription through either SMAD activation or non-SMAD pathways. Genomic analysis identifies multiple conserved SMAD4 binding sites in VPS72 promoter regions (Fig. 39). Together with the fact that tumor TME-induced VPS72 expression and TGF- p induced Tregs (iTreg) are defective in VPS72KO mice (Figs. 25 & 31), it follows that TGF- P induces VPS72 gene expression through activation of SMAD4 TF.
  • Hypoxia may be used to induce VPS72 expression in Tregs. Hypoxia often arises in the TME through the uncontrolled oncogene-driven proliferation of cancer cells. Tregs can be cultured under hypoxic (1% oxygen) or normoxic conditions for 12 hours. VPS72 expression can be measured with qPCR and western blotting. Given that multiple hypoxia-related TFs, including HIFla and SP1, are located in the VPS72 promoter region, it follows that hypoxia in the TME is involved in VPS72 induction to maintain FoxP3 stability. The molecular mechanism can be dissected and ChIP analysis can be performed to assess binding of HIFla or SP1 at the VPS72 promoter. Once direct binding of HIFla or SP1 to the VPS72 promoter is detected, it can show' an extent to which loss of HIFla in Tregs abolishes hypoxia-induced VPS72 expression.
  • a FOXO3 binding site is located in the VPS72 promoter region (Fig. 38).
  • the molecular mechanisms can be dissected by performing a ChIP analy sis to detect FOXO3 binding at the VPS72 promoter. It follows that treating Tregs with AMPK-specific inhibitors will abolish, at least partially, the increased VPS72 expression seen in Tregs in low glucose medium, (ii) Low amino acid levels involved in VPS72 induction in Tregs.
  • Tregs can be cultured in low amino acid medium and whether the expression of VPS72 is altered can be analyzed. It follows that VPS72 expression is induced in low amino acid conditions.
  • the mTOR mimmalian target of rapamycin pathway is often inhibited in cells cultivated in medium lacking sufficient amino acids. Rapamycin treatment is a potential treatment option for recapitulating the effect of low amino acids to induce Treg VPS72 expression.
  • TME-specific factors cytokines, hypoxia, and metabolic changes
  • TME-specific factors can be more particularly identified that reshape Tregs through selective induction of VPS72-mediated H2A.Z remodeling.
  • cytokines IL-1 P, IL-6 & TNF-a, etc.
  • IL-1 P, IL-6 & TNF-a, etc. inflammatory cytokines
  • a panel of inflammatory and antiinflammatory cytokines using in vitro culture to identify all cytokines that induce Treg VPS72 expression can be used.
  • the underlying molecular pathways can be dissected, (ii) Hypoxia can also induce gene expression through HIF- independent mechanisms, such as the AMPK-FOXO3 pathway.
  • FOXO3 can be activated by both hypoxia and low glucose, which increase VPS72 expression, (iii)
  • the Smad, HIFla, and AMPK-FOXO3 pathways can play a role in VPS72 induction in Tregs under TGF-P, hypoxia, and metabolic stress.
  • RNA-Seq and scRNA-seq analysis discussed herein can be used to identify additional pathways involved in VPS- 72/H2A.Z-induced Treg stability and function in the TME, revealing aspects of the molecular mechanisms, (v) Moreover, human Tregs can be used to validate results from TME-induced VPS72 expression in mouse Tregs.
  • CD4 T cells from PBMCs can be isolated and polarized into iTregs.
  • the effects of TME-specific factors, including cytokines (TGF-0, hypoxia, and metabolites (low glucose and amino acids) on human Treg VPS72 expression can be determined by qPCR and western blotting.
  • Tregs CD4+CD25+FR4+ or CD4+CD25+CD1271ow

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Abstract

The present disclosure relates to compositions and methods of altering an immune cell response in a subject in need thereof, said method comprising administering an inhibitory agent that inhibits VPS72 activity and/or expression in an immune cell of the subject.

Description

METHODS AND COMPOSITIONS FOR IMPROVING IMMUNOTHERAPY
TECHNICAL FIELD
[0001] The present disclosure relates to compositions and methods to treat immune related diseases such as autoimmune diseases and inflammation related diseases and conditions.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. Said XML copy, created on 9-Oct-2024, is named 1059-3049_Sequence-Listing and is 11 kilobytes in size.
BACKGROUND
[0003] The vacuolar protein sorting-associated protein 72 homolog (VPS72, also known as YL1) was initially identified in 1995 as a nuclear protein with DNA-binding ability in NIH3T3 cells. VPS72 localizes in nuclear specks and in the nucleoplasm. Available gene expression data (GeneCard) show that VPS72 is ubiquitously expressed in most organs, including the immune system. However, VPS72 is highly expressed in hematopoietic stem cells (HSC), suggesting that VPS72 may play a role in maintaining stem cell activity. Although few studies of VPS72 have been done in the past 27 years (<15 papers in PubMed), recent studies indicate that VPS72 functions as a histone chaperone for H2A to H2A.Z exchange in chromatin remodeling and belongs to two multi-subunit chromatin-remodeling complexes: the Snf2-related CBP-activator protein chromatin remodeling (SRCAP) complex (FIG.l); and the TRRAP/TIP60 complex (FIG. 1). VPS72-mediated H2A.Z exchange is required for nuclear reassembly after mitosis in HeLa cells, and VPS72-H2A.Z interaction with the help of Znhitl determine Lgr5+ stem cell fate (FIG. 1). Interestingly, VPS72 is also reported to regulate the acetylation of non-histone proteins, including autophagy gene ATG8a in Drosophila (FIG. 1).
[0004] Chromatin remodeling is an important layer of epigenetic regulation and plays a key role in immune cell development, homeostasis, and function. Nucleosomes are the basic unit of chromatin. The histone octamer packages DNA into nucleosomes, maintains the nucleosome morphology, and serves as a regulatory7 layer for gene expression. This octamer consists of histone proteins such as H2A, H2B, H3, and H4. Among histones, the most studied is H2A, which comprises an H2A variant known as histone H2A.Z. H2A.Z has two different isoforms, H2A.Z1 and H2A.Z2, which differ by only three amino acids and are encoded by tw o separate genes, H2AFZ and H2AFV, respectively. Interestingly, a recent study showed that the deletion of both isoforms is essential for decreased expression of Pten-mediated transcriptional control of HSC quiescence. Moreover, H2A.Z has been linked to diverse biological processes such as memory, epithelial-to-mesenchymal transition, microglial development, and neuronal survival through promoting nuclear-encoded mitochondrial gene expression and organelle function. Also, overexpression of H2A.Z is associated with a greater proliferative capacity in multiple cancers such as metastatic melanoma, colorectal, liver, and lung. Exchanging H2A for variant histone H2A.Z through the VPS72/SRCAP/TIP60 complex (Fig. 1) has been shown to modulate local chromatin structure to activate or repress target genes that regulate cellular processes, such as cell cycle progression, autophagy regulation, metabolic processes, and mitochondrial function especially in HSC and embryonic stem cell development. However, the functions of VPS72-mediated H2A.Z exchange in immune cells remain unknown.
SUMMARY
[0005] VPS72 is a chaperone protein for H2A.Z and deposits the H2A.Z in the nucleosome exchange. In some aspects, the present disclosure provides a method of altering an immune response, particularly in subjects having a malfunctioning immune activated state, for example, in autoimmune diseases and diseases where inflammation is a factor or cause. In these aberrant immune activated states, the subj ect is treated by administering a therapeutically effective amount of VPS72 inhibitor which decreases the expression of VPS72 in the immune cells that contribute to the hyper immune response or decreases the activity of VPS72 by acting as a blocking peptide with its cognate binding molecule H2A.Z. [0006] In other aspects, the present disclosure provides a VPS72 peptide inhibitor that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2.
[0007] In still other aspects, the present disclosure provides a VPS72 inhibitor that is a nucleic acid, for example, a DNAi or an RNAi molecule that inhibits the transcription or translation of VPS72 in the immune cell that contributes to the hyper immune state which causes the autoimmune and/or inflammatory disease or condition.
[0008] In yet other aspects, the present disclosure provides a combination of a VPS72 inhibitor and at least one of an anti-inflammatory, and an immune suppressive agent for the treatment of an autoimmune disease and/or diseases w here inflammation is at least a symptom or cause of a disease or condition. BRIEF DESCRIPTION OF FIGURES
[0009] The following figures are provided by way of example and are not intended to limit the scope of the invention.
[0010] FIG. 1 depicts a schematic representation of the historic studies involving the role and function of VPS72 in chronological order.
[0011] FIG. 2 CD4Cre mediated VPS72 deletion. (Panels A-B) Expression pattern of VPS72 in iNKT cells VPS72 expression from ImmGen RNA-seq(C57BL/6, 6wks old) (Panel A) and qPCR (C57BL/6, 8-12wks old) (Panel B) in sorted DP, CD4, CD8, and iNKT cells. (Panel C) Cytometry histogram shows VPS72 expression in DP, DN, CD4 T, CD8 T and iNKT cells. (Panel D) Generation of VPS72 floxed allele and conditionally deleted allele. (Panel D) Schematic diagram showing technical strategy to generate conditional knockout mice. (Panel E) q-RCR and (Panel F) western blot show the deletion of VPS72 in the thymocytes from VPS72 cKO mice.
[0012] FIG. 3 VPS72 is required for iNKT cell development in a cell-intrinsic manner. (Panels A-B) Flow cytometry plots shows thymic CD8, CD4, and iNKT cells. (Panel C) Frequency (up) and absolute number (down) of indicated subsets of T cells from thymus. (Panels D-E) Flow cytometry and bar graphs show the frequency and absolute number of iNKT cells at stage 0 (STO), STI, ST2, and ST3 in WT and VPS72 cKO. (Panels F-G) the frequency and absolute number of iNKT cells from peripheral organs. (Panel H) Flow cytometry plots show thymic iNKT cells and developmental stages of iNKT cells derived from BM. (Panel I) the percentages and absolute numbers of iNKT cells **P<0.01, ***p<0.001, and ****P<0.0001.
[0013] FIG. 4 VPS72 is required for iNKT cell homeostasis. Histogram and bar graph show Ki-67 expression in total iNKT cells and substages of iNKT. Flow cytometry and bar graph show iNKT cell early and later apoptosis in WT and VPS72cKO. (Panels C-D) Flow cytometry of iNKT 1/2/17 in total iNKT cells (Panel C) and NK1.1- subset (Panel D) from VPS72cKO and WT mice. (Panel E) bar graph depicting total iNKTl/2/17 frequency and absolute. (Panel F) Heatmaps show the iNKTl/2/17 signature expression in thymic iNKT cells from WT and VPS72cKO mice. *P<0.05, ***P<0.001, and ****P<0.0001.
[0014] FIG. 5 VPS72 is required for iNKT function in tamoxifen induced VPS72uKO mice. (Panel A) VPS72 expression. (Panel B) CD69 expression upon P/I stimulation. (Panel C) iNKT cells early apoptosis and later apoptosis post P/I stimulation. (Panel D) IFN-y and IL-4 production in iNKT cells. (Panel E) cell apoptosis and cytokine production in conventional CD4 and CD8 T cells. *P<0.05 (for many of the heatmap-style figures disclosed herein, the red or “hot” zone is indicated with a star).
[0015] FIG. 6 VPS72 deletion led to accumulation of cellular organelles in iNKT cells (Panel A) Heatmap shows gene signatures related to autophagy assembly in iNKT cells. (Panel B) Flow cytometry showing Mitotracker Deep Red and Mitotracker Orange dye in total iNKT cells, and subsets of iNKT cells.
[0016] FIG. 7 VPS72 deletion alters the gene expression program in thymic DP cells undergoing iNKT cell selection. (Panel A) IL-2 production from DN32.D3 iNKT cells with DP thymocytes from WT and VPS72cKO mice in the presence of a-GalCer. (Panel B) qPCR shows Val4-Jal8 rearrangement in thymic DP cells. (Panels C-D) Heatmap of DP RNA-Seq shows key TCR signaling pathways related to iNKT selection (Panel C) and key mitochondrial function pathway in DP thymocytes (Panel D).
[0017] FIG. 8 Defective iNKT cells in H2A.ZdKO mice. (Panel A) Co-IP showing VPS72 and H2A.Z interacting binding in iNKT cell line. (Panels B-C) Western blots; (Panel D) thymic T cells and iNKT cell development; (Panel E) Mitotracker Deep Red and Mitotracker Orange dye in iNKT cells. (Panel F) Pie chart of different genomic regions obtained H2A.Z CUT&RUN-seq analysis of thymic iNKT cells. (Panel G) Gene Ontology analysis of the gene identified; (Panel H) Enrich KEGG pathw ays. (Panel I) HOMER de novo motif analysis of H2A.Z binding peaks related to TCR and autophagy pathways in iNKT cells.
[0018] FIG. 9 VPS72 is required for MAIT cell development. (Panel A) Flow cytometry plots shows MAIT cells in thymus and peripheral organs. (Panel B) Frequency and absolute number of MAIT cells in indicated organs. Each dot on bar graph represents an individual mouse. *P<0.05, **P<0.0I. (Panel C) Flow cytometry plots shows developmental stages of MAIT cells in thymus. (Panel D) Frequencies and absolute numbers of indicated stages of MAIT cells. Each dot on bar graph represents an individual mouse. * P<0.05, p**<0 01, and ***P<0.001. (Panel E) Flow cytometry plots shows MAIT1 (Tbet+RORyt-), MAIT17 (T-bet-RORyt+), and MAIT0 (T-bet-RORyt-) cells in thymus and peripheral organs. (Panel B) Frequencies and numbers of MAIT1, MAIT17 and MAIT0 cells in indicated organs. *P<0.05, **P<0.01.
[0019] FIG. 10 Deletion of VPS72 alters MAIT cell CD4 and CD8 expression.
(Panel A) Flow cytometry plots show CD4 and CD8 expressing MAIT cells in thymus and peripheral organs. (Panel B) Frequencies and numbers of CD4+MAIT, CD8+MAIT, CD4+CD8+ DP MAIT, and CD4-CD8-DN MAIT cells in indicated organs. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
[0020] FIG. 11 Generation ofVPS72 floxed allele and conditionally deleted allele. (Panel A) Schematic diagram showing technical strategy to generate conditional knockout mice. (Panel B) Western blot shows deletion of VPS72 in CD4+, CD8+ and CD4+CD25+ cells from VPS72 cKO spleen.
[0021] FIG. 12 VPS72 is required for Treg homeostasis in periphery. (Panel A) Flow cytometry plots shows thymic CD8, CD4, DP and DN cells. (Panel B) Frequency (left) and absolute number (right) of indicated subsets of T cells from thymus. (Panel C) Flow cytometry and (Panel D) bar graphs showing the frequency (left) and absolute number (right) of Treg cells. (E-F) Flow cytometry (Panel E) and bar graphs show the frequency and absolute number of CD4 and CD8 T cells (Panel F) in spleen and LN. (G-H) Flow cytometry' (Panel G) and bar graphs showing the frequency and absolute number (Panel H) of Tregs in spleen and lymph node (LN). I) Flow cytometry (Panel J) and bar graphs show the frequency of naive CD4 and CD8 T cells (left) and effector CD4 and CD8 T cells (right) in. spleen. (Panel K) mixed bone marrow chimera model. Flow cytometry plots show splenic T cells (Panel L) and its frequency (Panel M). (Panel N) Flow cytometry plots show splenic Tregs derived from bone marrow, (Panel O) the percentages (left) and absolute numbers (down) of Treg cells in spleen derived from bone marrow. Bar graph represents mean± SEM (n=7-8).
*P<0.05. ***P<0.00L ****P<0.000I. WT: CD4cre-VPS72fl/fl, KO: CD4cre+VPS72fl/fl.
[0022] FIG. 13 Treg specific loss of VPS72 results in lethal, multi organ autoimmune disease. (Panel A) Gross anatomical analysis showing runted growth in FOXP3cre VPS72 KO compared to WT and heterozygote (Het) mice. (Panel B) Analysis of body weight of WT, Het and KO mice starting from 3 weeks (n=6-I0 each). (Panel C) Early death of VPS72 KO mice, Kaplan-Meier plots (n=12 each). (Panel D) Comparison of spleen, lymph node and thymus of WT, Het and KO mice (left) and cellularity of thymus, spleen and lymph nodes (n=5-8 mice per group). (Panel E) H and E staining of sections from liver, lung and heart. (Panel F) Flow cytometric analysis and (Panel G) frequency (left) and absolute number (right) of T cell subsets in thymus. (Panel H) Flow cytometric analysis and (Panel I) frequency (left) and absolute number (right) of T cell subsets in spleen and lymph nodes. (J- M) Treg deficient VPS72 mice developed normally till 2 weeks of age compared to control mice. (Panel J) Gross anatomical analysis of normal thymus, spleen and lymph node. (Panel K) Total cell numbers from thymus, spleen and lymph. Frequencies of T cell subsets in thymus (Panel L) and in spleen and LN (Panel M) in control and KO mice (for panels G, I, and K-M, the control is listed before FOXP3 VPS72 cKO). Bar graph represents mean SEM (n=5-8/group). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Control: FOXP3YFPcre-VPS72fl/fl, KO: FOXP3YFPcre+VPS72fl/fl.
[0023] FIG. 14 VPS72 deletion in FOXP3+ Tregs causes activation of conventional T cells. Compared with control WT controls, lack of VPS72 in Tregs show expansion of (Panel A) CD4+CD2LloCD44hi and (Panel B) CD8+CD62LloCD44hi effector cells in spleen and lymph nodes, with corresponding representative plots (left) and bar diagram showing their frequency (up) and absolute number (dowTi). (Panel C) CD4+Ki67+ and CD8+Ki67+ proliferating cells in spleen and LN by flow cytometry' (left) and bar graphs (right). Bar graph represents mean SEM (n=5-8/group). (D-E) Foxp3-cre vps72-deficient Tregs were unable to control the expansion of Thl, Th2 and Thl7 effector cells. Flow cytometric analysis and bar graph of IL-4, IFNy and IL-17 expressions within CD4+ T cells (D-E) and IFNy within CD8+ T cells (Panel F) from spleen of WT and Foxp3 vps72 KO mice following stimulation of total splenocytes with PMA and lonomycin (P/I) for 4 hours. (G-H) cell population with flow representing plot (left) and bar diagram showing frequency (Up) and absolute number (down) in each panel. (Panel I) bar diagram showing mean fluorescent intensity (MFI) of FOXP3 within CD4+FOXP3+ population in spleen and lymph node from control and Foxp3 vps72 KO mice (control is listed before FOXP3 VPS72 cKO in this figure). Bar graph represents mean SEM (n=5-8/group). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Control: FOXP3YFPcre-VPS72fl/fl, KO: FOXP3YFPcre+VPS72fl/fl.
[0024] FIG. 15 Deletion of VPS72 has protective role in melanoma tumors. (Panel A) Flowchart for analysis of Tregs cell in melanoma cancer. (Panel B) mice were sacrificed at 24 day and tumor size (Panel B) and weight (Panel C) measured. WT: Foxp3eCrFP'Cre'FRT2' VPS72fl/fl, KO: Foxp3eGFp-Cre-ERT2+VPS72fl/fl.
[0025] FIG. 16 VPS72 is required for HSC and its progenitors. (Panel A) Flowchart show ing the experiment design. Deletion of Vps72 shown by Western blot in bone marrow (Panel B) and by qPCR in LSK+ and Lin-c-Kit+ cells (Panel C). (Panel D) Bar graph showing the number of bone marrow cells in WT and KO. (E-G) Flow cytometric analysis (Panel E) and bar graph and absolute number (F-G) of hematopoietic stem cells and its progenitors. (Panel H) Flow cytometric analysis and bar graph showing frequency and absolute number myeloid progenitors. (Panel I) Summary' of deletion of Vps72 decreases lymphoid and increases myeloid progenitors. Bar graph represents mean SEM (n=5-7/group). *P<0.05. **P<0.0I, ***P<0.00I, and ****P<0.0001. LT-HSC: long term hematopoietic stem cells, ST-HSC: short term hematopoietic stem cells, MPP: multipotent progenitors, LMPP: lymphoid-primed multi-potent progenitor. CLP: common lymphoid progenitors, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythroid progenitor, CMP: common myeloid progenitor. WT: Mxlcre'VPS72fl/fl, KO: Mxlcre+VPS72fl/fl.
[0026] FIG. 17 VPS72 is required for the maintenance of body weight in adult mice. (Panel A) Scatter dot plot showing the body weight in gram (Panel B) Representation of mouse image from Csflricre+VPS72fl/fl (KO) and Csflricre-VPS72fl/fl (WT) mice. Each dot represents individual events as indicated and each bar showed mean ± SEM from the measurement n> 3 independent samples. * Significant differences (p < 0.05) were compared between KO and WT mice.
[0027] FIG. 18 VPS 72 is required for TRMs in organ specific manner from embryonic (E14.5) to adult mice after birth. Fate-mapping analysis for Csflrlcre+VPS72fl/fl expressing mice. (Panel A) Single cell suspension of epidermis, caudal skin/ dermis, brain, eye, lung, kidney, heart and liver starting E14.5 to 3 weeks of age were prepared from Csflriore+VPS72fl/fl (KO) and Csflricre-VPS72fl/fl (WT) mice. Flow cytometric analysis of TRMs after exclusion of dead cells (FSC-A vs SSC-A) with doublets (FSC-A vs FSC-H) followed by gating CD45+ live cells in epidermis (CD45hlF4/80lu from El 8.5 to P0 and CD45hlMHCIIhl in adult), caudal skin/dermis (CDl lbintF4/80hl from E14.5 to P0 and CD1 lbhiF4/80hi in adult), brain (CD1 lbintF4/80hi from E14.5 to P0, CD1 lbmtCD39hi adult), eye (CD1 lbintF4/80hi from E14.5 to P0 and CD1 lbintCD39hi in adult), lung (CD1 lbintF4/80hi from E14.5 to P0 and CD1 lchlSiglecFhl in adult), kidney (CD1 IbintF4/80hl from E14.5 to adult), heart (CD1 lbintF4/80hi E18.5 to adult) and liver (CD1 lbintF4/80hl from E14.5 and CD1 lbmt TIM4hl E17.5 to adult) as indicated, and (Panel B) the frequencies of all TRMs specific to epidermis, caudal skin/dermis, brain, eye, lung, kidney, heart and liver per time point as in 2a in KO and WT littermate mice. Each dot represents individual embryo or newborn, or adult samples as indicated, and bar represents mean ± SEM from the measurement n> 3 independent samples. *Significant differences (p < 0.05) were compared between KO and WT mice.
[0028] FIG. 19 VPS72 is required for CD45 in organ specific manner from embryonic (E14.5) to adult mice after birth. Fate-mapping analysis for Csflrlcie+VPS72flfl expressing mice. (Panel A) Single cell suspension of epidermis, caudal skin/dermis, brain, eye, lung, kidney, heart and liver starting E14.5 to 3 weeks of age were prepared from Csftrlcre+VPS72fl/fl (KO) and Csflrlcre-VPS72fl/fl (WT) mice. Flow cytometric analysis of CD45 after exclusion of dead cells (FSC-A vs SSC-A) with doublets (FSC-A vs FSC-H) followed by gating of CD45+ live cells in epidermis, caudal skin/dermis, brain, eye, lung, kidney, heart and liver in E14.5 to adult mice as indicated, and (Panel B) the frequencies of all CD45 specific to epidermis, caudal skin/dermis, brain, eye. lung, kidney, heart and liver per time point as in 3a in KO and WT littermate mice. Each dot represents individual embryo or newborn, or adult samples as indicated, and bar represents mean ± SEM from the measurement n> 3 independent samples. * Significant differences (p < 0.05) were compared between KO and WT mice.
[0029] FIG. 20 VPS72 required for post-natal maintenance of LCs and AMs. (Panels A-B) Single cell suspension of epidermis and lung were prepared from CDl lccre+VPS72fl/fl (KO) and CD1 lccre-VPS72fl/fl (WT) mice. Flow cytometric analysis of LCs and AMs after exclusion of dead cells (FSC-A vs SSC-A) with doublets (FSC-A vs FSC-H) followed by gating CD45+ live cells in epidermis (CD45hlMHCIIhl) and lung (CD1 lchlSiglecFhl) in adult as indicated, (Panel B) the frequencies of CD45 and corresponding epidermal LCs and Lung AMs per time point as indicated in 3a images and (Panel C) representative Western blot images of VPS72 and corresponding loading control GAPDH expression in KO and WT littermate mice. Each dot represents individual adult samples as indicated and bar represents mean ± SEM from the measurement n> 3 independent samples. *Significant differences (p < 0.05) were compared between KO and WT mice.
[0030] FIG. 21 (Panel A) depicts ribbon structure schematic drawings representing VPS72 and H2A.Z interaction crystal structures in human. (Panel B) VPS72 predicated crystal structure in mouse. (Panel C) Depicts the amino acid sequence of VPS72. and the VPS72/H2A.Z peptide inhibitor (SEQ ID NO: 2 and SEQ ID NO: 3) that blocks the interaction between VPS72 and H2A.Z. (Panel D) Bone marrow transplant strategy using an inhibitor of VPS72 to repress gene expression.
[0031] FIG. 22 VPS72-mediated H2A.Z exchange regulates peripheral Treg cell stability and function, and Treg adaptation to tumor microenvironment (TME).
[0032] FIG. 23 VPS72-mediated H2A.Z epigenetic axis is involved in the biological processes.
[0033] FIG. 24 FoxP3 is upregulated in tumor infiltrating Tregs. (Panel A) The frequency of CD4FoxP3+ Tregs in the spleen (tSp) and tumor (Tu) infiltrated T cells from B16 melanoma bearing mice and the spleens (Sp) from control tumor-free mice were analyzed by flow cytometry. (Panels B & C) The MFI of FoxP3 expression in Treg was analyzed. (Panel D) The surface expression of CD25, PD-1, CTLA-4 and GITR in Treg cells were analyzed. (N=7). Student t test was used for the statistical analysis. **p<0.01. ****p<0.0001. [0034] FIG. 25 VPS 72 is upregulated in tumor infiltrated Tregs in B16 melanoma.
(A) CD4+CD25+YFP+ Tregs in tumor (Tu) infiltrated T cells and spleen (tSp) from tumor bearing mice were sorted. The expression of VPS72 (Panel A) and H2AV-H2AZ (Panel B) were determined by real-time PCR. The VPS72 MFI in Tregs analyzed with represent images and average MFI (Panel C). VPS72 MFI in CD4+ and CD8+ T cells (Panel D). (N=2-7). *p<0.05, **p<0.01 and ****p<0.0001.
[0035] FIG. 26 Increase VPS72 expression in tumor infiltrated Tregs from human liver cancers and melanoma compared to that in Tregs from PBMCs based on sc-RNA-Seq data. *p<0.05, **p<0.01 and ****p<0.0001.
[0036] FIG. 27 Protein expression of VPS72 in splenic CD4+T cells in VPS72-TKO mice (Panel A) and CD4+CD25+ cells from VPS72-TregKO (Panel B) by western blots. [0037] FIG. 28 VPS72 is required for periphery Treg maintenance. (Panels A-B) Representative image (Panel A) and (Panel B) absolute number of T and Treg cells in thymus (Thy), spleen (Sp), and lymph node (LN) from WT and VPS72-TKO mice. (C) MFI of FoxP3 in Thy (top), Sp (middle) and LN (down), (N=8), Student t test, *p<0.05, **p<0.01, ***p<0.00I.
[0038] FIG. 29 Flow cytometry plots show thymic, splenic and lymph node (LN) Tregs derived from recipient BM (CD45.2). N=3-4.
[0039] FIG. 30 Reduced peripheral Tregs lead to T cells activation in VPS72-TregKO mice. CD4 T cells from WT and VPS72-TregKO were analyzed for CD4+YFP+ Tregs frequency and cell number (Panel A) and FoxP3 Treg frequency and MFI of FoxP3 in Tregs (Panel B). (Panel C) Representative image of Teff cells with their respective absolute numbers. (N=3-7). Student t test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
[0040] FIG. 31 VPS72 is required for iTreg differentiation from CD4+ naive T cells. CD4 Naive T cells were sorted from spleen WT and VPS72-TregKO mice and stimulated with anti-CD3 coated plate with anti-CD28, IL-2, anti-IL4, anti-IFNy and TGF-(3 for 5 days. Then cells were isolated and expression of FoxP3 was analysis by flow cy tometry'. (Panel A) Representative image and (Panel B) bar graphs show the frequency of Treg cells. (Panel C) Quantification of FoxP3 MFI of splenic iTreg cells. (N=3). Student t test. *p<0.05 and **p<0.01.
[0041] FIG. 32 H2AZ is not required for T cell and Treg development in thymus but required for peripheral Treg stability. (Panel A) H2AZ protein expression in total thymocytes and splenocytes by western blots. (Panel B) Representative image for T cells (left) and frequency of T cells (right) in Thy, Sp and LN from WT and H2AZ-TKO mice. (Panel C) Representative image for Tregs (left) and their frequencies (right) in thymus, spleen and LN from WT and H2AZ-TKO mice. (N=4-6). Student t test. *p<0.05 and ****p<0.0001.
[0042] FIG. 33 H2A.Z CUT&RUN-seq analysis of spleen Tregs cells. (Panel A) Pie chart: genomic distribution of H2A.Z binding. (Panel B) Gene Ontology (GO) enrichment analy sis. (Panel C) KEGG enriched pathways. (Panel D) HOMER de novo motif analysis of H2A.Z binding peaks related to Tregs development. (Panel E) Genomic track of peaks in the FoxP3 and Tgfbl regions.
[0043] FIG. 34 Deletion of VPS72 in Treg cells enhances the antitumor response against B16 melanoma. Tumor grow th (Panel A) and tumor w eight (Panel B) w as reduced in VPS72-TregiKO mice. Tumor infiltrated lymphocytes were analyzed in CD45+ cells by flow cytometry (Panel C). The levels of GZMB (Panel D) and IFN-y (Panel E) were analyzed by intracellular staining after PMA/Ionomycin stimulation. (Panels F-G) CD4+FoxP3+ Tregs in the infiltrated CD4 cells were analyzed. The Treg percentage (right) and MFI (left) of FoxP3+ Tregs are shown (Panel G). (N=5-7). Student t test was used for the statistical analysis. *p<0.05, **p<0.01 and ****p<0.000I.
[0044] FIG. 35 The effect of glucose-deprivation on VPS72 expression. Sorted Tregs were cultured for 24h in glucose-restricted (0.5mM) condition or normal medium (1 ImM glucose) supplemented with 10% of glucose-free FBS and Ing/mL IL -2. The expression level of VPS72 was determined by qPCR. (N=3). **p<0.01.
[0045] FIG. 36 VPS72-mediated H2A.Z epigenetic axis directly (Panel A) or indirectly (Panel B) regulates FoxP3.
[0046] FIG. 37 Skin Graft model. (Panel A) Schematic for BrafV600EPTEN mice.
(Panel B) Diagram of skin graft approach. Approximately lcm2 skin section from BrafV600EPTEN mice were transplanted into WT and VPS72/H2A.Z-TregiKO mice. One week later, one of the skin grafts was treated by topical application of tamoxifen. (Panel C) Two pieces of skin graft from BrafV600EPTEN mice were transplanted into WT mice. At day 7, one graft w as treated by topical application of tamoxifen (bottom) and w as untreated as a control (top). The melanoma development was photographed daily. Images at day 7. 35. and 50 from one representative mouse are shown.
[0047] FIG. 38 TME regulates VPS72 expression. The binding sites of Smad, FOxO3, HIFla, Spl are located within lOOObp at promoter of VPS72.
DETAILED DESCRIPTION
[0048] Definitions [0049] 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 disclosure belongs. Accordingly, the following terms are intended to have the following meanings:
[0050] As used in the specification and claims, the singular form “a”, “an” and “the” includes plural references unless the context clearly dictates otherwise.
[0051] As used herein, “administration” of a disclosed polypeptide encompasses the delivery to a subject of a polypeptide or composition of the present invention, as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, using any suitable formulation or route of administration, e.g., as described herein.
[0052] As used herein, the term “and/or.” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as comprising components A, B, and/or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B. and C in combination.
[0053] As used herein, “treatment” and “treating”, are used interchangeably herein, and refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underly ing disorder. The term “treat”, in all its verb forms, is used herein to mean to relieve, alleviate, prevent, and/or manage at least one symptom of a disorder in a subject.
[0054] A “subject,” as used herein, can refer to any animal which is subject to a viral infection, e.g., a mammal, such as an experimental animal, a farm animal, pet, or the like. In some embodiments, the animal is a primate, preferably a human. As used herein, the terms “subject” and “patient” are used interchangeably. The terms “subject” and “patient” refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), specifically a “mammal” including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzee and a human), and more specifically a human. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a “human’".
[0055] As used herein, the term “linked” refers to unifying two molecules having the same or different function or structure, and the methods of fusing may include any physical, chemical or biological method capable of binding the peptide to the protein, the smallmolecule drug, the nanoparticle or the liposome. Preferably, the fusion may be mediated by a linker peptide, and for example, the linker peptide may be fused to the C-terminus of a peptide.
[0056] The terms, “disease”, “disorder”, and “condition” may be used interchangeable.
[0057] As used herein, the terms “treat” and “treatment” and “‘treating” 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 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. “Treatment” as used herein covers any treatment of a disease in a subject, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom. Those in need of treatment include individuals already diagnosed with a disease, as well as those in which the disease is to be prevented. Thus, the terms “treat” or “treatment” or “treating” refer to both therapeutic and prophylactic treatments. For example, therapeutic treatments includes the reduction or amelioration of the progression, severity and/or duration of a disease symptom, or the amelioration of one or more symptoms.
[0058] In specific embodiments, the therapeutic treatment includes the amelioration of at least one measurable physical parameter. In other embodiments the therapeutic treatment includes the inhibition of the progression of a disease or worsening symptom, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g.. stabilization of a physical parameter, or both.
[0059] The terms “prophylaxis” or “prophylactic use” and “prophylactic treatment” as used herein, refer to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease. As used herein, the terms “prevent”, “prevention” and “preventing” refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a person with the disease. The term “chemoprophylaxis” refers to the use of medications, e.g. small molecule drugs (rather than “vaccines”) for the prevention of a disorder or disease.
[0060] As used herein, an “effective amount” refers to an amount sufficient to elicit the desired biological response. The desired biological response is the reduction or amelioration of disease severity, duration, progression, reoccurrence or delayed onset of disease or symptoms associated with the disease, or to enhance or improve the prophylactic or therapeutic effect(s) of another therapy used to treat the disease (e.g., cancer). The precise amount of the therapeutic composition administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When coadministered with other immunotherapies or chemotherapies, e.g., when co-administered with an anti-proliferation medication, an “effective amount” of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a polypeptide described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed. For example, compounds described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.
[0061] The term “peptide” is used herein to refer to a chain of amino acid residues. The terms oligonucleotide or nucleic acid are used interchangeably herein to refer to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term “polymer” is used used interchangeably herein to refer to a chain of covalently linked amino acid or nucleotide (RNA or DNA) monomers.
[0062] The term “reduce” or other forms of the word, such as “reducing” or “reduction.” generally refers to the lowering of an event or characteristic (e.g., one or more symptoms, or the numbers of cells). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. In some embodiments, the term “reducing,” is used in the context of “reducing the functional response of an immune cell”.
[0063] The term “inhibit” or “inhibitor” as used in the context of “inhibiting VPS72” refers to a decrease in the activity or function of VPS72. For example, in some embodiments. a peptide binds to a particular target epitope, e.g., a protein dimer, or any thereof, with a given affinity, and thereby inhibits VPS72 protein binding and therefore it’s chaperone activity. Measuring the affinity of binding is well known in the art. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a dissociation constant (KD or Kd) [0064] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding target or partner (e.g., peptide). The affinity of a molecule for its target can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Briefly, the strength, or affinity of binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents a greater affinity. The binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigenbinding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The ratio of Koff/Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant KD. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
[0065] Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity' can be measured by well-established methods known in the art, including those described herein. Thus, in some embodiments, “reduced binding” refers to a decrease in affinity for the respective interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction. [0066] In some embodiments, a recombinant peptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is <1 pM. In some embodiments, a recombinant peptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is <100 nM. In some embodiments, a recombinant peptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is <10 nM. In some embodiments, a recombinant peptide of the present invention can specifically bind to an epitope when the equilibrium binding constant (KD) is <100 pM to about 1 pM, as measured by assays such as Surface Plasmon Resonance (SPR), Octet assays, or similar assays known to those skilled in the art. In some embodiments, a KD can be 10 5 M or less (e.g., 10 6 M or less, 10 7 M or less. 10 s M or less. 10 s M or less, 10 l0 M or less, 10 1 1 M or less, 10 l2 M or less, 10 15 M or less, 10 14 M or less, 1 13 M or less, or 10 16 M or less).
[0067] In some embodiments, there can be a reduction of binding of 1%, 2%, 3%, 4%, 5%, 6%. 7%, 8%, 9%. 10%. 20%. 30%. 40%. 50%, 60%, 70%, 80%, 90% or 100% in a recombinant polypeptide compared to a control. For example, in recombinant peptides having reduced heparin or heparan sulfate binding, there can be there can be a reduction of binding of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in a recombinant peptide compared to a control.
[0068] An "isolated'’ peptide or oligonucleotide as used herein, is obtained directly from a synthetic or recombinant generated peptide or oligonucleotide sequence that is identical, substantially related to, complementary, or modified from a known protein or polynucleotide or a fragment thereof. The term “substantially related”, as used herein, means that the protein or oligonucleotide may have been modified by chemical, physical or other means (e.g. sequence modification).
[0069] “Excipient” refers to any pharmacologically inactive, natural, or synthetic, component or substance that is formulated alongside (e.g., concomitantly), or subsequent to, the active ingredient of the present invention. In some embodiments, an excipient can be any additive, adjuvant, binder, bulking agent, carrier, coating, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle, or combination thereof, with which a recombinant polypeptide of the present invention can be administered, and or which is useful in preparing a composition of the present invention. Excipients, include any such materials known in the art that are nontoxic and do not interact with other components of a composition. In some embodiments, excipients can be formulated alongside a recombinant polypeptide when preparing a composition for the purpose of bulking up compositions (thus often referred to as bulking agents, fillers or diluents). In other embodiments, an excipient can be used to confer an enhancement on the active ingredient in the final dosage form, such as facilitating absorption and/or solubility. In yet other embodiments, an excipient can be used to provide stability, or prevent contamination (e.g., microbial contamination). In other embodiments, an excipient can be used to confer a physical property7 to a composition (e.g., a composition that is a dry granular, or dry7 flowable powder physical form). Reference to an excipient includes both one and more than one such excipients. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.
[0070] ■‘Carrier” as used herein refers to a delivery system. A carrier delivery system functions to increase circulation time in the subject, increase solubility to enhance bioavailability, prevent protease or nuclease degradation, and allow drug targeting to specific cell types. Various types of carriers are described below, each with unique advantages.
[0071] “Operable” refers to the ability to be used, the ability to do something, and/or the ability to accomplish some function or result. For example, in some embodiments, “operable” refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other oligonucleotide sequence to bind a complementary nucleic acid sequence (e.g., antisense oligonucleotide).
[0072] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers. [0073] VPS72 Inhibitors
[0074] In various embodiments, the present disclosure provides an inhibitor for use to reduce the expression of VPS72 in targeted or exposed cells, or for use in inhibiting the activity of VPS72 in immune cells. In various embodiments, an inhibitor of the present disclosure includes a peptide or protein that binds to VPS72 and inhibits its binding to its binding partner H2A to H2A.Z exchange in chromatin remodeling. In some embodiments, a peptide inhibitor comprises a peptide having at least 80%, or 85%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or at least 100% amino acid identity to a peptide of SEQ ID NO: 2 over the entire length of the peptide of SEQ ID NO: 2.
[0075] Amino Acid sequence (homo sapiens) of SEQ ID NO: 2: GRKKRRQRRRPP- EEEDEFYQTTYGGFTEESGDDEYQGD. In various embodiments, a peptide inhibitor of the present disclosure comprises one, two, three, four or five amino acid substitutions in the sequence of SEQ ID NO: 2. In further embodiments, a peptide inhibitor of the present disclosure comprises one, two, three, four or five ammo acid conservative amino acid substitutions in the sequence of SEQ ID NO: 2. In further embodiments, a peptide inhibitor of the present disclosure comprises one, two, three, four or five amino acid conservative amino acid substitutions in the sequence of SEQ ID NO: 2 ranging from amino acid position 13 to 55 of SEQ ID NO: 2. In further embodiments, a peptide inhibitor of the present disclosure comprises one, two, three, four or five amino acid conservative amino acid substitutions in the sequence of SEQ ID NO: 2 ranging from amino acid position 13 to 55 of SEQ ID NO: 2. with the exception of amino acids F29, Y30, Y34. F37, D43, E35, and Y64 of SEQ ID NO: 2. In further embodiments, a peptide inhibitor of the present disclosure comprises a peptide having 23 to 100 amino acids, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 2 ranging from amino acid position 13 to 55 of SEQ ID NO: 2.
[0076] Relative to the sequence of VPS72, the VPS72 inhibitor peptide of SEQ ID NO: 2 comprises a region of VPS72 as underlined below
[0077] MSL AGGRAPRKTAGNRLS GLLEAEEEDEFYQTTYGGFTEESGDDEYQ
GDQSDTEDEVDSDFDIDEGDEP - Partial amino acid sequence of VPS72 (homo sapiens) - SEQ ID NO: 3, which is a modified form of SEQ ID NO: 2.
[0078] The method of any one of claims 1-18, wherein a peptide inhibitor blocks VPS72 from binding to H2A.Z-H2B dimer by simulating the critical amino acids in the N- terminal binding domain of VPS72 that interacts with H2A.Z-H2B as shown underlined relative to the VPS72 amino acid sequence of SEQ ID NO: 3, which includes F29, Y30, Y34, F37, D43, E35. and Y64 of SEQ ID NO: 3. In various embodiments, an inhibitor peptide comprises an amino acid sequence that comprises at least a region of the VPS72 amino acid sequence comprising positions F29, Y30, Y34, F37, D43, E35, and Y64 of SEQ ID NO: 3. In various embodiments, the inhibitor peptide ranges from 20 amino acids to 60 amino acids, more preferably from 25 amino acids to 50 amino acids, most preferably from 30 amino acids to 40 amino acids in length, wherein the inhibitor peptide has amino acids F29, Y30, Y34, F37, D43, E35, and Y64 relative to the entire amino acid sequence of SEQ ID NO: 3. In various embodiments, an exemplary7 inhibitor peptide of VPS72 comprises a peptide having at least 95%, or 96%, or 97%, or 98%, or 98%, or 99%, or at least 100% sequence identity to a peptide of SEQ ID NO: 2, along its entire length. In some preferred embodiments, the inhibitor peptide of VPS72 comprises a peptide having at least 95% sequence identity to a peptide of SEQ ID NO: 2, wherein the inhibitor peptide has 5, or 4, or 3, or 2 or 1 amino acid substitutions relative to SEQ ID NO: 2 along its entire length.
[0079] In various embodiments, the VPS72 inhibitor peptide when administered to a subject or exposed to a cell, for example, an immune cell, for example, an immune cell selected from the group: invariant natural killer T cells (iNKT) cells, mucosal associated invariant T cells (MAIT) cell development and activity, Tregs, hematopoietic stem cells (HSC), Langerhans cells (LC), macrophages, and dendritic cell (DC) inhibits the VPS72 activity of that cell by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, of the VPS72 activity in that cell.
[0080] In some embodiments, a VPS72 inhibitor of the present disclosure is a nucleic acid that inhibits the transcription or expression or translation of VPS72 DNA or VPS72 mRNA.
[0081] VPS72 RNA Interfering Nucleic Acids and Constructs Containing Same. [0082] RNA interference (RNAi), also called post-transcriptional gene silencing (PTGS), is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Without being bound by theory, it appears that, in the presence of an antisense RNA molecule that is complementary to an expressed message (i.e., a mRNA), the two strands anneal to generate long double-stranded RNA (dsRNA), which is digested into short (<30 nucleotide) RNA duplexes, known as small interfering RNAs (siRNAs), by an enzyme know n as Dicer. A complex of proteins known as the RNA Induced Silencing Complex (RISC) then unwinds siRNAs, and uses one strand to identify and thereby anneal to other copies of the original mRNA. RISC cleaves the mRNA within the complementary sequence, leaving the mRNA susceptible to further degradation by exonucleases, which effectively silences expression of the encoding gene.
[0083] Several methods have been developed that take advantage of the endogenous machinery to suppress the expression of a specific target gene and a number of companies offer RNAi design and synthesis services (e.g., Life Technologies, Applied Biosystems). In mammalian tissues, the use of RNAi can involve the introduction of long dsRNA (e g., greater than 50 bps) or siRNAs (e.g., 12 to 23 bps) that have complementarity to the target gene, both of which are processed by the endogenous machinery. Alternatively, the use of RNAi can involve the introduction of a small hairpin RNA (shRNA); shRNA is a nucleic acid that includes the sequence of the tw o desired siRNA strands, sense and antisense, on a single strand, connected by a “loop” or “spacer” nucleic acid. When the shRNA is transcribed, the two complementary portions anneal intra-molecularly to fonn a “hairpin,” which is recognized and processed by the endogenous machinery.
[0084] A RNAi nucleic acid molecule as described herein is complementary to at least a portion of a target mRNA (i.e., a VPS72 mRNA), and typically is referred to as an “antisense strand”. Typically, the antisense strand includes at least 15 contiguous nucleotides of the DNA sequence (e.g., the VPS72 nucleic acid sequence shown in SEQ ID NO:4 (homo sapiens and/or mus musculus for SEQ ID NOS: 4-9)); it would be appreciated that the antisense strand has the “RNA equivalent” sequence of the DNA (e.g., uracils instead of thymines; ribose sugars instead of deoxyribose sugars).
[0085] A RNAi nucleic acid molecule can be, for example, 15 to 500 nucleotides in length (e.g., 15 to 50, 15 to 45, 15 to 30, 16 to 47, 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74. 17 to 162, 17 to 305, 18 to 60. 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides in length).
[0086] In some embodiments, the ‘"antisense strand” (e.g., a first nucleic acid) can be accompanied by a “sense strand” (e.g., a second nucleic acid), which is complementary to the antisense strand. In the latter case, each nucleic acid (e.g., each of the sense and antisense strands) can be between 15 and 500 nucleotides in length (e.g., between 15 to 50, 15 to 45, 15 to 30, 16 to 47. 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60. 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides in length). [0087] In some embodiments, a spacer nucleic acid, sometimes referred to as a loop nucleic acid, can be positioned between the sense strand and the antisense strand. In some embodiments, the spacer nucleic acid can be an intron (see, for example, Wesley et al., 2001, The Plant J., 27:581-90). In some embodiments, although not required, the intron can be functional (i.e., in sense orientation; i.e., spliceable) (see, for example. Smith et al., 2000, Nature, 407:319-20). A spacer nucleic acid can be between 20 nucleotides and 1000 nucleotides in length (e.g., 25-800, 25-600, 25-400, 50-750, 50-500, 50-250, 100-700, 100- 500, 100-300, 250-700, 300-600, 400-700, 500-800, 600-850, or 700-1000 nucleotides in length).
[0088] In some embodiments, a construct can be produced by operably linking a promoter that is operable in mammalian cells; a DNA region, that, when transcribed, produces an RNA molecule capable of forming a hairpin structure; and a DNA region involved in transcription termination and polyadenylation. It would be appreciated that the hairpin structure has two annealing RNA sequences, where one of the annealing RNA sequences of the hairpin RNA structure includes a sense sequence identical to at least 20 consecutive nucleotides of the VPS72 nucleotide sequence, and where the second of the annealing RNA sequences includes an antisense sequence that is identical to at least 20 consecutive nucleotides of the complement of the VPS72 nucleotide sequence. In addition, as indicated herein, the DNA region can include an intron (e.g., a functional intron). When present, the intron generally is located between the two annealing RNA sequences in sense orientation such that it is spliced out by the cellular machinery (e.g., the splicesome). Such a construct can be introduced into one or more plant cells to reduce the phenotypic expression of a VPS72 nucleic acid (e.g., a nucleic acid sequence that is normally expressed in a plant cell).
[0089] In some embodiments, a construct (e.g., an expression construct) can include an inverted-duplication of a segment of a VPS72 gene or portion thereof, where the inverted- duplication of the VPS72 gene segment includes a nucleotide sequence substantially identical to at least a portion of the VPS72 gene and the complement of the portion of the VPS72 gene. It would be appreciated that a single promoter can be used to drive expression of the inverted- duplication of the VPS72 gene segment, and that the inverted-duplication typically contains at least one copy of the portion of the VPS72 gene in the sense orientation. Such a construct can be introduced into one or more plant cells to delay, inhibit or otherwise reduce the expression of a VPS72 gene in the plant cells.
[0090] The components of a representative RNAi nucleic acid molecule directed toward VPS72 are shown in SEQ ID NO:4 (a sense strand to VPS72); SEQ ID NO: 5 (an antisense strand to VPS72); and SEQ ID NO:6 (a spacer or loop sequence).
[0091] It would be appreciated by the skilled artisan that the region of complementarity, between the antisense strand of the RNAi and the mRNA or between the antisense strand of the RNAi and the sense strand of the RNAi, can be over the entire length of the RNAi nucleic acid molecule, or the region of complementarity can be less than the entire length of the RNAi nucleic acid molecule. For example, a region of complementarity' can refer to, for example, at least 15 nucleotides in length up to, for example. 500 nucleotides in length (e.g., at least 15, 16, 17, 18, 19, 20, 25, 28, 30. 35. 49. 50. 60. 75, 80, 100, 150. 180, 200, 250, 300, 320, 385, 420, 435 nucleotides in length up to, e.g., 30, 35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200, 225, 250, 280, 300, 325, 350, 400, 450, or 475 nucleotides in length). In some embodiments, a region of complementarity can refer to, for example, at least 15 contiguous nucleotides in length up to, for example, 500 contiguous nucleotides in length (e g., at least 15, 16, 17, 18, 19, 20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200, 250, 300, 320, 385, 420. 435 nucleotides in length up to, e.g., 30, 35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200. 225, 250, 280, 300, 325. 350, 400. 450, or 475 contiguous nucleotides in length).
[0092] It would be appreciated by the skilled artisan that complementary can refer to, for example, 100% sequence identity' between the two nucleic acids. In addition, however, it also would be appreciated by the skilled artisan that complementary’ can refer to, for example, slightly less than 100% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity). In calculating percent sequence identity, two nucleic acids are aligned and the number of identical matches of nucleotides (or amino acid residues) between the two nucleic acids (or polypeptides) is determined. The number of identical matches is divided by the length of the aligned region (i.e.. the number of aligned nucleotides (or amino acid residues)) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both nucleic acids up to the full-length size of the shortest nucleic acid. It also will be appreciated that a single nucleic acid can align with more than one other nucleic acid and hence, can have different percent sequence identity values over each aligned region.
[0093] The alignment of two or more nucleic acids to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31( 13): 3497- 500. ClustalW calculates the best match between a query and one or more subject sequences (nucleic acid or polypeptide), and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty7: 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0; gap extension penalty’: 5.0: and weight transitions: yes. For fast pairw ise alignment of polypeptide sequences, the following parameters can be used: word size: 1; window size: 5; scoring method: percentage: number of top diagonals: 5; and gap penalty7: 3. For multiple alignment of polypeptide sequences, the following parameters can be used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly. Pro, Ser, Asn, Asp, Gin, Glu, Arg. and Lys; and residue-specific gap penalties: on. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.
[0094] The skilled artisan also would appreciate that complementary can be dependent upon, for example, the conditions under which two nucleic acids hybridize. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclose suitable Southern blot conditions for oligonucleotide probes less than about 100 nucleotides (Sections 11.45-11.46). The Tm between a nucleic acid that is less than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Section 11.46. Sambrook et al. additionally disclose Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54). The Tm between a nucleic acid greater than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al.
[0095] The conditions under which membranes containing nucleic acids are prehybridized and hybridized, as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe, can play a significant role in the stringency of the hybridization. Such hybridizations and washes can be performed, where appropriate, under moderate or high stringency conditions. For example, washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the w ashes are performed. Simply by w ay of example, high stringency conditions typically include a w ash of the membranes in 0.2*SSC at 65° C.
[0096] In addition, interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium. It will be readily appreciated by those of ordinary skill in the art that although any number of hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids under identical hybridization, washing, and exposure conditions. Preferably, the target nucleic acids are on the same membrane. A nucleic acid molecule is deemed to hybridize to a nucleic acid, but not to another nucleic acid, if hybridization to a nucleic acid is at least 5-fold (e g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50- fold, or 100-fold) greater than hybridization to another nucleic acid. The amount of hybridization can be quantified directly on a membrane or from an autoradiograph using, for example, a Phosphorlmager or a Densitometer (Molecular Dynamics, Sunnyvale, Calif.). [0097] A construct (also known as a vector) containing a RNAi nucleic acid molecule is provided. Constructs, including expression constructs, are described herein and are known to those of skill in the art. Expression elements (e.g., promoters) that can be used to drive expression of a RNAi nucleic acid molecule are known in the art and include, without limitation, constitutive promoters such as, without limitation, the cassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the actin promoter, or the glyceraldehyde-3-phosphate dehydrogenase promoter, or tissue-specific promoters such as, without limitation, root-specific promoters such as the putrescine N-methyl transferase (PMT) promoter or the quinolinate phosphosibosyltransferase (QPT) promoter. It would be understood by a skilled artisan that a sense strand and an antisense strand can be delivered to and expressed in a target cell on separate constructs, or the sense and antisense strands can be delivered to and expressed in a target cell on a single construct (e.g.. in one transcript). As discussed herein, a RNAi nucleic acid molecule delivered and expressed on a single strand also can include a spacer nucleic acid (e.g., a loop nucleic acid) such that the RNAi forms a small hairpin (shRNA).
[0098] Methods of Stimulating Immunosuppression via Inhibition of HSC, LC, macrophages, and DC development and function.
[0099] In various embodiments of the present disclosure, methods and compositions are provided to inhibit the expression and/or activity of the protein vacuolar protein sorting- associated protein 72 homolog (VPS72, also known as YL1). VPS72 from human is characterized and the amino acid sequence for human VPS72 can be found in the NCBI database under accession No. NP 0012580I6.1, the disclosure of which is incorporated by reference herein in its entirety.
[0100] VPS72 functions as a histone chaperone for H2A to H2A.Z exchange in chromatin remodeling and belongs to two multi-subunit chromatin-remodeling complexes: (1) the Snf2 -related CBP-activator protein chromatin remodeling (SRCAP) complex; and (2) the TRRAP/TIP60 complex. Exchanging H2A for variant histone H2A.Z through the VPS72/SRCAP/TIP60 complex has been show n to modulate chromatin structure to activate or repress target genes, modify the local chromatin structure, and regulate cellular processes and mitochondrial function. The present disclosure provides methods for down regulating or abolishing the activity of VPS72 using different mouse models showed that VPS72 is critical for invariant natural killer T cells (iNKT) cells, mucosal associated invariant T cells (MAIT) cell development and activity, Tregs, hematopoietic stem cells (HSC), Langerhans cells (LC), macrophages, and dendritic cell (DC) development and function. Mechanism studies disclosed herein showed that VPS72 interacts with H2AZ in regulation of cell development. In some embodiments, inhibitors of VPS72 function, for example, the disclosed VPS72|H2A.Z blocking inhibitor peptide of SEQ ID NO: 2, it is possible to inhibit VPS72 expression and/or activity which results in therapeutic applications for the treatment and/or prevention of autoimmune diseases (for example, allergic contact dermatitis, lupus, scleroderma, inflammatory bowel disease colitis (e.g., ulcerative colitis) systemic lupus erythematosus (i.e., lupus) multiple sclerosis and arthritis, and symptoms associated with these conditions and diseases), inflammation (for example, asthma, lung infections, and skin inflammation among other forms of inflammation), hematological diseases and others for example, wound healing.
[0101] In one embodiment, the present disclosure provides a method of altering an immune cell response in a subject in need thereof, said method comprising administering an agent that inhibits VPS72 activity and/or expression in an immune cell of the subject. The methods of the present disclosure anticipate inhibiting the expression of VPS72 in immune cells and/or inhibiting the expression of VPS72 in the target immune cells, for example, iNKT cells and MAIT cells.
[0102] The present disclosure therefor provides methods and compositions for reducing the immune response as provided by iNKT cells by administering to a subject in need thereof, agents that down reg ulate the stimulation of immune function, particularly by inhibiting iNKT cells by administering an agent to the subj ect in need thereof, that inhibits VPS72 or inhibits the activity of VPS72 or both. This results in a corresponding benefit to subjects with autoimmune diseases, subjects with local or systemic inflammation and other diseases of hyperimmune function.
[0103] Thus in various embodiments, compositions of the present disclosure provide inhibitor}’ agents that selectively inhibit the expression and/or activity’ of VPS72, which in turn when certain immune cells are exposed to said compositions, VPS72 expression levels and or activity is reduced compared to not exposed immune cells, and wherein those exposed immune cells are produced in lower numbers, are stunted developmentally, or fail to express one or more pro-inflammatory or immunostimulatory Thl and Th2 cytokines, for example, interferon-y, IL-1 beta. IL-4. IL-13, and fail to modulate cytokine production by bystander cells. [0104] iNKT cells arise in the thymus from the CD4+CD8+ double-positive (DP) stage and are selected by CD Id-expressing DP thymocytes. In contrast to the selection of conventional T cells by thymic epithelial cells, iNKT cell selection requires stronger TCR signal strength. After iNKT cells (CD24+CD44loNKl.r) are selected (stage 0, STO), subsequent events elicit proliferation and a progression of maturation from immature CD24" CD44loNKl. l (stage 1. STI) to semi-mature CD24 CD44hlNKl. T(stage 2, ST2) to mature CD44hlNKl. l+(stage 3, ST3) iNKT cells. ST2 iNKT cells possess higher proliferation potential, and most iNKT cells at this stage emigrate to peripheral organs. Recent studies suggest that thymic iNKT cells terminally differentiate into at least three distinct lineages of iNKT effector cells (iNKTl, iNKT2, and iNKT 17) that phenot pically differ, having distinct transcription factor expression and cytokine production: iNKTl cells are PLZFloCD44hlNKl. l+ (i.e., ST3), express T-bet, and mainly produce IFN-y; iNKT2 cells are PLZF1"CD44I"NK I . I (i.e., ST2), express GATA3, and produce abundant IL-4; and iNKT17 cells resemble iNKT2 cells in being PLZF'"tCD441"NK I . I but express RORyt and produce IL- 17. Recent studies identified that some transcription factors regulate specific stages of iNKT cell development and that distinct mitochondrial metabolic programs and autophagy are specifically required for iNKT cell differentiation and function. However, how these transcriptional factors and mitochondrial metabolic pathways are integrated with chromatin remodeling programs that promote iNKT positive selection, lineage specification, acquisition of functional activity, and homeostasis remains poorly understood.
[0105] During immune cell development, epigenetic programming is required to ensure the establishment of proper chromatin organization for regulating lineage-specific gene expression. The epigenetic regulation of iNKT cell development through the miRNA pathway has long been recognized, and the present inventors were the first to uncover that Dicer-deficient mice (miRNA deficiency) completely lack iNKT cells. Studies have shown roles for multiple miRNAs, including miR-183-96-182, Let-7, and the miR-150, miR-155, miR-181, miR-17-92 family cluster in regulating iNKT cell development. The role of histone post-transcriptional modifications in iNKT regulation, such as the addition or removal of methyl and acetyl groups, has also been illuminated. The present inventors and others have identified that histone demethylases (UTX and JMJD3) are required for iNKT cell development and that histone deacetylase HDAC3 regulates iNKT cell development and differentiation by targeting autophagy. However, the role of nucleosome dynamics in the form of histone H2A-H2A.Z exchange in T cells, especially in iNKT cells, remains completely unknown. Using T cell specific VPS72 and H2A.Z deletion mouse models (VPS72cKO and H2A.ZcK0, respectively), we have found that loss of VPS72 severely impairs thymic iNKT cell early selection and development, without interrupting thymic conventional T cells. Furthermore, loss of VPS72 interrupted iNKT cell survival and autophagy and dysregulated global chromatin accessibility. As expected, mice lacking H2A.Z resemble VPS72cKO mice in having defective thymic iNKT cells.
[0106] Mucosal-associated invariant T (MAIT) cells are innate-like T cells defined by their semi -invariant a[3 T cell receptor (TCR) which recognizes small-molecule biosynthetic derivatives of riboflavin synthesis presented on the restriction molecule major histocompatibility7 complex (MHC)-related protein- 1 (MR1) . MAIT cells were described in 1999 based on their TCR comprising a semi-invariant TCR-a chain — usually Va7.2- Ja33/12/20 in humans. Val9-Ja33 in micepredominantly associated with the (3-chains V 2/V 13 in humans and V 6/V 8 in mice.
[0107] MAIT cells contrast with conventional T cells which have highly variable TCRs, capable of targeting a vast array of peptide epitopes produced by viruses, bacteria and malignant cells. Conventional T cells therefore have exquisite specificity for individual peptides, and individual clones may undergo massive expansion, to provide T cell memory . However, at the first encounter with a pathogen the frequency of any individual peptidespecific T cell will be very low. In contrast, the MAIT cell TCR provides an innate capacity7 to respond to a specific set of ligands without the need for expansion.
[0108] Several properties of MAIT cells imply fundamental roles in mammalian immunity7. First, MAIT cells have an intrinsic effector-memory phenoty pe, usually CD45RA-CD45RO+ CD95HiCD62LLoCD44Hi , with capacity7 for rapid secretion of several pro-inflammatory7 cytokines. Second, MAIT cells are remarkably abundant in human tissues, typically comprising 1-4% of all T cells in peripheral blood and up to 10% of airway T cells and 20-40% of liver T cells. Moreover, as each TCR recognizes the same ligand, early in an immune response, MAIT cells will markedly exceed the numbers of conventional antigen-specific T cells responding to cognate antigens. Third, the MR1-MAIT cell axis is strikingly conserved across 150 million years of mammalian evolution, with -90% sequence homology for MR1 between mouse and human, implying a strong evolutionary7 pressure maintaining the MAIT cell repertoire. Nonetheless identifying the critical functional role(s) played by these cells has not proved straightforward, perhaps because these cells perform not a single, but several distinct functions. Indeed, several new MAIT cell functions have recently been discovered, representing distinct transcriptional programs which can be triggered via distinct activation pathways. [0109] Conventional T cells are implicated as effectors in many organ-specific autoimmune diseases such as type-1 diabetes or multiple sclerosis, but strong HLA associations in a range of systemic autoimmune diseases imply a pathogenic role in these diseases as well. As the MAIT cell ligands are not synthesized by human cells, MAIT cell activation in these conditions would be presumed to be via the cytokine-mediated TCR- independent pathway. Changes in MAIT cell frequencies and phenotype are observed in a range of autoimmune conditions.
[0110] Blood MAIT cells are decreased in children with type 1 diabetes mellitus. In the non-obese diabetic mouse MR1 mice have accelerated diabetes and increased gut permeability, suggesting MAIT cells may be protective against diabetes by supporting intestinal mucosal integrity, although the data are complicated by evidence in mice and humans of MAIT cell activation, exhaustion and capacity for islet-cell killing. Similarly, in type 2 diabetes peripheral blood MAIT cells are reduced in frequency, associated with increased caspase-3 -dependent apoptosis.
[OHl] In rheumatoid arthritis MAIT cell frequencies are increased in synovial tissue and so may contribute to maturation and cross-differentiation of T cells locally. Consistent with a pathogenic role, inflammation is reduced in murine collagen-induced arthritis in MR1 mice. MAIT cells are increased in inflammatory lesions in human multiple sclerosis. Likewise, inflammation is suppressed by inhibitory MAIT cell ligands in an animal model of systemic lupus erythematosus. In the gut, MAIT cells are found in proximity to Helicobacter pylori in human gastric mucosa, and in mice, MAIT cells were associated with accelerated H. pylori gastritis. In inflammatory bowel disease, several studies show decreases in peripheral blood MAIT cell frequencies and generally an increase in intestinal tissue, with increased IL- 17 and IL-22 production by blood MAIT cells, although it remains to be seen if these changes are causally linked and whether they are pathogenic or protective by restoring mucosal integrity.
[0112] In various embodiments of the present disclosure, methods are provided to treat diseases associated with a hyperimmune disease or condition, such as for example, an autoimmune disease, inflammation, graft-versus-host-disease (GVHD), poor wound healing and the like by administering to a subject with an autoimmune disease, local or systemic inflammation, a transplant recipient, or suffers from poor would healing, for example a diabetes patient, a therapeutically effective amount of a VPS72 inhibitor of expression and/or activity to the subject in need. In some embodiments, the VPS72 inhibitor is a peptide as described herein that competes for VPS72 and does not enable the binding of cellular VPS72
T1 with H2A.Z. In these embodiments, suppression of at least partial immune responses that are believed to play a role in the etiology, or progression or maintenance of the immune related disease or condition involves the inhibition, suppression, or elimination of one or more immune cells selected from iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSC, LC, macrophages and dendritic cells (DC) cell development. Immune suppression required for the treatment of autoimmune diseases, local or systemic inflammation and wound healing is believed to be attributed to the suppression of VPS72 to bind to its functional binding partner H2A and most notably H2A.Z involved in chromatin remodeling. Inhibiting the expression and/or activity' of VPS72 prevents the formation of the complex between VPS72 and H2A.Z.
[0113] As used herein, inhibiting the activity and/or expression of VPS72 that leads to a result in an improvement in the diseases and conditions, or symptoms associated thereto, which are mediated by VPS72, such as autoimmune diseases, inflammation and other diseases associated with expression and upregulation of the immune response generally refers to an inhibition of VPS72 activity and/or expression in the target immune cells, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more. In some embodiments, exemplary' target immune cells that result in the inhibition of the undesirable immune function can include any one or more of iNKT cells, MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) as described herein.
[0114] In various embodiments, administration of an inhibitor of VPS72 results in the reduced hematopoiesis of one of more immune cells selected from: iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC). In addition, administration of an inhibitor of VPS72 results in altering hematopoiesis for example, the modification of specific hematopoietic stem cell lineages. [0115] Methods of treatment in accordance with the present disclosure results in the suppression of immune cells or the activity' of immune cells, for example, iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC), wherein the reduction in immune cell numbers or activity of the immunosuppressed immune cells results in the reduction of tissue resident macrophage response by reducing tissue resident macrophage numbers. Such reduction in macrophage numbers aids in the treatment of autoimmune diseases, inflammation and other diseases associated with increased or overexpression and upregulation of the immune response, for example, w herein the reduction of the immune cells comprises reducing the numbers of Langerhan cells, a tissue resident macrophage of skin. The reduction of these cells can find utility in the treatment of skin inflammatory diseases, for example, wherein the skin disease is Langerhans cell Histiocytosis or other forms of inflammatory skin disease such as allergic contact dermatitis, hives, eczema or psoriasis.
[0116] Methods of treatment in accordance with the present disclosure results in the suppression of immune cells or the activity of immune cells, for example, immune cells selected from iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC), wherein the reduction in immune cell numbers or activity of the immunosuppressed immune cells results in the reduction of tissue resident macrophage response by reducing tissue resident macrophage numbers which is a therapeutic treatment for an autoimmune disease, for example, systemic sclerosis, systemic lupus erythematosus, or other organ specific autoimmune disease.
[0117] In some embodiments, reducing the activity and/or numbers of tissue resident macrophages in a subject that has an inflammatory' disease or condition mediated by systemic inflammation, or an acute inflammatory state can result in the treatment or amelioration of symptoms and conditions related to metabolic syndrome. In some embodiments, the methods of the present disclosure can be used to treat a metabolic syndrome or symptoms of metabolic syndrome wherein the symptom or condition associated with the metabolic syndrome can include non-alcoholic fatty liver disease (NAFLD), heart disease, diabetic retinopathy, stroke risk, obesity, hypertension, or a neurodegenerative disease associated with metabolic syndrome.
[0118] Dysregulation of TRM function can have multiple consequences in many diseases, including cardiovascular and metabolic condition, obesity, cancer, amyloidosis, and infections. Reestablishment of functional macrophages may become an opportunity for treating those metabolic disease. Accumulating evidence suggest that post-natal microglial activation may become chronic inflammatory source to drive progressive neurodegeneration and induce diabetic retinopathy. Further, Kupffer cells play important roles in iron metabolism, cholesterol metabolism, and immune surveillance. In case, if liver KC become dysfunctional or diminished, this may lead to pathogenic situation by increased lipogenesis, released inflammatory cytokines, and activated stellate cells to generate fibrosis condition like non-alcoholic fatty' liver disease (NAFLD). Hence, elimination and maintenance of tissue specific macrophage can be important. In that context, regulation of resident macrophage population may become an important tool to treat the symptoms of metabolic syndrome.
After understanding these dynamics of macrophage and the required role of VPS72 in maintaining resident macrophages (See Figs. 18-20), inhibitors of VPS72 using a pharmacological or genomic-based inhibition of VPS72 reduces the macrophage population to treat metabolic syndrome in the subject in need thereof.
[0119] In some embodiments, reducing the activity and/or numbers of tissue resident macrophages in a subject that has an inflammatory' disease or condition mediated by a systemic inflammation, or an acute inflammatory state can result in the treatment or amelioration of symptoms and conditions related to age-related macrophage dysfunction (ARMD). In various embodiments, administering a composition containing an inhibitor of VPS72 increases immunity' to infections, improves vaccine response, or reduces susceptibility to autoimmune disease or cancer.
[0120] In addition to reducing the numbers, activity or activation of immune cells selected from iNKT cells, MAIT cells, HSCs, LCs, macrophages and dendritic cells (DC), inhibiting the expression of VPS72 or its activity reduces Treg cell functional response. In some embodiments, reducing Treg functional response can be advantageous in the treatment of certain autoimmune diseases.
[0121] In some embodiments, the VPS72 inhibitors and compositions containing such inhibitors can be used to effectively treat or reduce chronic graft versus host disease (cGVHD) in a patient. In some instances, the patient has received a bone marrow transplant, a hematopoietic stem cell transplant, or a progenitor cell transplant, from a donor. In some instances, the patient has received a solid organ transplant (e.g., kidney, liver, heart, lung, etc.) from a donor. In addition, the methods described herein can be used to treat an autoimmune or alloimmune disease (e.g., chronic alloimmune or autoimmune responses).
[0122] Pharmaceutical Compositions
[0123] The peptides and oligonucleotides described herein can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention relates to a phannaceutical composition comprising a peptide of the invention described above, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a phannaceutical composition comprising an effective amount of a peptide of the present invention or a pharmaceutically' acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable excipient include, for example, phannaceutical diluents, excipients or excipient suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. [0124] An “effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount”. The term “therapeutically effective amount” refers to an amount effective in treating and/or ameliorating a symptom of disease.
[0125] A pharmaceutically acceptable carrier may contain inert ingredients which do not unduly inhibit the biological activity of the poly peptides. The pharmaceutically acceptable excipient should be biocompatible, e.g., non-toxic, non-infl ammatory, non- immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.
[0126] The pharmaceutically acceptable excipient, adjuvant, or vehicle, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various excipient used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the polypeptides described herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. As used herein, the phrase “side effects” encompasses unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., prophylactic or therapeutic agent) might be harmful or uncomfortable or risky'. Side effects include, but are not limited to fever, chills, lethargy, gastrointestinal toxicities (including gastric and intestinal ulcerations and erosions), nausea, vomiting, neurotoxicities, nephrotoxicities, renal toxicities (including such conditions as papillary necrosis and chronic interstitial nephritis), hepatic toxicities (including elevated serum liver enzyme levels), myelotoxicities (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste, prolongation of gestation, weakness, somnolence, pain (including muscle pain, bone pain and headache), hair loss, asthenia, dizziness, extra-pyramidal symptoms, akathisia, cardiovascular disturbances and sexual dysfunction.
[0127] Some examples of materials which can serve as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository' waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
[0128] In some embodiments, a composition of the present invention comprises a pharmaceutically acceptable salt.
[0129] The term "pharmaceutically acceptable salts” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral fonn of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic. citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
[0130] Thus, the peptide or oligonucleotides, also described as polymers, of the present invention may exist as salts, such as with pharmaceutically acceptable acids. The present invention includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, propri onates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art.
[0131] The neutral forms of the polymers are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the poly mer may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
[0132] In addition to salt forms, of the disclosure provides polymers, which are in a prodrug form. Prodrugs of the polymers described herein are those that readily undergo chemical changes under physiological conditions to provide increased bioavailability or decreased degradation of the polymers. Prodrugs of the polymers described herein may be converted in vivo after administration. Additionally, prodrugs can be converted to the polymers of the present invention by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
[0133] Certain polymers of the disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain polymers of the disclosure may exist in multiple cry stalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. [0134] In vivo carriers of the isolated antisense oligonucleotide or peptide inhibitor, or pharmaceutical salt thereof, or encapsulated pharmaceutical composition is linked with, or covalently or non-covalently bonded to a carrier selected from the group consisting of organic nanoparticles or microparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, extracellular vesicles, exosomes, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, polyplexes; polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, roN-e-caprolactone (PCL), starch hydroxyethyl (HES, MEA), polyglycolate (PGA), poly (lactic-co-glycolide) , poly lactide (PLA), poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), (2- Hydroxypropyl) methacrylamide (poly (HPMA) or PHPMA); and dextran; dendrimers, selected from the group consisting of: polyether-hydroxylamine (PEHAM), polyamidoamine (PAMAM), polyesteramine, polypropylene imine and polyglycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA) nanofibers, of polylactide (PLA), polyethylene oxide and poly-s-caprolactone (PCL); or inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD. Formulations described herein as being useful for pulmonary delivery’ are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
[0135] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0. 1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and. optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0. 1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
[0136] Other delivery systems can include time-release, delayed release or sustained release delivery' systems. Such systems can avoid repeated administrations of the active compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly (lactide-glycolide), copolyoxalates, poly caprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and poly anhydrides.
Microcapsules of the foregoing polymers containing drugs are described in, for example. U.S. Pat. No. 5,075.109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty7 acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings: compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active compound is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
[0137] Use of a long-term sustained release implant may be desirable. Long-term release, are used herein, means that the implant is constructed and arranged to delivery7 therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Longterm sustained release implants are known to those of ordinary skill in the art and include some of the release systems described above.
[0138] Administration Methods
[0139] In some embodiments, the compositions of the present invention may be administered to a subject in need thereof. In some embodiments, the compositions of the present invention may be co-admimstered with one or more additional therapies.
[0140] The terms “administration” or “administering” refer to the act of providing an composition of the present invention, e.g., a polypeptide or pharmaceutically acceptable salt thereof, to a subject in need of treatment thereof.
[0141] In some embodiments, the compositions of the present invention can be administered as follows: oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g.. a mini- osmotic pump, to a subject. Accordingly, administration can be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
[0142] By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of additional therapies. The therapeutic drugs can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the components individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances. As used herein, “sequential administration” includes that the administration of two agents (e.g., the agents described herein) do not occur on a same day.
[0143] As used herein, “concurrent administration” includes overlapping in duration at least in part. For example, when two compositions (e.g., any of the compositions described herein) are administered concurrently, their administration occurs within a certain desired time. The administration of the compositions may begin and end on the same day. The administration of one composition can also precede the administration of a second composition by day(s) as long as both compositions are taken on the same day at least once. Similarly, the administration of one composition can extend beyond the administration of a second composition as long as both agents are taken on the same day at least once. The composition do not have to be taken at the same time each day to include concurrent administration.
[0144] As used herein, “intermittent administration includes the administration of an agent for a period of time (which can be considered a “first period of administration”), followed by a time during which the composition is not taken or is taken at a lower maintenance dose (which can be considered “off-period”) followed by a period during which the composition is administered again (which can be considered a “second period of administration”). Generally, during the second phase of administration, the dosage level of the agent will match that administered during the first period of administration but can be increased or decreased as medically necessary. [0145] The VPS72 inhibitors, whether protein, peptide or nucleic acid based inhibitors and pharmaceutically acceptable compositions described above, can be administered to humans and other animals orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. [0146] In some embodiments of the invention, when treating a subject, an inhibitory agent is administered by systemic intravenous (IV) or by a local intranasal route, such as an intranasal spray, a metered-dose inhaler, a nebulizer, or a dry powder inhaler. Formulations for delivery' by a particular method (e.g., solutions, buffers, and preservatives, as well as droplet or particle size for intranasal administration) can be optimized by routine, conventional methods that are well-known in the art. For inhibitory agents that are in the form of aerosol formulations to be administered via inhalation, the aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen or the like.
[0147] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active polypeptides, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1.3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
[0148] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[0149] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0150] In order to prolong the effect of a polypeptide described herein, it is often desirable to slow the absorption of the polypeptide from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the polypeptide then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered polypeptide form is accomplished by dissolving or suspending the polypeptide in an oil vehicle. Injectable depot forms are made by fonning microencapsule matrices of the polypeptide in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of polypeptide to polymer and the nature of the particular polymer employed, the rate of polypeptide release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the polypeptide in liposomes or microemulsions that are compatible with body tissues.
[0151] Compositions for rectal or vaginal administration are specifically suppositories which can be prepared by mixing the polypeptides described herein with suitable nonirritating excipients or excipient such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active polypeptide.
[0152] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the polypeptide (i.e., active polypeptide) is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, I) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
[0153] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[0154] The active polypeptides can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active polypeptide may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
[0155] Dosage forms for topical or transdermal administration of a polypeptide described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a polypeptide to the body. Such dosage forms can be made by dissolving or dispensing the polypeptide in the proper medium. Absorption enhancers can also be used to increase the flux of the polypeptide across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the polypeptide in a polymer matrix or gel.
[0156] The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Specifically, the compositions are administered orally, intraperitoneally or intravenously.
[0157] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
[0158] The pharmaceutical compositions described herein comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality' of VPS72 inhibitors may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, excipient commonly used include, but are not limited to, lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
[0159] Alternatively, the pharmaceutical compositions described herein comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include, but are not limited to. cocoa butter, beeswax and polyethylene glycols.
[0160] The pharmaceutical compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
[0161] Topical application for the lower intestinal tract can be effected in a rectal suppository' formulation (see above) or in a suitable enema formulation. Topically- transdermal patches may also be used. For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more excipient. Excipient for topical administration of the polypeptides of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable excipient. Suitable excipient include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.
[0162] For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, specifically, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzyl al konium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum. [0163] The pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
[0164] The polypeptides for use in the methods of the invention can be formulated in unit dosage form. The term "unit dosage form” refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g.. about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. [0165] Pulmonary/Nasal Administration
[0166] For pulmonary administration, preferably, compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS 72 inhibitors is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. According to the invention, compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors as disclosed herein can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices are capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of polypeptides are also known in the art. Many of such devices can use formulations suitable for the administration for the dispensing of polypeptides in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles.
[0167] Metered dose inhalers like the Ventolin® metered dose inhaler, typically use a propellant gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like TURBUHALER™ (Astra), ROTAHALER® (Glaxo), DISKUS® (Glaxo), SPIROS™ inhaler (Dura), devices marketed by Inhale Therapeutics, and the SPINHALER® powder inhaler (Fisons), use breath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No. 5,458.135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference). Nebulizers like AERX™ Aradigm, the ULTRAVENT® nebulizer (Mallinckrodt), and the ACORN II® nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376). the above references are entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols. These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.
[0168] In some embodiments, a composition comprising compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors as disclosed herein is delivered by a dry powder inhaler or a sprayer. There are several desirable features of an inhalation device for administering at least one polypeptide of the present invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device can optionally deliver small dry7 particles, e.g., less than about 10 pm, preferably about 1-5 pm, for good respirability.
[0169] A spray including the compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors can be produced by forcing a suspension or solution of at least one polypeptide through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of at least one polypeptide delivered by a sprayer have a particle size less than about 10 pm, in some embodiments, in the range of about 1 pm to about 5 pm, of from about 2 pm to about 3 pm.
[0170] Formulations having compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors suitable for use with a sprayer typically include a polypeptide composition in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one polypeptide per ml of solution or mg/gm, or any range, value, or fraction therein. The formulation can include agents, such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors compositions include albumin, protamine, or the like. Typical carbohydrates useful in formulating polypeptide compositions include sucrose, mannitol, lactose, trehalose, glucose, or the like. The compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality7 of VPS72 inhibitors formulation can also include a surfactant, which can reduce or prevent surface- induced aggregation of the polypeptide composition caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80. polysorbate 20. or the like. Additional agents known in the art for formulation of compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an RNAi nucleic acid molecule or plurality of VPS72 inhibitors, such as preservatives and protease and nuclease inhibitors, can also be included in the formulation.
[0171] Administration of the Inhibitor Peptide or Nucleic Acid Compositions by a Nebulizer
[0172] Peptide or oligonucleotide compositions of the disclosure can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws a solution of polypeptide composition through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy7 is transmitted to the formulation of the polypeptide composition either directly or through a coupling fluid, creating an aerosol including the polypeptide composition. Advantageously, particles of the polypeptide composition delivered by a nebulizer have a particle size less than about 10 gm, in some embodiments, in the range of about 1 pm to about 5 pm, or from about 2 pm to about 3 pm.
[0173] Formulations of compositions comprising a VPS72 inhibitor, whether a peptide inhibitor or an nucleic acid inhibitor of VPS72, for example, a RNAi nucleic acid molecule or plurality of VPS72 inhibitors suitable for use with a nebulizer, either jet or ultrasonic, typically include a concentration of about 0.001 mg to about 100 mg of at least one VPS72 inhibitor per ml of solution. The formulation can include agents, such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the at least one polymer composition, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating at least one polymer compositions include albumin, protamine, or the like. Typical carbohydrates useful in formulating at least one polymer include sucrose, mannitol, lactose, trehalose, glucose, or the like. The at least one polymer formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one polymer caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between about 0.001 and 4% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like.
[0174] Administration of the VPS72 Inhibitor Compositions by a Metered Dose Inhaler
[0175] In a metered dose inhaler (MDI), a propellant, at least one VPS72 inhibitor as disclosed herein, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 pm, in some embodiments, about 1 pm to about 5 pm, or from about 2 pm to about 3 pm. The desired aerosol particle size can be obtained by employing a formulation of the VPS72 inhibitor composition produced by various methods known to those of skill in the art, including jetmilling, spray drying, critical point condensation, or the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant. Formulations of at least one polypeptide for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one VPS72 inhibitor as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane- 227), or the like. Preferably, the propellant is a hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one polypeptide as a suspension in the propellant, to protect the active agent against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases, solution aerosols are preferred using solvents, such as ethanol. Additional agents known in the art for formulation of a polypeptide or nucleic acid can also be included in the formulation. One of ordinary' skill in the art will recognize that the methods of the current invention can be achieved by pulmonary administration of at least one VPS72 inhibitor composition via devices not described herein.
[0176] Methods of Treatment
[0177] The preparations of the invention are administered in effective amounts. An effective amount is that amount of a VPS72 inhibitor agent that alone stimulates the desired outcome. In some embodiments, the desired outcome is a decrease in the number and/or activity of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC). In some embodiments, the desired outcome is a decrease or elimination of symptoms associated with a condition characterized by increased number and/or activity of iNKT cells, MAIT cells. T-regulatory T-cells (herein referred to as "Tregs"). HSCs, LCs, macrophages and dendritic cells (DC).
[0178] In some embodiments, the desired outcome is a decrease in the number and/or activity of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC). In some embodiments, the desired outcome is a decrease or elimination of symptoms associated with a condition characterized by increased number and/or activity of MAIT cells.
[0179] The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age. physical condition, size, weight, and the stage of the disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
[0180] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every' third day, every' week, every' two weeks, every three weeks, or every' four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g.. two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). [0181] In certain embodiments, an effective amount of a VPS72 inhibitor or a preparation for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a VPS72 inhibitor per unit dosage form.
[0182] In certain embodiments, the VPS72 inhibitor agents may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
[0183] It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
[0184] The invention contemplates in vitro and in vivo uses of the VPS72 inhibitor immuno-inhibitory molecules and preparations provided herein. When used in vivo, the VPS72 inhibitor and preparations may be formulated as pharmaceutical compositions (or preparations), intending that they are suitable for administration to a subject. A pharmaceutical composition need not be therapeutic or prophylactic however (i.e. , it may not eradicate an existing condition or prevent a condition from ever occurring in a subject). Instead, it may be used to modulate an aberrant immune response such as an increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) based immune response, or an increase in Treg activity, and thereby optionally modulate symptoms resulting from the underlying iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell based immune response. Such in vivo uses may be in subjects being treated for a particular condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell activity with the intention of providing some therapeutic or prophylactic benefit. Alternatively, such the VPS72 inhibitor and preparations may be used in vivo for research purposes, inter alia, typically in non-human subjects. The VPS72 inhibitors and preparations may be used in vitro to modulate immune responses involving activated iNKT cells, MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cells. Whether in vivo or in vitro, the VPS72 inhibitor or preparations may be used in screening assays to identity' iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) stimulatory agents or inhibitory- agents.
[0185] In some embodiments, the VPS72 inhibitor or preparations may be used in a method that involves contacting the VPS72 inhibitor (s) or preparation with an antigen presenting cell, and contacting the "loaded" antigen presenting cell with an iNKT cell, MAIT cell, T-regulatory T-cell (herein referred to as “Tregs”), HSC, LC, macrophage and dendritic cell (DC). The antigen presenting cells typically will express CD Id on their surface. A "loaded" antigen presenting cell intends an antigen presenting cell that has an immunoinhibitory molecule of the invention bound to its CDld and is therefore able to present such molecule to an iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs. LCs, macrophages and dendritic cells (DC). The contacting may occur in the presence of an agent that stimulates iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC)such as the VPS72 inhibitor peptide of SEQ ID NO: 2. The contacting may occur in the absence of such an immunostimulatory agent, and instead the iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) may be activated iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC). Such cells may have been activated in vitro prior to the contacting step or they may have been obtained from a subject having an increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell based immune response. It is to be understood that these methods may be carried out in vivo or in vitro.
[0186] Conditions
[0187] The VPS72 inhibitor of the invention as well as preparation and combination treatments containing such VPS72 inhibitor may be used to treat conditions that are characterized by increased levels of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cells and/or activity, including elevated cytokine profile, for example, elevated levels of pro- inflammatory or immunostimulator}' Thl and Th2 cytokines, for example, interferon-y, IL-1 beta, IL-4, IL-13 or by-stander cells that produce these Thl and Th2 cytokines relative to normal levels from a healthy adult or child subject. An increased level of iNKT and/or MAIT cells or activity is measured relative to a normal subject (or a normal population of subjects) not having an autoimmune or inflammatory condition and nor at increased (or elevated, intending above-normal) risk of developing such a condition (e.g., as may be the case if the condition is inherited). iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”). HSCs, LCs, macrophages and dendritic cells (DC) and/or activity may be measured in a blood sample or a biopsy such as a colonic (e.g., lamina propria) biopsy. Serum levels of proinfl ammatory cytokines, such as IFN-gamma, may be measured from the blood sample, for example. In some instances, persons having a family medical history or a personal medical history of a condition characterized by increased levels of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) and/or activity, such as for example colitis, arthritis, asthma, and the like, may be presumed to have or be at risk of developing the condition even if they are not experiencing symptoms at or near the time of treatment. In some instances, the subject may have an allergy or an allergic disorder. In some instances, activated iNKT cells. MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) are identified by the presence of CD Id tetramers, and may be detected and/or measured using for example flow cytometry or other immunostaining methods.
[0188] Such conditions treatable with the VPS72 inhibitors of the present disclosure include inflammatory conditions. Inflammatory conditions are conditions caused by, resulting from, or resulting in inflammation. An inflammatory condition may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) leading to abnormal tissue damage and/or cell death. An inflammatory condition can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. An inflammatory condition may be an autoimmune disease or it may be a non-autoimmune disease.
[0189] Inflammatory conditions include, without limitation, atherosclerosis, arteriosclerosis, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, giant cell arteritis, polymyositis, dennatomyosis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, mixed connective tissue disease, sclerosing cholangitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respirator}’ Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis, rheumatic fever, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomylitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fascihtis, and necrotizing enterocolitis. Conditions charactenzed by increased levels of iNKT cells and/or activity may be autoimmune diseases. Autoimmune diseases are diseases arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is ty pically with immunosuppression, e.g., medications that decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, multiple sclerosis, inflammatory bowel diseases such as ulcerative colitis, Crohn's disease, and ileitis, glomerulonephritis, Goodpasture's disease or syndrome, Graves' disease, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid, arthritis, psoriatic arthritis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis. anti-phospholipid antibody syndrome, scleroderma, perphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), urveitis, Sjogren's syndrome, Reiter's syndrome, ankylosing spondylitis, Lyme arthritis, GuillainBarre syndrome, Hashimoto's thyroiditis, and cardiomyopathy.
[0190] The term "treat", "treated," "treating" or "treatment" is used herein to mean to relieve, reduce or alleviate at least one symptom of a condition characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) or activity, or lowered Treg cell numbers and/or activity in a subject. For example, treatment can be diminishment of one or several symptoms of such a condition or complete eradication of the condition. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a condition) and/or reduce the risk of developing or worsening a condition. The term "protect" is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a condition in a subject.
[0191] A "subject" to which administration is contemplated includes, but is not limited to, humans and other non-human animals including, for example, companion animals such as dogs, cats, domesticated pigs, ferrets, hamsters, and the like; primates such as cynomolgus monkeys, rhesus monkeys, and the like; and agricultural animals such as cattle, pigs, horses, sheep, goats, birds (e.g., chickens, ducks, geese, and/or turkeys), and the like. In important embodiments, the subject is a human subject.
[0192] The subject may be of any age ranging from newborn to elderly. In some important embodiments, the subject is a pediatric subject such as a neonate, infant, child or adolescent. In such embodiments, the invention contemplates administering the active agents of the invention in order to render the subject resistant to conditions characterized by increased iNKT cells, MAIT cells. T-regulatory T-cells (herein referred to as “Tregs”). HSCs, LCs, macrophages and dendritic cells (DC) numbers or activity. As discussed herein, such conditions include but are not limited to asthma and autoimmune diseases such as but not limited to ulcerative colitis. Thus, the invention contemplates prophylactic treatment of a subject to prevent such conditions from manifesting. Accordingly, in some embodiments, the subject may be less than 10 years of age, less than 5 years of age, less than 1 year of age, less than 6 months of age, or less than 1 month of age, or 4 to 12 years of age, 12-18 years of age or 18-80 years of age and any age within these ranges. The invention further contemplates administration of older subjects such as adults, 18 to 80 years of age. The subject may be a pregnant subject or a female subject of child-bearing age, either of which may optionally be at increased risk of developing a condition characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or activity’ (e.g., an autoimmune disease, asthma, and the like). These latter embodiments are premised, at least in part, on the surprising finding that it was possible to impart resistance to offspring by administering VPS72 inhibitor to their mother during pregnancy. This finding, among others, suggested that iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) iNKT cells, MAIT cells, T-regulatory' T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers may be set early7 in life, thereby dictating whether a person is more likely or less likely to develop conditions characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as "Tregs”). HSCs, LCs, macrophages and dendritic cells (DC)cell numbers and/or activity. [0193] The invention further contemplates that subjects may be treated once, twice or more times, over a period of time. This period of time may be days, weeks, months, or years. As an example, the agents may be administered daily or weekly in a subject experiencing symptoms associated with a condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as "Tregs"). HSCs, LCs, macrophages and dendritic cells (DC) cell numbers or activity7, until such symptoms are reduced or eliminated. As another example, the agents may be administered one or more times in the early years of life of a subject and then may be administered again after several years, as a "boost" to the original administration. This latter administration schedule could be similar to that used in more traditional vaccination schemes.
[0194] Some embodiments of the invention involve treatment of subjects having asthma or treatment of subjects prior to the onset of asthma (e.g., children). A "subject having asthma" is a subject that has a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms. An "initiator" as used herein refers to a composition or environmental condition which triggers asthma. Initiators include, but are not limited to, allergens, cold temperatures, exercise, viral infections, and the like.
[0195] In another aspect, the invention provides a method comprising administering to a subject having or at risk of developing a condition characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers or activity any of the foregoing VPS 72 inhibitors, peptide or nucleic acids in an effective amount to decrease iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as "Tregs"). HSCs, LCs. macrophages and dendritic cells (DC) cell numbers or activity.
[0196] In some embodiments, the condition is an inflammatory condition. In some embodiments, the condition is asthma. In some embodiments, the condition is an autoimmune disease. In some embodiments, the condition is inflammatory bowel disease. In some embodiments, the condition is colitis (e.g., ulcerative colitis). In some embodiments, the condition is systemic lupus erythematosus (i.e., lupus). In some embodiments, the condition is multiple sclerosis. In some embodiments, the condition is arthritis.
[0197] In some embodiments, the VPS72 inhibitor is administered locally such as to the lungs or to the colon or gut. Local administration to the lungs may be carried out via nebulization. as an example. In some embodiments, the isolated VPS72 inhibitor is administered systemically.
[0198] In some embodiments, the subject is human. In some embodiments, the subject is less than 5 years of age, less than 1 year of age, less than 6 months of age, or less than 1 month of age. In some embodiments, the subject is a pregnant subject and optionally is at high risk of developing a condition characterized by increased iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or activity. In some embodiments, the subject is a female subject of child-bearing age (e.g., in humans, approximately 15-55 years of age), and optionally is at increased (i.e.. above-normal) risk of developing a condition characterized by increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers or activity.
[0199] In some embodiments, the subject is administered a second active agent such as an immunosuppressant or an anti-inflammatory agent.
[0200] In some embodiments, the method further comprises identifying a subject having or at risk (including increased risk) of developing the condition.
[0201] As used herein, an immunoinhibitory molecule or preparation intends that the molecule or preparation is able to inhibit or reduce iNKT cells, MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell activity. It is therefore to be understood that the immunoinhibitory molecules and preparations of the invention are immunoinhibitory in the context of activated iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cells. For example, the molecules and preparations are able to reduce the number and activity of iNKT cells. MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs. macrophages and dendritic cells (DC) cells, including activated iNKT cells, MAIT cells, T- regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC), and/or are able to prevent the activation of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”). HSCs, LCs, macrophages and dendritic cells (DC) in the presence of an agent that stimulates iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) such as an VPS72 inhibitor such as for example a peptide having at least 95% sequence identity to a peptide of SEQ ID NO:2. Thus, in some instances, the immunoinhibilory VPS72 inhibitor of the invention are able to compete and/or interfere with the expression and/or activity of VPS72, such as an inhibitor peptide having at least 95% sequence identity7 to a peptide of SEQ ID NO: 2, thereby preventing or reducing the degree of immunostimulation that would otherwise occur in the absence of an inhibitor peptide having at least 95% sequence identity to a peptide of SEQ ID NO:2 or a VPS72 expression inhibitor, for example, a nucleic acid that inhibits the transcription, or translation ofVPS72. Where iNKT cells, MAIT cells, T-regulatory T- cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and activity levels are normal (e.g., the levels in a subject that does not have an inflammatory or autoimmune diseases, conditions or symptoms related thereto and/or is not at elevated risk of developing an inflammatory or autoimmune diseases, conditions or symptoms related thereto (as a result of heredity', for example)), then the immunoinhibilory molecules and preparations may manifest no immunoinhibitory effect essentially because there is no observable background iNKT and/or MAITcell based immune stimulation. In some instances, however, they may manifest no immunoinhibitory effect in the short term but may function to prevent immunostimulatory in the long term by rendering a subject or the offspring of a subject, such as an infant or a child, resistant to future aberrant iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) based immunostimulation.
[0202] Assays for measuring iNKT cells, MAIT cells. T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) numbers are known in the art. Assays for measuring iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as “Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) activity are also known in the art and are described in the Examples herein. These assays include cytokine production assays such as IFN-gamma, IL-13 and IL- 1 -beta production assays. In some instances, a reduction or inhibition of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as "Tregs”). HSCs, LCs, macrophages and dendritic cells (DC) cell numbers and/or iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as ‘'Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) cell activity is measured by symptoms that result from increased numbers of iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as '‘Tregs”), HSCs. LCs, macrophages and dendritic cells (DC) and/or increased iNKT cells, MAIT cells, T-regulatory T-cells (herein referred to as ‘'Tregs”), HSCs, LCs, macrophages and dendritic cells (DC) activity. Such symptoms include the symptoms associated with inflammatory conditions and autoimmune diseases and conditions. An exemplary' but not limiting inflammatory condition is asthma. An exemplary but not limiting autoimmune disease is colitis.
[0203] Combination Therapy
[0204] An effective amount can be achieved in the method or pharmaceutical composition of the VPS72 inhibitor or a pharmaceutically acceptable salt or solvate (e.g., hydrate) thereof alone or in combination with an additional suitable anti-inflammatory or immunosuppressing therapeutic agent, for example, a chemotherapeutic or immunotherapeutic. When ‘'combination therapy” is employed, an effective amount can be achieved using a first amount of the polymer, or a pharmaceutically acceptable salt or solvate (e.g., hydrate) thereof, and a second amount of an additional suitable therapeutic agent.
[0205] In other embodiments, the polymer and the additional therapeutic agent, are each administered in an effective amount (i.e., each in an amount which would be therapeutically effective if administered alone). In another embodiment, the polymer and the additional therapeutic agent are each administered in an amount which alone does not provide a therapeutic effect (a sub-therapeutic dose). In yet another embodiment, the polymer can be administered in an effective amount, while the additional therapeutic agent is administered in a sub-therapeutic dose. In still another embodiment, the polymer can be administered in a sub-therapeutic dose, while the additional therapeutic agent, for example, a suitable immunotherapeutic agent is administered in an effective amount.
[0206] As used herein, the terms “in combination” or ''co-administration” can be used interchangeably to refer to the use of more than one therapy7 (e.g., one or more prophylactic and/or therapeutic agents). The use of the terms does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject.
[0207] Co-administration encompasses administration of the first and second amounts administered in an essentially simultaneous manner, such as in a single pharmaceutical composition, for example, capsule or tablet having a fixed ratio of first and second amounts, or in multiple, separate capsules or tablets for each. In addition, such co-administration also encompasses use of each composition in a sequential manner in either order.
[0208] The invention contemplates the administration of one or more VPS72 inhibitors with one or more additional active agents. The additional active agents include but are not limited to immunosuppressants or anti-inflammatory agents, asthma medicaments, allergy medicaments, and the like. The additional active agents may be blocking antibodies although they are not so limited.
[0209] Immunosuppressants or anti-inflammatory agents are agents that suppress or reduce an immune response. General classes of anti-inflammatories include steroids, nonsteroid anti-inflammatory drugs (NSAIDS), as well as various classes listed herein.
[0210] Non-limiting examples of immunosuppressants or anti-inflammatory agents include without limitation Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride: Bromelains; Broperamole: Budesonide: Carprofen;
Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate: Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;
Desoximetasone: Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; FluoromethoIone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;
Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;
Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol;
Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin;
Glucocorticoids; Zomepirac Sodium.
[0211] The additional active agent may be an asthma medicament, meaning a medicament that reduces the symptoms, inhibits the asthmatic reaction, or prevents the development of an asthmatic reaction. Various ty pes of medicaments for the treatment of asthma are described in the Guidelines for The Diagnosis and Management of Asthma. Expert Panel Report 2. NIH Publication No. 97/4051. Jul. 19. 1997, the entire contents of which are incorporated herein by reference. Asthma medicaments include, but are not limited, PDE-4 inhibitors, Bronchodilator/beta-2 agonists, K+ channel openers, VLA-4 antagonists, Neurokin antagonists, TXA2 synthesis inhibitors, Xanthanines, Arachidonic acid antagonists, 5 lipoxygenase inhibitors. Thromboxin A2 receptor antagonists, Thromboxane A2 antagonists, Inhibitor of 5-lipox activation proteins, and Protease inhibitors.
[0212] Bronchodilator/beta-2 agonists are a class of compounds which cause bronchodilation or smooth muscle relaxation. Bronchodilator/beta-2 agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerol methylxanthines and orciprenaline Long-acting beta-2 agonists include, but are not limited to, salmeterol and albuterol. These compounds are usually used in combination with corticosteroids. Methylxanthines, including for instance theophylline, have been used for long-term control and prevention of symptoms. Short-acting beta-2 agonists include, but are not limited to, albuterol, bitolterol, pirbuterol, and terbutaline.
[0213] Allergy medicaments include, but are not limited to, anti-histamines, steroids, and prostaglandin inducers. Anti-histamines include, but are not limited to, loratidine, cetirizine, buclizine, ceterizine analogues, fexofenadine, terfenadine, desloratadine, norastemizole, epinastine, ebastine, ebastine, astemizole, levocabastine, azelastine, tranilast, terfenadine, mizolastine, betatastine, CS 560, and HSR 609. Prostaglandin inducers include, but are not limited to, S-5751. The steroids include, but are not limited to, beclomethasone, fluticasone, tramcinolone, budesonide, corticosteroids and budesonide.
[0214] Corticosteroids include, but are not limited to, beclomethasome dipropionate, budesonide, flunisolide, fluticaosone, propionate, and triamcinoone acetonide. Systemic corticosteroids include, but are not limited to, methylprednisolone, prednisolone and prednisone.
[0215] Immunomodulators include, but are not limited to, the group consisting of anti-inflammatory agents, leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, immunosuppressants (such as Tolerizing peptide vaccine), anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists, CCRS antagonists, VLA-4 inhibitors, and, and Downregulators of IgE.
[0216] Leukotriene modifiers include, but are not limited to, zafirlukast tablets and zileuton tablets. Zileuton tablets function as 5 -lipoxygenase inhibitors.
[0217] Other immunomodulators include neuropeptides such as substance P that have been shown to have immunomodulating properties. Substance P is a neuropeptide first identified in 1931 by Von Euler and Gaddum and see Chang et al. 1971. Nature (London) New Biol. 232:86-87 (1971).
[0218] Another class of compounds useful as secondary active agents includes the down-regulators of IgE. These compounds include peptides or other molecules with the ability to bind to the IgE receptor and thereby prevent binding of antigen-specific IgE. Another type of downregulator of IgE is a monoclonal antibody directed against the IgE receptor-binding region of the human IgE molecule. Thus, one type of downregulator of IgE is an anti-IgE antibody or antibody fragment. Anti-IgE is being developed by Genentech. One of skill in the art could prepare functionally active antibody fragments of binding peptides which have the same function. Other types of IgE downregulators are polypeptides capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound.
[0219] These types of asthma medicaments are sometimes classified as long-term control medications or quick-relief medications. Long-term control medications include compounds such as corticosteroids (also referred to as glucocorticoids), methylprednisolone, prednisolone, prednisone, chromolyn sodium, nedocromil, long-acting beta2-agonists, methylxanthines, and leukotriene modifiers. Quick relief medications are useful for providing quick relief of symptoms arising from allergic or asthmatic responses. Quick relief medications include short-acting beta2 agonists, anticholinergics and systemic corticosteroids. Anticholinergics include, but are not limited to, ipratrapoium bromide.
[0220] When two or more agents (e.g. a VPS72 inhibitor and a secondary antiinflammatory or immunosuppression agent) are administered to a subject, these can be administered simultaneously (e.g., where they are pre-mixed and administered together), substantially simultaneously (e.g., where they are administered one after another in the time it would take a medical practitioner to administer two agents to a subject), or sequentially with a period of time lapsing between the administrations. The two or more agents can also be administered by the same route or by a different route. For example, the agents may be all administered by inhalation. As another example, one agent may be administered by injection and another may be administered by inhalation.
[0221] When co-administration involves the separate administration of the first amount of the polymer and a second amount of an additional therapeutic agent, the polypeptides are administered sufficiently close in time to have the desired therapeutic effect. For example, the period of time between each administration which can result in the desired therapeutic effect, can range from minutes to hours and can be determined taking into account the properties of each composition such as potency, solubility, bioavailability. plasma half-life and kinetic profile. For example, a polymer and the second therapeutic agent can be administered in any order within about 24 hours of each other, within about 16 hours of each other, within about 8 hours of each other, within about 4 hours of each other, within about 1 hour of each other or within about 30 minutes of each other.
[0222] More, specifically, a first therapy (e.g., a prophylactic or therapeutic agent such as any one of the disclosed polymers) can be administered prior to (e.g.. 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks. 3 w eeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks. 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent such as an immunotherapy) to a subject.
[0223] It is understood that the method of co-administration of a first amount of the polymer composition and a second amount of an additional therapeutic agent can result in an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect that would result from separate administration of the first amount of the polymer composition and the second amount of the additional therapeutic agent.
[0224] As used herein, the term "synergistic" refers to a combination of the disclosed polymer composition and another therapy (e.g., a prophylactic or therapeutic agent), which is more effective than the additive effects of the therapies. A synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can permit the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies to a subject. The ability to utilize lower dosages of a therapy (e.g., a prophylactic or therapeutic agent) and/or to administer said therapy less frequently can reduce the toxicity associated with the administration of said therapy to a subject without reducing the efficacy of said therapy in the prevention, management or treatment of a disorder. In addition, a synergistic effect can result in improved efficacy of agents in the prevention, management or treatment of a disorder. Finally, a synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.
[0225] The presence of a synergistic effect can be determined using suitable methods for assessing drug interaction. Suitable methods include, for example, the Sigmoid-Emax equation (Holford, N.H.G. and Scheiner, L.B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H , Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied with experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. [0226] Kits
[0227] The invention also encompasses a packaged and labeled pharmaceutical product. This article of manufacture or kit includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or plastic ampoule or other container that is hermetically sealed. Preferably, the article of manufacture or kit further comprises instructions on how to use including how to administer the pharmaceutical product. The instructions may further contain informational material that advises a medical practitioner, technician or subject on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instructions indicating or suggesting a dosing regimen for use including but not limited to actual doses, monitoring procedures, and other monitoring information.
[0228] In some embodiments, the unit dosage form comprising an inhibitor of VPS72 should be suitable for pulmonary delivery for example by aerosol.
[0229] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. [0230] The kits may include agents in sterile aqueous suspensions that may be used directly or may be diluted with normal saline for intravenous injection or use in a nebulizer, or dilution or combination with surfactant for intratracheal administration. The kits may therefore also contain the diluent solution or agent, such as saline or surfactant. The kit may also include a pulmonary delivery device such as a nebulizer or disposable components therefore such as the mouthpiece, nosepiece, or mask.
EXAMPLES
[0231] The Examples in this specification are not intended to. and should not be used to, limit the invention; they are provided only to illustrate the invention.
[0232] Introduction
[0233] VPS72 and chromatin remodeling The vacuolar protein sorting-associated protein 72 homolog (VPS72, also known as YL1) was initially identified in 1995 as a nuclear protein with DNA-binding ability in NIH3T3 cells 1. VPS72 localizes in nuclear specks and in the nucleoplasm. Available gene expression data (GeneCard) show7 that VPS72 is ubiquitously expressed in most organs, including the immune system. However, VPS72 is highly expressed in hematopoietic stem cells (HSC), suggesting that VPS72 may play a role in maintaining stem cell activity. Although few studies of VPS72 have been done in the past 27 years (<15 papers in PubMed), recent studies indicate that VPS72 functions as a histone chaperone for H2A to H2A.Z exchange in chromatin remodeling and belongs to two multisubunit chromatin-remodeling complexes: (1) the Snf2 -related CBP-activator protein chromatin remodeling (SRCAP) complex; and (2) the TRRAP/TIP60 complex.6 VPS72- mediated H2A.Z exchange is required for nuclear reassembly after mitosis in HeLa cells, and VPS72 interact with H2A.Z with the help of Znhitl determine Lgr5+ stem cell fate. Interestingly, VPS72 is also reported to regulate the acetylation of non-histone proteins, including autophagy gene ATG8a in Drosophila. Key findings and milestones ofVPS72 research are summarized (Fig 1).
[0234] Chromatin remodeling is an important layer of epigenetic regulation and can play a key role in immune cell development, homeostasis, and function. Nucleosomes are the basic unit of chromatin. The histone octamer packages DNA into nucleosomes, maintains the nucleosome morphology, and serves as a regulatory layer for gene expression. This octamer consists of histone proteins such as H2A, H2B, H3, and H4 (Fig 1). Among histones, the most studied is H2A, which comprises an H2A variant known as histone H2A.Z15. H2A.Z has two different isoforms, H2A.Z1 and H2A.Z2, which differ only by 3 amino acids and are encoded by two separate genes, H2AFZ and H2AFV, respectively. Interestingly, a recent study showed that the deletion of both isoforms is essential for decreased expression of Pten- mediated transcriptional control of HSC quiescence. Moreover, H2A.Z has been linked to diverse biological processes such as memory', epithelial-to-mesenchymal transition, microglial development, and neuronal survival through promoting nuclear-encoded mitochondrial gene expression and organelle function; also, overexpression of H2A.Z is associated with a greater proliferative capacity in multiple cancers such as metastatic melanoma, colorectal, liver, and lung. Exchanging H2A for variant histone H2A.Z through the VPS72/SRCAP/TIP60 complex has been shown to modulate chromatin structure to activate or repress target genes, modify the local chromatin structure, and regulate cellular processes and mitochondrial function, which are required for cell cycle progression, autophagy regulation, metabolic processes, especially in HSC and embryonic stem cell development. However, the functions of VPS72-mediated H2A.Z exchange in immune cells remain totally unknown. We generated T cell specific VPS72cKO and H2A.Zl/Z2cKO mice and found that VPS72 and H2A.Z are specifically required for thymic iNKT cell development, but not for conventional T cells.
[0235] H2A.Z and human diseases
[0236] H2AZ histone variant plays major roles in the control of gene expression, its acetylation and ubiquitination to positive and negative regulation of gene expression, respectively. The deposition of H2A.Z into chromatin genome-wide has been shown to be associated with SRCAP, and mediated by VPS72. H2A.Z and SRCAP complex have been linked to several disease, most notably cancer. Many reports have suggested that upregulation of H2AZ in metastatic melanoma, breast, prostate, colorectal, liver, bladder, and lung cancer. [0237] In uterine leiomyoma, the inactivating mutations in SRCAP complex genes and a consequent H2A.Z loading defect w ere considered as a potential driver of neoplasia. The defective of H2A.Z deposition could initiate tumorigenic differentiation program including overexpression of CBX2/4/8 and developmental transcription factors such as SATB2 and HOXA13. In prostate cancer, H2AZ function as a pro-oncogenic regulator. It promotes activation of oncogenes and repression of tumor suppressor genes. In breast cancer, it has been reported that H2A.Z strongly impedes estrogen-dependent growth of breast cancer cell as well as affect ER-target gene expression. In metastatic melanoma, H2A.Z controls the transcriptional output of E2F target gene in melanoma cells and H2A.Z deficiency sensitize melanoma cells to chemotherapy and targeted therapies [0238] Methods
[0239] Animal All mice were housed under pathogen free conditions, allowed ad libitum food and water and maintained on standard conditions (25°C, 14: 10-h light- dark cycle). All experimental procedures were performed according to institutional guidelines for animal care and use committee of Henry Ford Health Sy stem, which conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. To generate VPS72 conditional knockout mice, the LlL2_Bact_P cassette was inserted at position 95117966 of Chromosome 3 upstream of the critical exons (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance gene under the control of the human beta-actin promoter, SV40 poly A, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon(s) at position 95119493. The critical exon(s) is/arethus flanked by loxP sites. A "conditional ready" (VPS72fl/fl) allele was created by flp recombinase expression in mice carrying this allele. Mice carry ing a floxed allele of VPS72 (VPS72fl/fl) were crossed to CD4cre mice and Foxp3YFPcre, to generate CD4cre+VPS72fl/fl, Foxp3YI Pcre VPS7211 11 conditional T cell specific and Treg specific knockout mice, respectively. Additionally, VPS72 (VPS72ftfl) mice were crossed to Csflricre and CD1 lccre mice to generate myeloid lineage specific Csflricre+VPS72fl'fl and dendritic cell lineage specific CD1 1 crcrc VPS7211 11 knockout mice, respectively. We also used the timely inducible deletion with the Cre-ERT2 model to study Tregs. By crossing FoxP3eGFP-Cre-ERT2 mice to VPS72flfl mice to allow for specific deletion of VPS72 in Treg cells (FoxP3eGFP-Cre-ERT2VPS72fl fl) following tamoxifen treatment. For inducible deletion, 6-8 weeks of control and knockout mice were treated w ith tamoxifen i.p. route for five successive days and then allowed to rest for 9. To study role of VPS72 in hematopoietic stem cell, VPS72fl/fl mice were bred with MxlCre mouse. Mxlcre+vps72fl/fl mice were treated with dsRNA poly (I: C) to induce deletion of VPS72 throughout all hematopoietic lineages and hematopoietic stem cells. Generally, myeloid lineage cell expresses Csflrcre in all organs at early embry onic stages (>E9.5d) and dendritic cell express CD1 lccre in lung as well as epidermis after birth. Based on this, Csflrlcre VPS72fl/fl knockout mice were utilized for assessing TRMs and CD45 cells in all organs from embryonic to adult stage, whereas
[0240] CD1 lccreVPS72fl/fl KO mice were used for assessing CD45 along with TRM in lung and epidermis (Fig. 17-19). For embryonic experiment, both male and female mice were kept for timed matting. The precise age of each offspring litters obtained from timed mattings were determined by regular monitoring of vaginal plug around 6: 00 AM-8: 00AM morning. The moment of observed plug was considered 0.5 embryonic (E0.5) day. After birth, all mouse colony of respective genotypes were maintained and utilized. [0241] Genotyping PCR genotyping for single and double mutant genes for Csflricre, CD1 Iccre and VPS72fl/fl. mice were performed using the following PCR primer pair.
Figure imgf000065_0001
[0242] Isolation of single cell suspension and flow cytometry
[0243] Spleen, thymus and lymph nodes were mechanically disrupted between ground glass slides and washed with wash buffer (1XPBS, 2% FBS and ImM EDTA). Bone marrow cells were isolated from all four hing-leg bones ((femurs and tibiae) and 2 fore-leg bones (humeri) and collected in PBS. The single cell suspension was then washed and filtered to remove debris. Erythrocytes were lysed, washed with wash buffer then resuspended with staining buffer (IxPBS and 2% FBS). Single cell suspensions were blocked with purified anti F-cyRII/III antibody (clone 2.4G2) for 15 min at 4C and incubated with different fluorescence-labeled surface antibodies. For the analysis of transcription factors, intracellular staining was performed using a Biolegend intracellular staining kit. Briefly, cells were fixed, washed with permeabilization buffer and stained with intracellular antibodies for 30 min on ice. Then cells were washed two times with permeabilization w ash buffer and cells were collected in PBS. Flow cytometry was performed with BD FACSCelesta (BD Biosciences) and data were analyzed using Flow Jo (10.0.7, Tree Star, BD Biosciences). The cytokine production were measured following with PMA and lonomycin (P/I) for 4 hours and analysed with flow' cytometry. Apoptotic cells were identified by using Annexin V staining. Cells were first stained for surface markers for 30 min on ice follow'ed by w'ashing. Then cell pellet was resuspended in Ixbinding buffer (eBioscience) then stained with Annexin V for 12 min at room temperature. Cells were washed with binding buffer and immediately analyzed by flow cytometry. For cell proliferation, cells were stained with intracellular ki67 and analyzed.
[0244] Isolation of TRM cells from embry o and adult tissue
[0245] Isolation of embryo ranging from E14.5 to El 7.5d were removed from the uterine horns of pregnant female mice under CO2 exposure. All mouse embry os were decapitated and washed using cold phosphate-buffered saline (PBS), (Invitrogen, Carlsbad, CA). All organs including yolk sac, fetal liver, brain, lungs, liver, eye, pancreas, kidney, heart and skin (E14.5- E17.5) were collected optimally with age of embryo, then cut into fine pieces and transferred in a tube containing tissue digestion buffer [Img/ml collagenase D (Roche, Basel, Switzerland), 0.01% DNase I (Worthington Biochemical Corp., Lakewood, NJ) and 3% fetal bovine serum (FBS, Hy clone, San Angelo, TX) in PBS], Following this, the tubes were shaken for collagen break down using an orbital shaker for 30 mins at 37°C and dissociated into a form of cellular suspension. Likewise, cells from adult tissue organ were also isolated in similar fashion as embryonic tissues. For illustration, adult lung tissues were first flushed, harvested, minced, incubated in tissue digestion buffer and were shaken on orbital shaker for 45mins at 37°C, whereas the liver, brain, kidney, and heart tissues were digested for 30 mins followed by density’ gradient centrifugation using 33% percoll (GE Healthcare. MA, USA) prepared in RPMI media (Sigma- Aldrich, St. Louis, USA) at 2500 rpm in room temperature for 25 mins. Moreover, the skin tissue before E17.5 were processed as other embryonic tissue. Nevertheless, the skin after E17.5 to adult were collected, scrapped out gently for all connective tissue, then incubated completely in flattened and floated way by keeping dermal side down in petri dishes filled with 0.5% dispase II buffer (Thenno Fisher Scientific, Waltham, MA) for 60 min at 37°C. Following incubation, epidermal sheets were cautiously separated from dermis, then cut into tiny pieces in the petri dish, then dissociated epidermal single cells in the digestion buffer [0.25% trypsin and 0.01% Dnase I (Worthington)] made in PBS using Precision™ shaking water baths (ThermoFisher Scientific, MA, USA) with 50 rpm for 10 min at 37°C. Collected dermal layers separated from skin (E17.5- P0) were crumbled, incubated and shaken with tissue digestion buffer for 60 min at 37°C using orbital shaker. In adult mice, ears were collected for preparing dermal cell suspension. Thus, collected ear were first scrapped and then peeled off for epidermal layer from dermal layer to incubate into the dispase II buffer. Thereafter, dermis was collected by scrapping out epidermal layer, then processed comparably like other dermal tissues (El 7.5- PO). For preparing eye cell suspension in adult mice, retinal samples were collected, pipetted and were processed using Accumax (Sigma- Aldrich, St. Louis, USA) digestion for 10 min. Whenever necessary, lysis buffer (0.83% NH4C12 buffer) were used for 3 mins to remove erythrocytes from isolated embryo or adult cell suspension. Later, all cell suspensions from tissue including peritoneal lavage were filtered through a 70-micron followed 40-micron size cell strainer (BD Biosciences, San Jose, CA). During isolation, wash buffer (1% FBS and 0.5M EDTA in PBS) was used to stop the digestion. Rest filtration as well as for erythrocyte lysis procedure, followed by immediate centrifugation at 450 x g for 7 min at 4°C. After isolation of cell from both embroy as well as from adult mice, cell were blocked with purified anti-F-cyRII/III antibody (clone 2.4G2) for 15 min at 4C and incubated with different fluorescence-labeled surface antibodies. Flow cytometric carried out using BD FACSCelesta (BD Biosciences) and sorting procedure were carried out using BD FACSAria (BD Biosciences).
[0246] Histology
[0247] Different tissues were isolated from Treg-deficient VPS72 mice and fixed in 10% formalin overnight, preserved in 70% EtOH. Then, tissues were embedded in paraffin, cut and sections stained with hematoxylin and eosin (H&E).
[0248] Western blotting Total protein was isolated using radioimmunprecipitation assay (RIP A) buffer (Thermo Fisher Scientific, Waltham, MA) and freshly added complete protease/phosphatase inhibitor cocktail. Protein concentration was measured by Bradford protein assay kit (Bio-Rad, Hecules, California). Equal amount of protein were separated on 12% on sodium dodecyl sulfate polyacrylamide gel and elecro-transferred onto nitrocellulose membrane. The membrane is blocked with 5% bovine serum albumin for one hour and probed with primary anti -rabbit VPS 72 antibody (#PA5-36321. Thermo Fisher Scientific) for overnight at 4°C followed by washing with TBST buffer (Bio Rad). Then the membrane is incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (#AAI46P, Bio-Rad) followed by washing and visualized with an enhanced chemiluminescence detection system (GE Healthcare. NJ) using ChemiDocTM MP imaging system and associated software (Bio-Rad, Hercules, CA).
[0249] Real time RT-PCR
[0250] Total RNA was extracted from sorted cells using Gene Elute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, USA) following manufacturer's instruction. RNA concentration was measured by NanoDrop-2000 (Thermo Scientific, MA, USA). mRNA was reverse transcribed by reverse transcriptase using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was carried out using Universal SYBR Green Master mix (Roche) on QuantStudio 7 Flex Real- Time PCR system (Applied Biosystems). Beta-actin was used as internal housekeeping control gene. Expression of target genes was normalized against beta-actin expression levels. Then changes in gene expression were calculated using the AACt method.
[0251] Bone Marrow transfer experiment
[0252] To generate a bone marrow suspension for transfer, CD4cre+vPS72fl/fl and CD4cre-VPS72fl/fl (CD45.2) or SJL (CD45.1) donor animals were euthanized. Tibia and femurs were harvested and marrow was flushed from bone using PBS. After Red blood cells lysis, cell were counted and resuspended in PBS. Cells from CD45.2 (WT and KO separately) were mixed with the cells from CD45. 1 in 1: 1 ratio. Lethally irradiated (10G, Cs - 137 Irradiator) adult mice (SJL/B6 background containing CD45. 1/CD45.2) were anesthetized with xylazine/ketamine. Then bone marrow cells (10xl06/animal in lOOpl PBS) were injected retro-orbitally into recipient mice. Animals were maintained for 7-8 weeks and then sacrificed. Tissues were harvested and prepared for cellular isolation and stained. Cells were assessed for the expression of CD45. 1 (SJL derived leukocyte marker) and CD45.2 (WT or KO derived leukocyte marker).
[0253] RNA-seq
[0254] To determine the genome wide effect of Treg specific VPS72 deletion on gene expression, bulk RNA-Seq was perfonned in sorted splenic Tregs cells from Treg deficient VPS72 mice or WT mice. cDNA synthesis and preamplification were performed to achieve optimal library7 quality' using SMART-seq v4 Ultra Low Input RNA Kit (Clontech, Mountain View, CA) following the manufacturer’s instruction. Briefly, pre-amplification of cDNA was done using a thermal cycler (Applied Biosystems) for 12-14 cycles and fragmented into 150- 400bp using a Bioruptor Pico Sonicator (Diagenode, Denville, NJ). The DNA ends were repaired using End-ItTM DNA End-Repair Kit (Epicentre, Charlotte, SC). The end-repaired DNA fragments were ligated to adaptors by T4 ligase (New England Biolabs, Ipswich, MA) after a single “A” was added at 3” by Klenow Fragment (3’— >5’ Exo-). MinElute Reaction Cleanup Kit (Qiagen, Gaithersburg, MD) was used for DNA cleanup after each step. Illumina P5 and P7 primers with indicated adaptors were used for final library amplification. The 150- 400bp final product was purified on a 2% E-gel (Thermo Fisher Scientific). Sequencing was performed by the DNA sequencing core Facility at University of Michigan using a 50bp single end read setup on the Illumina Hiseq 4000 platform.
[0255] Statistical Analysis [0256] Statistical analysis was performed with GraphPad Prism7 software (GraphPad, La Jolla, CA). All data are shown as mean± SEM obtained from at least two independent experiments. Statistical significances were displayed as * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.
[0257] VPS72 function as a chaperone and mediates H2A.Z deposition. Our study showed VPS72 plays critical role in multiple immune cell development, and we propose that VPS72 involved in H2A.Z deposition can be useful therapeutic target to reduce immune activity in subjects with autoimmune and inflammatory diseases and conditions.
[0258] VPS72 regulates iNKT cells
[0259] iNKT cells arise in the thymus from the CD4+CD8+ double-positive (DP) stage and are selected by CD Id-expressing DP thymocytes. In contrast to the selection of conventional T cells by thymic epithelial cells, iNKT cell selection requires stronger TCR signal strength. After iNKT cells (CD24+CD441oNKl. l-) are selected (stage 0, STO), subsequent events elicit proliferation and a progression of maturation from immature CD24- CD441oNKl. l -(stage 1, STI) to semi-mature CD24-CD44hiNKl .1 -(stage 2, ST2) to mature CD44hiNKl. l+(stage 3, ST3) iNKT cells. ST2 iNKT cells possess higher proliferation potential, and most iNKT cells at this stage emigrate to peripheral organs. Recent studies suggest that thymic iNKT cells terminally differentiate into at least three distinct lineages of iNKT effector cells (iNKTl, iNKT2, and iNKT 17) that phenotypical ly differ, having distinct transcription factor expression and cytokine production: iNKTl cells are PLZFloCD44hiNKl.l+ (i.e., ST3), express T-bet, and mainly produce IFN-y; iNKT2 cells are PLZFhiCD44hiNKl.l- (i.e., ST2), express GATA3, and produce abundant IL-4; and iNKTl 7 cells resemble iNKT2 cells in being PLZFintCD44hiNKl. l- but express RORyt and produce IL-17. Recent studies identified that some transcription factors regulate specific stages of iNKT cell development and that distinct mitochondrial metabolic programs and autophagy are specifically required for iNKT cell differentiation and function. However, how these transcriptional factors and mitochondrial metabolic pathways are integrated with chromatin remodeling programs that promote iNKT positive selection, lineage specification, acquisition of functional activity, and homeostasis remains poorly understood.
[0260] During immune cell development, epigenetic programming is required to ensure the establishment of proper chromatin organization for regulating lineage-specific gene expression. The epigenetic regulation of iNKT cell development through the miRNA pathway has long been recognized, and we were the first to uncover that Dicer-deficient mice (miRNA deficiency) completely lack iNKT cells. Our studies and others have shown roles for multiple miRNAs, including miR-183-96-182, Let-7, and the miR-150, miR-155, miR-181, miR-17-92 family cluster in regulating iNKT cell development. The role of histone post- transcriptional modifications in iNKT regulation, such as the addition or removal of methyl and acet l groups, has also been illuminated. We and others have identified that histone demethylases (UTX and JMJD3) are required for iNKT cell development and that histone deacetylase HDAC3 regulates iNKT cell development and differentiation by targeting autophagy. However, the role of nucleosome dynamics in the form of histone H2A-H2A.Z exchange in T cells, especially in iNKT cells, remains completely unknown. Using T cell specific VPS72 and H2A.Z deletion mouse models (VPS72cKO and H2A.ZcK0, respectively), we have found that loss of VPS72 severely impairs thymic iNKT cell early selection and development, without interrupting thymic conventional T cells. Furthermore, loss of VPS72 interrupted iNKT cell survival and autophagy and dysregulated global chromatin accessibility. As expected, mice lacking H2A.Z resemble VPS72cKO mice in having defective thymic iNKT cells.
[0261] VPS72 in iNKT cells
[0262] VPS72 is highly expressed in thymic iNKT cells in adult mice. VPS72 mRNA expression is slightly higher in iNKT cells, as compared with conventional CD4, CD8, DP T cells, (www. immgen.org) (Fig. 2A, C57B/6 mice at 6 weeks old). Our TaqMan RT-PCR data showed that VPS72 significant highly expressed in thymic iNKT cells as compared to CD4, CD8, and DP T cells from B6 mice at 8-12 weeks old (Fig. 2B). Our flow cytometry data further indicated that VPS72 protein was expressed at a low level on DP thymocytes, but was significantly upregulated upon selection, especially in iNKT cells (Fig. 2C), suggesting that VPS72 might be involved in iNKT fate determination.
[0263] Generating VPS72fl/flCd4Cre conditional KO mutant mice.
[0264] To investigate the functions of VPS72 in iNKT cell development and homeostasis, we generated VPS721oxp mice by crossing VPS72 targeted allele mice (MGI: 5008033) with Flp recombinase expression (Jax 016226) mice. VPS721oxp mice were then crossed with CD4Cre (Jax 004194) to generate Vps72fl/flCd4Cre conditional KO (VPS72cKO) mice (Fig.2D). VPS72 contains 6 exons, and exons 2-4 are flanked by loxP sites. Expression of VPS72 mRNA in thymic T cells, including iNKT cells, measured by RT- PCR (Fig.2E) and protein in DP thymocytes measured by Western blot (Fig.2F) were dramatically reduced in VPS72cKO mice.
[0265] VPS72 is critical for thymic iNKT cell development, but not for thymic conventional T cells. [0266] We found that frequencies and cell numbers of conventional CD4, CD8, DP, and DN T cells in thymus were comparable between WT and VPS72 cKO littermates (Fig. 3A,C). To our surprise, analysis of iNKT cells showed a severe impairment of thymic iNKT cell development in KO mice compared to controls as assessed by TCR(3 and CDld-tetramer (Fig.3B,C). Thus, deletion of VPS72 impairs thymic iNKT cells but not thymic conventional T cells.
[0267] VPS72 deletion blocks iNKT cell maturation.
[0268] After positive selection, iNKT CD24+ precursors progress through CD441o and CD44hi stages and lastly acquire NK1.1 expression during their final maturation.44 Therefore, the CD24/CD44/NK1.1 profiles of CDld-tetramer+ thymocytes were analyzed to determine if iNKT cell development and maturation were defective in VPS72cKO mice. We found that the frequencies of immature thymic iNKT cells at ST 0-2 in VPS72cKO mice were relatively increased, but mature iNKT cells at ST3 were dramatically reduced. The absolute numbers at STO-2 in KO mice were comparable to control mice, but they were dramatically reduced in ST3 compared to WT controls (Fig.3D-E). iNKT cells at STI and ST2 have a great capability to migrate to peripheral organs. We next assessed the frequencies and numbers of iNKT cells in the spleen, liver, and lymph node, and found dramatic reductions of iNKT cells in all peripheral organs (Fig.3F-G). Taken together, lack of VPS72 impairs thymic iNKT cell early stage development and late stage maturation and subsequently reduces peripheral iNKT cells.
[0269] VPS72 regulates iNKT cell development in a cell-intrinsic manner.
[0270] iNKT cell development requires positive selection by DP thymocytes through
CDld and SLAM family receptors. Thus, thymic environmental changes in VPS72 cKO mice may impact iNKT cell development. To determine if VPS 72 regulates iNKT cell development in a cell-intrinsic manner, we generated bone marrow (BM) chimeras by reconstituting lethally irradiated CD45.1+ CD45.2+SJL/B6 recipients with a 1:1 mixture of CD45.1+SJL and CD45.2+ WT or VPS72 cKO BM. Analysis of mixed chimeras after 8 wks revealed that VPS72 cKO donor BM poorly reconstituted the iNKT cell compartment in thymus (Fig. 3H-I), suggesting that VPS72 acts as a cell-intrinsic factor in iNKT cell development.
[0271] Residual iNKT cells in VPS72cKO mice are hyperproliferative but undergo enhanced apoptosis.
[0272] Since iNKT cells have an intra-thymic proliferation wave before acquiring maturity we first tested if the defect in iNKT cells in VPS72cKO mice was due to impaired cell proliferation. Surprisingly, we found more Ki-67-labeled total iNKT cells and iNKT cells at both STI and ST2 from VPS72cKO mice (Fig. 4A), suggesting that loss of VPS72 may not impair iNKT cell proliferation. We next tested iNKT cell apoptosis and found that VPS72-deficient iNKT cells consistently exhibited a higher percentage of early apoptotic cells (Annexin V+ 7AAD-) and late apoptotic cells (Annexin V+ 7AAD+) (Fig.4B), suggesting augmented apoptosis in VPS72 cKO iNKT cells. As expected, apoptosis in thymic conventional CD8 and CD4 T cells was not altered (data not shown).
[0273] VPS72 regulates thymic iNKT cell differentiation. Unlike conventional T cells, thymic iNKT cells are differentiated to iNKTl, iNKT2, and iNKT17 cells byexpressing lineage-specific transcription factors. As shown in Fig.4C-E, the VPS72 deficiency resulted in a severe loss of the PLZFloT-bet+ iNKT l subset, with significantly increased frequency of PLZFhiT-bet- iNKT2 cells and moderately decreased PLZFint RORrt+ iNKT 17 cells in gated total iNKT cells. The absolute numbers of all three lineages were reduced. Given the iNKT cell maturation block in VPS72KO mice, to rule out the maturation basis, we further gated NK1. 1- (STI and ST2) iNKT cells and found that T- bet+NKTl. l- NKT1 precursors were dramatically reduced, while PLZFhiT-bet- iNKT2 cells were increased (Fig.4D). To further define the potential genes and pathways targeted by VPS72, we perfonned RNA-seq on FACS-sorted thymic iNKT cells from VPS72cKO and WT mice. Using known signatures for iNKTl, iNKT2, and iNKT17, we found that iNKTl and iNKTl 7 signatures were markedly- reduced in VPS72cKO mice; however, iNKT2 signature markers were relatively increased in VPS72cKO mice (Fig.4F). These results suggest that VPS72 regulates iNKT lineage differentiation, especially for iNKTl and iNKT2 cells.
[0274] VPS72 is required for well-developed iNKT cell IFN-y secretion, but not for conventional T cells.
[0275] To bypass early developmental defects and delineate the impact of VPS72 on iNKT cell function, we generated an inducible VPS 72 KO mouse model (VPS72fl/flUbcCre+, VPS72uKO) by breeding VPS721oxp mice with a tamoxifen inducible UbcCreER mouse (Jax 007001), where VPS72 was deleted in well-developed T cells, including iNKT cells. After tamoxifen treatment, we observed a marked reduction of VPS72 expression in splenic iNKT cells in VPS72uKO mice (Fig.5 A). Upon PMA and ionomycin (P/I) stimulation for 4h, iNKT cells from VPS72uKO mice showed a dramatic upregulation of activation marker CD69 (Fig.5B) and showed markedly increased apoptosis (Fig.5C). indicating the VPS72 deficient- iNKT cells are more sensitive to activation-induced cell death (AICD).. More interestingly, VPS72 deficient-iNKT cells, showed dramatically- reduced IFN-y production, but comparable IL-4 production compared to WT controls (Fig. 5D). However, those scenarios were not observed in CD4 and CD8 T cells (Fig. 5E).
[0276] VPS72 regulates iNKT cell autophagy.
[0277] Multiple cellular processes, including cell development, surv ival, and differentiation are mediated through autophagy, which removes unnecessary or dysfunctional cellular components. Defective autophagy can cause cellular apoptosis. By RNA-seq analysis, we found that autophagy assembly pathway-related genes were significantly reduced in VPS72cKO iNKT cells (Fig. 6A). It has been shown that defective autophagycauses accumulation of cellular organelles, ultimately leading to T cell apoptosis. Using organelle-specific dyes and flow cytometry, we observed increased labeling of Mitotracker DeepRed (mitochondrial mass) and Mitotracker Orange (mitochondria membrane potential) in total iNKT cells and subpopulations with VPS72 deletion (Fig. 6B), but not in conventional T cells (Data not shown), reflecting dysregulated autophagy in iNKT cells. Thus, VPS72 regulates iNKT cell autophagy-.
[0278] Defective rearrangement of Val4-Jal8 gene segments in thymic DP cells from VPS72cKO mice.
[0279] We did not find defective CD1 expression on DP cells from VPS72cKO mice. To explore whether the absence of VPS 72 results in defective presentation by DP thymocytes, we cocultured DP thymocytes from WT or VPS72cKO mice with the iNKT cell hybridoma cell line DN32.D3 in the presence of a-GalCer for 24 hours. IL-2 secreting iNKT cells measured by flow cytometry were comparable between WT and VPS72cKO mice (Fig. 7A), implying a normal glycolipid presentation by VPS72-deficient DP cellsThe rearrangement of Val4-Jal8 gene segments in thymic DP cells is necessary- for iNKT cell TCR-positive selection. This process is temporally regulated during ontogeny and requires adequate survival of the DP population, related to stronger TCR signaling and specific mitochondrial metabolism. We therefore tested Val4-Jal8 rearrangement by RT-PCR in DP thymocytes, sorted after exclusion of Tetramer+ iNKT cells. As showed in Fig. 7B, the rearrangement was significantly reduced in VPS 72 cKO DP thymocytes. We then performed RNA-Seq and found that the key components of TCR signaling, mitochondrial function, and autophagy- pathways were dramatically- downregulated in DP thymocytes from KO mice (Fig. 7C-D). These data suggest that the defect in iNKT cells from VPS72-deficient mice is likely due to the reduced lifespan of DP thymocytes, defective TCR signaling, and autophagy- related apoptosis, potentially impairing the Vo.14-Ja.18 rearrangement. [0280] H2A.Z is required for iNKT cell development.
[0281] Although the role of VPS72 interact with H2A.Z in SCARP complex has been elucidated in yeast and HeLa cells, it has not been defined in immune cells. We therefore performed Co-IP using two different H2A.Z antibodies in mouse iNKT cell line DN32.D3 against VPS72 antibody. As shown in Fig. 8A, we confirmed that VPS72 indeed interacts with H2A.Z in iNKT cells. To further confirm the functional specificity’ of VPS72 as a histone chaperone that recognizes and exchanges H2A.Z, we crossed
H2A.Zlfl/fl.H2A.Z2fl/fl with CD4Cre mice to generate CD4cre.H2A.Zl.H2A.Z2 double KO mice (H2A.ZdKO). As shown in Fig. 16B, we confirmed H2A.Z deletion in the thymocytes and splenic T cells by western blot (H2A.Z antibody recognizes both H2A.Z1 and Z2 isoforms). We found that loss of H2A.Z did not impact thymic conventional CD4 and CD8 cells, but almost entirely depleted thymic iNKT cells and totally blocked iNKT cell maturation (Fig. 8D-E), which is phenotypically similar to VPS72cKO mice. Furthermore, H2A.Z-deficient iNKT cells had increased Mito-Red and Mito-Orange labeling (Fig. 8E), reflecting dysregulated autophagy, which was found in VPS72cKO mice. See FIG. 81 for binding motifs (see SEQ ID 10 for STAT3; FOXO3 (TGTAAACA); SEQ ID 1 1 for NRF1; SEQ ID 12 for NUR77).
[0282] Mapping of H2A.Z binding sitNes across the genome in iNKT cells.
[0283] Next, we used CUT&RUN (cleavage under targets and release using nuclease) sequencing analysis to identify genes that are directly bound by H2A.Z in thymic iNKT cells from B6.Val4-Jal8 transgenic mice (also called rec-Val4Tg), which closely mimic the endogenous TCR locus but have abundant iNKT cells (a gift from Dr. Derek Sant' Angelo). In line with previous studies, most H2A.Z binding sites (50%) were found at gene promoters flanking the transcription start site (TSS) and promoter-TSS regions (Fig.8F). These peaks mapped back to 2626 genes from gene ontology (GO) analysis. Consistently, H2A.Z binding peaks were highly enriched in the GO terms of mitochondrial metabolism (transport, organization, protein complex, outer membrane, and nuclear envelope), transcription coactivator, translation regulator, and DNA-binding transcription factor (Fig.8G). KEGG pathway analysis also indicates that the enriched peaks were found mostly in autophagy /mitophagy and TCR signaling pathway gene regions (Fig.8H). Interestingly, we observed significant enrichment of H2A.Z binding in iNKT cells within motifs for transcription factors in mitochondrial function, such as FOXO3, STAT3, and NRF-1, and in TCR signaling, such as NUR77 (Fig.81). These data indicate that H2A.Z likely regulates mitochondrial metabolism and TCR signaling pathways in iNKT cell development. [0284] iNKT cells function in tumor
[0285] The tumor microenvironment is created by tumor cells as well as the infiltrating immune cells. iNKT cells have the ability to activate and steer other immune cells via rapidly cytokine secretion. iNKT cell are postulated to strongly contribute to the tumor immunosurveillance. And more importantly, iNKT cells have been explored as targets in cancer therapy. Their protection against cancer has been found mainly dependent on production of Thl cytokines, especially IFN-r, and their lytic activity, which potentially directly lyse tumors that express CDld. Indeed, a-GalCer was observed to have potent antitumor activity7. Dendritic cell pulsed with a-GalCer were also found to be therapeutic against established liver metastases of B16 melanoma. In our study, we observed in UBC Cre- VPS72 KO mouse model, IFN-r production of iNKT cells were dramatically reduced, which lead to a hypothesis that VPS72 may contribute the protection of tumor immunity mediated by iNKT cells.
[0286] VPS72 function in MAIT Cell
[0287] Although MAIT cells were identified alongside iNKT cells about 25 years ago, much less is know n about MAIT cell development compared to iNKT cells. MAIT cells are positively selected by MR1 -expressing DP cortical thymocytes, and they recognize microbial riboflavin-derivative antigens restricted by the MHC class I-like protein MR1. They can acquire innate-like effector function in the thymus and secrete cytokines upon activation, comparable to iNKT cells, and they play a critical role in various inflammatory diseases, including ulcerative colitis, autoimmune disease, and several cancers. While their frequency is relatively constant in the thymus, MAIT cells undergo a large population expansion in the periphery. The developmental pathway of MAIT cells is defined by stages of CD24 and CD44 expression. The least mature cells, termed stage 1-CD24+CD44-. give rise to stage 2 CD24-CD44- and then stage 3 CD24-CD44+ 44 cells. This is remarkably similar to the linear differentiation model of iNKT cell development. Moreover, miRNAs are required beyond stage 1 for MAIT cell development, while PLZF is necessary7 for MAIT cell maturation from stage 2 to stage 3 Stage 3. MAIT cells in mice comprise two subsets: T- bet+RORyt- (MAIT1) and RORyt+T-bet- cells (MAIT17), which produce IFN-y and IL-17, respectively. Recent data even show that T-bet+RORyt+ MAIT2 cells also exist in stage 2. Several studies have shown that there are a few7 factors that regulate the differentiation steps, such as LEF1, SATB1. TCF1 (Tcf7), BACH2, ZAP70, SAP, PLZF, and cytokines including IL-7 and IL-15. Recent studies showed that epigenetic factors also in controlling MAIT cells and we reported that miR-155 regulates thymic MAIT1 and MAIT17 development. However, a gap still remains in our understanding of how epigenetic regulators control MAIT cell development and function. Using CD4cre.VPS72 mice, we have found that loss of VPS72 in the T cell lineage dramatically reduces thymic MAIT cells and dysregulates MAIT1 and MAIT 17 differentiation.
[0288] VPS72 in MAIT cells
[0289] VPS72 regulates MAIT cell development and maturation. MAIT cells share some characteristics with iNKT cells. Surface staining of TCR0 and 5-OP-RU-MR1 tetramers showed a significant reduction in the frequency and absolute number of thymic and peripheral (splenic, lymph node, liver, lung, and skin) MAIT cells in VPS72cKO mice (Fig. 9A-B). Furthermore, the frequencies of stage 1 (CD24+CD44-) MAIT cells were significantly increased; however, the frequencies of stage 3 (CD24-CD44+) MAIT cells were significantly reduced in VPS72 deficient mice, (Fig. 9C-D). In addition, as in iNKT cells, VPS72 regulates MAIT cell development in a cell-intrinsic manner (data not shown).
[0290] VPS72 regulates MAIT1 and MAIT17 cell differentiation.
[0291] Thymic MAIT cells complete their functional differentiation in the thymus. We found that the frequency and absolute number of T-bet+MAITl cells and RORyt+ MAIT17 cells were reduced in VPS72 deficient thymus, liver, and spleen compared to WT (Fig 9E). Additionally. CD4CD8 double negative (DN) MAIT cells population were almost absent in VPS72 deletion MAIT cells.
[0292] MAIT cells function in lung infection
[0293] The immune responses in the lung mucosa need to be finely regulated in a time- strength-dependent manner. The enrichment within lung mucosa for cells endowed with potent immunoregulatory function is paramount to combat infections and maintain tissue function. Innate like T cells, especially iNKT cells and MAIT cells are the central player in lung immunity. A large body of evidence in both preclinical and clinical setting has suggested that MAIT cells in host response against lung pathogens. MAIT cells are readily capable of cytokine/chemokine secretion and cytolysis rapidly. The lungs are particularly enriched for MAIT17 cells. VPS72 deletion results in severe MAIT 17 (Fig 9) subset defection. The role of MAIT cells in preclinical model of TB has been explored. The adjuvant properties of MAIT cells could be exploited in the design of more efficient vaccines. The replacement of conventional adjuvants by MAIT cell Ags (ribloflavin metabolites) in classical vaccines could be used to optimize the magnitude and duration of the adaptive immune. Through the ability of MAIT cells to subsequently activate/mature accessory cells including DCs. this strategy is likely to improve the development of the memory response. [0294] MAIT cells function in cancer
[0295] MAIT cells share many features with iNKT cells. MAIT cells constitute between 1% and 8% of human blood T lymphocytes, and they are more enriched in mucosal tissues. MAIT cells recognize antigens in the context of MRI. and upon activation, they instantly release cytokines and mediate cytolytic function on tumor cells. A deficiency in circulating MAIT cells was observed for mucosal-associated cancers, such as gastric, colon, lung cancer. Vps72 plays a critical role in MAIT cells development (Fig 9), therefore the potential of VPS72 on MAIT cells mediated tumor immunity is promising.
[0296] VPS72 function in Tregs Cell
[0297] Among immune cells, regulatory T cells (Tregs) have been widely studied for their role to induce self-tolerance and maintain peripheral immune homeostasis under multiple inflammatory conditions. Under physiological conditions, Treg is known to manage underlying active inflammation by peripheral tolerance mechanisms including immunosuppression. Nevertheless, majority of natural Treg immune cells are thymus-derived (thymus-derived Treg or tTreg cells) and produced functionally as a mature and distinct T- cells that are specialized for immune suppression to the periphery. In this scenario, expression of Foxp3 is critical in thymic development and suppressive capacity of Tregs. During this development, some of FoxP3+ Treg cells differentiate in the periphery from conventional T (Tconv) cells under certain conditions (peripherally derived Treg or pTreg cells) or by antigen stimulation in the presence of TGF-P and IL -2 (induced Treg or iTreg cells). Both pTreg and tTreg cells appear to be highly stable in the expression of FoxP3 and other Treg signature genes to mediate stable immune-suppressive function. However, studies have shown that Tregs are unstable and plastic, and their phenotype and function change with the specific environment. Indeed, upon losing or attenuating FOXP3 expression, Tregs lose their suppressive capacity7 and can adopt a proinfl ammatory phenoty pe that exacerbate pathogenesis of human diseases such as the allergic diseases, the autoimmune disorders, and cancer. Therefore, the equilibrium between phenotypic plasticity and stability' of Treg cells is important for maintaining the fine-tuned transcriptional and epigenetic events that required to ensure stable expression of Foxp3 in Treg cells. Most recently, the epigenetic regulation and function of FOXP3 has been linked to the histone methyltransferase (HMT) EZH2, histone deacetylases and acetyltransferase. Further, the role of histone deacetylases i.e. HDAC3 in the development and function of Tregs has been studied. Additionally, protein deacetylase inhibitors have been studied to enhance Tregs production and suppressive activity for the prevention and treatment of autoimmune disease and transplant rejection. Likewise, the inhibition of the histone acetyltransferase p300 impairing FOXP3 acetylation and Tregs function has shown to promote immune response in tumor-bearing host. Following the importance of FOXP3 in Tregs, another component of HAT complex, Tip60 was known to acetylate Foxp3 protein and enhanced the repression activity. In that context, VPS72, recently known component of HAT complex also acts as a key epigenetic regulator and contains acetyltransferase activity’. Therefore, we hypothesize that VPS72 may serve as a critical epigenetic factor in controlling Tregs homeostasis and functions, through regulating chromatin remodeling and fine-tuning the expression of specific target genes. By employing this, we have analyzed the effect of VPS 72 in Tregs phenotype and function, reasoning that VPS72 is required for Treg homeostasis that might represent a valuable pharmacologic target for modulation of Tregs functions.
[0298] VPS72 in Tregs development and function
[0299] Generation of VPS72 conditional knockout mice.
[0300] To investigate the functions of VPS72 in T cells development and homeostasis, we generated Cd4Cre-VPS72 conditional knock out mice (VPS72fl/fl Cd4Cre, VPS72fl/flcKO) (Fig. 11 A). The LlL2_Bact_P cassette was inserted at position 95117966 of Chromosome 3 upstream of the critical exons (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance gene under the control of the human beta-actin promoter, SV40 poly A, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon(s) at position 95119493. The critical exon(s) is/are thus flanked by loxP sites. A "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele. VPS72 ft/fl were crossed with transgenic mice expressing Cre recombinase driven by the Cd4 promoter, resulting in T cell specific deletion of the VPS72 gene starting at the DP thymocyte stage. The knockout efficiency was confirmed through the examination of VPS72 protein level in splenic CD4+CD25+ Treg cells (Fig. 11 B) [0301] VPS72 is required for Treg homeostasis in periphery.
[0302] Based on CD4 and CD8 staining, we first analyzed conventional T cells in the thymus and found that their frequencies and total cell numbers are comparable between VPS72cKO and WT (Fig. 12A, B). To determine whether VPS72 contributed to the development of thymus-derived regulatory T cells (tTregs). we used Foxp3 and CD25 as markers to identify Foxp3+ tTregs. The tTregs were comparable between WT and VPS72 cKO mice. Further, the frequencies and total cell numbers of conventional CD4 and CD8 T cells in thymus were comparable between WT and VPS72 cKO littermates (Fig. 12 C, D). These results suggest that that deletion of VPS72 does not impair thymic conventional T cells and tTregs. However, we find that the frequencies and cell number of KO splenic and LN CD4+ and CD8+ was significantly decreased compared to WT (Fig. 12 E, F). Further, frequencies and number of the inducible CD4+ inducible Tregs (iTregs) from KO spleen and lymph nodes were significantly decreased compared to WT (Fig. 12 G, H). Given the loss of conventional T cells in periphery, we investigated whether peripheral T cells displayed an activated phenotype in Vps72fl/flcKO mice and found that the total frequency activated CD62LloCD44hi effector memory T cells in the spleen was significantly increased without affecting its absolute number (not shown) of (Fig. 12 I, J).
[0303] To determine whether VPS72 regulates homeostasis in a cell-intrinsic manner, we generated bone marrow chimeras by reconstituting lethally irradiated CD45.1+CD45.2+ SJL/B6 recipients with a 1 : 1 mixture of CD45. 1+ SJL and CD45.2+ WT or Vps72 fl/fl cKO bone marrow cells. Analysis of mixed chimeras after 8 weeks of reconstitution revealed that Vps72fl/fl cKO donor bone marrow cells poorly reconstituted the T cells and Treg cell compartment in spleen (Fig. 12 K-O), which confirmed a cell intrinsic defect of Treg homeostasis in the absence of VPS72.
[0304] Treg specific loss of VPS72 results in lethal, multi-organ autoimmune disease.
[0305] To understand the Treg homoeostasis defect in the absence of VPS72 whether due to loss of Treg number or a loss of Treg suppressive function, we generated a mouse strain in which VPS72 was deleted specifically in the Treg expressing cells, using a Foxp3YFP/Cre recombinase expressed under the control of the Treg specific Foxp3 promoter. Treg specific deletion of VPS72 knockout mice (FOXP3YFPcre+VPS72fl/fl, Foxp3 VPS72 KO) manifested smaller size and their body weight was significantly reduced compared age/sex matched control mice at 3 weeks of age and the most Treg deficient mice succumbed to death before 5 weeks of age (Fig. 13 A-C). Further, these knockout mice presented with severe thymic atrophy, splenomegaly, and lymphadenopathy, suggestive of ongoing inflammation (Fig. 13 D). Upon disease onset, the total number of cells in thymus were significantly reduced, in contrast to a significant increase in total cell numbers in the lymph node (Fig. 13 D). However, WT and heterozy gote mice have similar characteristics. Histological examination of multiple organs revealed significant inflammatory cell infiltration in the liver, lungs and heart (Fig. 3 E). VPS72 deletion in Tregs reduced overall cellularity and decreased double positive and increased single positive thymocytes (Fig. 3 F,G), leading to gross disruption of normal thymic T cell development and consistent with thymic destruction by autoreactive T cells. The number of total CD4+ and CD8+ T cells was increased in the spleen but variable in the peripheral lymph nodes (Fig. 3 H,I). However, by comparing mice around 2 weeks after birth, we did not find significant changes in size and total cell numbers from the thymus, spleen, and lymph nodes between WT and Treg specific Foxp3 VPS72 KO mice (Fig. 3 J,K). Further, frequencies of CD4 and CD8 T cells in thymus, spleen and lymph nodes were comparable (Fig. 3 L,M), suggesting that FOXP3 KO mice were normally developed till 2 weeks after birth. Overall, these data suggest that Treg- specific deletion of VPS72 manifested with a spontaneous severe and lethal multi-organ autoimmune disorder, suggesting a loss of Treg suppressive function.
[0306] Treg-specific deletion of VPS72 results in the activation of peripheral T cells leading to break in immune tolerance. Given the severe immunopathology, we studied whether peripheral T cells displayed an activated phenotype in Foxp3 VPS72 KO mice. Flow cytometric analysis showed compared to control, CD4+ and CD8+ T cells of VPS72 KO mice had increased expression of CD44hi CD62Llo (Fig. 14 A, B), and Ki67 proliferation marker (Fig. 14 C) in peripheral organs in spleen and lymph node, which is suggestive of an inability of VPS72-deficient Tregs to appropriately regulate T cell response. To determine whether VPS72-defici ent Tregs were effective in controlling Thl, Th2 and Thl7 responses, we measured the cytokine production within CD4+ T cells from the spleen of WT and Foxp3 vps72 KO mice following stimulation of total splenocytes with PMA and lonomycin (P/I) for 4 hours. The expressions of IFNy, IL-4 and IL-17 in CD4+ T cells from Foxp3 VPS72- deficient mice were markedly increased compared to WT controls (Fig. 14 D,E). Further, the proportion of IFNy producing CD8+T cells was increased in the spleen of Foxp3 vps72- deficient mice (Fig. 14 F). These data support that VPS72-deficient Tregs were unable to control the expansion of Thl, Th2 and Thl7 effector cells specifically. The presence of activated T cells in Foxp3 VPS72-deficient mice is suggestive of a break in immune tolerance, might be caused by Tregs that lack suppressive function in absence of VPS72. The proportion of CD4+FOXP3+ Tregs was significantly decreased from spleen and LN of Foxp3 VPS72 KO mice (Fig. 14 G,H). There is a significant reduction in the expression of Treg- associated surface marker FOXP3 in absence of vsp72 in spleen, with no appreciable in lymph node (Fig. 4 I). These results suggest an inability for VPS72-defi cient Treg to restore expression of Treg marker that regulate their suppressive function.
[0307] Loss of VPS72 alters the Treg genetic signature and skews cells towards a Th2 profile profound effects of VPS72 deletion on gene expression in Tregs. Severe defects in Treg homoeostasis in the absence of VPS72 suggested that VPS72 is required maintaining Treg function and stability. To address whether VPS72 maintains a functional genetic signature, we performed RNA-seq analyses in splenic CD4+CD25+YFP+ (YFP+ Tregs) of Foxp3 VPS72 KO and heterozygote mice to assess global changes in gene expression regulated by VPS72. RNA-seq analysis shows a significant upregulation of Th2 signature genes such as IL4, Socs3, IL7R, IrF4, Gata3 and IL10, and Thl signature genes (Tbx21, Irfl and IFNy) in VPS72-deficient Tregs (Table 1 A), suggesting that VPS72- deficient Tregs might adopt a phenotype that more resembled to Thl and Th2 cells. Similarly, genes that are involved in regulating cytokine also found to increase the production of proinfl ammatory cytokines such as IL-10, IL-16 and IL-15ra, and chemokines such as CCL1, CXC13, CXCL10, and chemokine receptors and adhesion molecules (Slprl, Slpr4 and CCr4) in VPS72-deficient Tregs (Table 1 B), suggesting that Tregs lose suppressive function and failed to control against severe inflammation. Further, RNA-seq analysis shows that mitophagy (P62, Bnip, Smurf, Optn, Pinkl, Pari, Tfeb, Mfn), autophagy' (Atgs, Becnl, ULK3, BNIP3. Rptor). and apoptosis (Casp3, Cas, Bircs, Bad, Bid) associated genes are regulated in VPS72-deficient Tregs cells (Table 1 C-E). The increased expression of apoptotic marker in Treg in Foxp3 VPS72 KO mice suggest the exacerbation of apoptosis and need clearance. Importantly, autophagy and mitophagy supports their lineage stability and survival fitness in Tregs by removing defecting organelles, proteins and correcting T cell malfunction 146. Supporting this role, increased autophagy and mitophagy machinery in VPS72-defi cient Tregs cells seemed active and were unable to do clearance of all defecting Treg cell and other unwanted proteins, might be due to over-malfunction of Treg cells. [0308] Protective role of deletion of VPS72 in tumors.
[0309] Cancer is a leading cause of death worldwide accounting for nearly 10 million deaths in 2020. With this global burden, prevention of cancer is one of the most significant public health challenges these days. Cancer cells can evade the immune response and establish a very' complex balance for driving tumor progression, metastasis and resistance to therapy. Among skin cancers, melanoma is a highly aggressive cancer and metastasize to different organs, making it difficult to target via classical cancer therapies. Immunotherapy represents a novel emerging strategy7 for targeting aggressive cancers, however a major hurdle remains with the immunosuppressive environment within the melanoma tumor mediated by regulatory T cells (Tregs). Foxp3+Tregs, an immunosuppressive subset of CD4+ T cells, are highly infiltrated within the melanoma tumor that results into immunosuppression leading towards poor clinical prognosis. Therefore, targeting of Tregs in cancers may represent a promising avenue for a potential immunotherapeutic option. Despite several therapies targeting Tregs have been developed, prognosis of cancer is still poor.
[0310] Deletion of VPS72 in T cells showed no defect in T effector cell activation, indicating VPS72 as an ideal therapeutic target for modulating immunosuppression within the tumor microenvironment. As our VPS72/Treg KO mice die in early age, we used the timely inducible deletion with the Cre-ERT2 model. By crossing FoxP3eGFP-Cre-ERT2 mice to VPS72-floxed mice to allow for specific deletion of VPS72 in Treg cells (FoxP3eGFP-Cre- ERT2 VPS72fl/fl) following tamoxifen treatment. For inducible deletion, 6-8 weeks of control and knockout mice were treated with tamoxifen i.p. route for five successive days and then allowed to rest for 9 and challenged with Bl 6 melanoma cells (Fig. 15 A). Mice were sacrificed at 24 day and tumor weight measured. Deletion of VPS72 in Tregs have protective function as evident by smaller tumors and low- tumor weight in KO mice (Fig. 15 B,C) [0311] Tregs Application
[0312] Potential role of VPS72 in autoimmunity. Tregs are critical for the induction and maintenance of peripheral tolerance to prevent excessive immune responses and autoimmunity, and their failure contributes to autoimmune diseases such as such as multiple sclerosis, type 1 diabetes and systemic lupus erythematosus. Tregs have been extensively studied as a potential new7 tool for the treatment of autoimmune diseases. We have discovered a dominant role of VPS72 in maintenance of peripheral Tregs survival and function. Conditional knockout of VPS72 in Tregs significantly decreases the Tregs population in the peripheral immune organs, with consequent activation of immune cells and their infiltration and damage to host tissues leading to a spontaneous fatal autoimmune disease similar to those seen in scurfy mice. Our study demonstrates the critical role of VPS72 in maintaining peripheral Tregs and limiting autoimmune responses, therefore providing anew target for regulating immune responses therapeutically. Our study may possibly be able to develop selective and effective therapies based on targeting VPS72 dependent pathways to modulate Tregs for the treatment of autoimmune disorders in future.
[0313] Potential therapeutic role of VPS72 in cancer immunotherapy. Tregs infiltrate the tumor microenvironment which is a hurdle in treating cancer cells. Tregs inhibit tumorspecific T cell response and promote tumor growth. Therefore, there are clinical studies that report the depletion of Tregs may enhance the antitumor response in cancer patients 147-149. With this regard, our data shows that VPS 72 is required for peripheral Tregs homeostasis and deletion of VPS72 decreases melanoma tumor burden. Therefore. immunotherapy aimed at Tregs depletion through VPS72 may be potential immunotherapy strategy against cancer.
[0314] VPS72 function in HSC development and blood cancer
[0315] Hematopoietic stem cells (HSCs) are a rare cell population, primarily reside in bone marrow and they have the capacity to self-renew and to differentiate to give rise to all blood cell lineages. HSC have the potential to replenish the blood system for the lifetime of organism. The most important characteristic of HSC is their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation. Transplants of blood stem cells have become widely used to treat disease such as leukemia and myeloma. Therefore, they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene therapy. Role of histone demethylase Lsdl and lysine acetyltransferase Tip60 of HAT complex has been studied in hematopoietic stem cell maintenance. Novel gain-of-function screening approach identified VPS72 having a role for maintaining hematopoietic stem cell activity. However, the role of VPS72 in hematopoiesis in largely known. Given that the role VPS72, we hypothesize that VPS72 may serve as a therapeutic target to maintain HSC functions in different blood related diseases by regulating chromatin and gene expression.
[0316] VPS72 is required for HSC and its progenitors.
[0317] To test whether the deletion of VPS72 during adult hematopoiesis results in hematopoietic stem cell defects, VPS72fl/fl mice were bred with MxlCre mouse, which allows inducible deletion of VPS72 throughout all hematopoietic lineages and hematopoietic stem cells. Injection of Mxlcre-vps72fl/fl and Mxlcre+vps72fl/fl mice with dsRNA poly (I: C) (Fig. 16 A) resulted in efficient VPS72 inactivation as shown by western blot, qPCR and flow cytometry (Fig. 16 B.Cj.The overall cell numbers in the Mxlcre vps72 KO bone marrow was significantly decreased (Fig. 16 D) whereas frequencies of c-Kit+ Lineage (Lin-) Sca-1+ (LSK) cells increased (Fig. 16 E-F), with the increase in the frequencies of long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) with comparable their cell numbers (Fig. 16 E-G). Although the frequency of multipotent progenitors (MPP) was augmented, their total number was reduced (Fig. 16 E-G). However, frequency of lymphoid-primed multi- potent progenitor (LMPP) was decreased with increase in cell number in Mxlcre vps72 KO (Fig. 16 E-G). Both granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) showed increased frequencies, however, the frequency and number of megakaryocyte-erythroid progenitor (MEP) were reduced (Fig. 16 H). VPS72 loss in hematopoietic lineage causes early expansion of stem cells progenitors and perturbations of hematopoietic differentiation with a myeloid bias (Fig. 16 I).
[0318] HSC Application
[0319] Potential therapeutic role of VPS72 in hematological diseases.
[0320] HSC transplantation have been widely used to treat certain ty pes of cancer, such as leukemia, myeloma, and lymphoma, and other blood and immune system diseases that affect the bone marrow. These transplanted HSC exert rapid migration to bone marrow and give rise to specialized blood cells. Our data shows that VPS72 is required for the development of HSC and its progenitors. Targeting VPS72 for the proliferation and maintenance of HSC provides a potential therapeutic window of opportunities to treat hematological diseases.
[0321] Role of VPS72 in macrophage development and function
[0322] Tissue resident macrophages (TRMs) are a self-renewing mononuclear hematopoietic cell that maintain innate immune function, support tissue development and homeostasis, repair tissue damage as well as provide protection against injury’ or infection. Recent fate mapping studies from our group and others have revealed that TRM originates from primitive yolk sac and fetal liver hematopoiesis, self-maintained throughout life at steady stage and are regenerated by bone marrow (BM) under stress conditions. Depending on organs of residence, TRMs are known as Langerhans cells (LCs) in epidermis, dermal macrophage (DMs) in dermis, alveolar macrophage (AMs) in lung, Kupffer cell (KCs) in liver, microglia in brain and eye, as well as further defined as kidney and heart TRMs. Generally, TRMs resides in all organs and provides constant surveillance or scavenging of their tissue. By doing this, TRM maintains homeostatic functions by sensing their tissue specific surrounding and recycles macromolecules, cellular debris as well as invading pathogens. During that process, TRM produces a wide range of growth factors for tissue development and achieve endocytic as well as phagocytic activity’ for cellular/tissue specific defense. Additionally, study showed that TRMs is necessary' embryogenesis and organogenesis in numerous organs including lung, kidney. Following these diversified roles of TRM, macrophage mediated immunotherapies may become a potential tool against derailed tissue specific physiological and compromised pathological conditions. Therefore, any differentiated TRM cells or any important regulatory’ protein of TRMs can be utilized as a possible treatment regime for various diseases including cancer. Hence, our aim is to provide current experimental evidence on the role of VPS72 protein on TRMs during embryonic development and adult mice after birth. By providing this evidence and the important role of TRM in pathophysiology of various disease, we can further justify for the potential role of VPS72 based immune therapy. To accomplish that, our research utilized myeloid lineage (Csflricre) and dendritic cell specific (CDl lccre) VPS72 deletion mouse model for the first time and assess the role of VPS72 in different TRM of various organs including heart, liver, kidney, epidermis, dermis, brain.
[0323] VPS72 in myeloid and dendritic lineage development
[0324] Generation of VPS72 myeloid and dendritic lineage based conditional knockout mice.
[0325] To investigate the role of VPS72 in TRMs development and homeostasis, we generated Csflricre-Vps72 and CDl lccre-Vps72 conditional knock out mice (Fig. 17 A). While generating those mice, the LlL2_Bact_P cassette was inserted at position 95117966 of Chromosome 3 upstream of the critical exons (Build GRCm38). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance gene under the control of the human beta-actin promoter, SV40 poly A, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon(s) at position 95119493. The critical exon(s) is/are thus flanked by loxP sites. A "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele. Vps72 fl/fl mice were crossed with transgenic mice expressing Cre recombinase driven by the CD11c promoter, resulting in dendritic cell specific deletion of the Vps72 gene. Likewise, Vps72 fl/fl mice were further crossed with transgenic mice expressing Cre recombinase driven by the Csflricre promoter, resulting in myeloid lineage cell specific deletion of the Vps72 gene The knockout efficiency was confirmed through the examination of Vps72 protein level in LCs and AMs (Fig. 20). In addition to that, the effect of VPS72 deletion on body mass were measured by recording individual body weight in Csflricre+VPS72fl/fl (KO) and Csflricre-VPS72fl/fl (WT) mice before sacrifice. Our result the myeloid lineage specific deletion of VPS72 KO showed significant decrease in body weight after mice compared to WT littermates (Fig.17).
[0326] VPS72 is required for TRMs in organ specific manner from embryonic (E14.5) to adult mice after birth.
[0327] To test whether VPS72 regulates TRMs in all organs during embryonic development, we generated CsflricreVPS72fl/fl conditional knockout (CKO) mice, in which VPS72 was ablated in macrophage colony stimulating factor 1 receptor (Csflr) expressing manner within myeloid cells. Next, we examined the phenotypic profile of TRMs in mouse embryo (E14.5-17.5d) including CD45hiCDl Ibhi expressed epidermal Langerhans cells (LCs); CD1 lbintF4/80hi expressed dermal macrophage (DMs), microglia in the brain and retinal microglia, lung alveolar macrophage (AMs), kidney and heart TRMs; and CD1 lbintF4/80hi/ TIM4hi expressing liver Kupffer cells (KCs) from CsflricreVPS72fl/fl (CKO) mice and compared to wild type (WT) littermates. The frequencies of all TRMs in epidermis, dermis, brain, eye, liver, kidney was remarkably decreased at some or all specific time at embryonic stage in CKO mice compared to WT littermate embryo (Fig. 18 A,B, Table 2). Collectively, these observations suggest that VPS72 is required for TRMs in organ specific manner that derived from the development of yolk sac and fetal liver during embryogenesis. Next, we investigated whether the ablation of VPS72 has any post-natal effects on epidermal LCs (CD45hiCDl Ibhi at PO. CD45hiMHCIIhi in adult), dermal macrophage (DM) (CD1 lbintF4/80hi), brain and eye microglia (CD1 lbintF4/80hi at PO. CD1 lbintCD39hi in adult), lung AMs (CD1 lbintF4/80hi at PO, CD1 IchiSiglecFhi in adult), kidney, pancreas, heart TRMs (CDllbintF4/80hi at PO and in adult) and liver KCs (CDllbintF4/80hior CD1 lbintTIM4hi) in CKO mice compared to WT littennate. The frequencies of all TRMs in epidermis, dermis, liver, lung, and kidney were dramatic decreased in CKO mice at some or all specific time compared to WT littermate after birth (Fig. 18 A, B, Table 2).
[0328] Table 2. Myeloid lineage specific deletion of VPS72 leads to organ specific depletion of CD45/TRM
Figure imgf000086_0001
[0329] These observations suggest that VPS72 is required for TRMs in organ specific manner for its development and post-natal maintenance VPS72 is required for CD45 in organ specific manner from embryonic (E14.5) to adult mice after birth. As shown in Fig. 18, all TRM in all organs are derived from CD45 positive live cells population. Hence, the frequencies of CD45 have a cmcial role for the residing frequencies of TRM in various organs. To test whether VPS72 regulates CD45 positive cells in all organs during embryonic development, we examined CD45 populations between CsflricreVPS72fl/fl (CKO) mice and wild type (WT) littermates. As shown in Figure 3. the frequencies of CD45 population were significantly decreased in epidermis, dermis, liver, kidney heart and eye at some specific embryonic time point as indicated (Fig. 19 A,B, Table 2). Collectively, these observations suggest that VPS72 is required for CD45 cells during embry ogenesis. Next, we investigated whether the ablation of VPS 72 has any post-natal effects on CD45 population in CKO mice compared to WT littermate. The frequencies of all CD45 population were significantly decreased in epidermis, dermis, liver, kidney, heart, and eye in CKO mice at some or all specific time compared to WT littermate (Fig. 19 A,B, Table 2). These observations suggest that VPS72 is required for CD5 live cell in organ specific manner for its development and post-natal maintenance.
[0330] VPS72 required for post-natal maintenance of LCs and AMs.
[0331] Next, we examined whether VPS72 is required for the post-natal maintenance of LCS and AMs. To understand this, we utilized dendritic cell specific (CD11c ere) VPS72 deletion mouse model and examine the TRM in both epidennis and lung in adult CD1 lccreVPS72fl/fl (CKO) mice by comparing to wild type (WT) littermates. As shown in Fig. 20, the frequencies of LCs and AMs were dramatically reduced after the depletion of VPS72 as indicated by cell specific expression of protein, indicating that VPS72 is required for post-natal maintenance of AM and LCs (Fig. 20).
[0332] Potential therapeutic role of VPS72 in skin diseases.
[0333] VPS72 in LC histiocytosis. Langerhans cell histiocytosis (LCH) is a rare neoplastic histiocytic disorder in which excess immune LCs established in the body.
[0334] Generally, these normally residing LCs protect epidermis or skin against infection and destroy foreign invading substances in the body. LCH affects bones and skin, but it can also involve the bone marrow, liver, spleen, lungs, central nervous system, and other organs. Moreover, if any' therapy modulates this excessive disorder from LCs, this may become an important tool to treat this disease. In that context, our experiment data suggest that VPS72 is necessary for the development and maintenance of LCs (Fig. 18). We believe, if any intervention resulting in the reduction or lack of expression of VPS72, either pharmacological or genetic based inhibition of VPS72 is carried out, this rare LCH disease can be treated and eliminated.
[0335] VPS72 in Allergic contact dermatitis and other skin inflammatory diseases. [0336] Allergic contact dermatitis (ACD) is the most common occupational disease that might irritate the skin or trigger an allergic reaction. ACD is hypersensitivity response by an individual's immune system with direct skin contact with chemicals and appears as a red rash on irritant exposed skin site. During this condition, there is reduced number of LCs in ACD patients. Moreover, if any therapy modulates reestablishing LCs, this may become an important tool to treat this disease. In that context, our experiment data suggest that VPS72 is necessary’ for the development and maintenance of LCs (Fig. 18). We believe, if any intervention either pharmacological or genetic based topical activation of VPS72 is carried out, we can treat this allergic contact dermatitis.
[0337] VPS72 in skin cancer.
[0338] Skin cancer is the most common type of cancer mostly consists of squamous cell carcinoma, basal cell carcinoma, and melanoma. Among them, melanoma is uncommon cancer with high mortality rate and is highly malignant to other tissue areas. During having melanoma, various studies indicated that there is a decline in LCs in the epidermis of skin, suggesting association of LC in advancing melanoma cancer. Likewise, the basal cell carcinoma has increased number of epidermal LCs in the periphery and decreases frequency of LC in tumor indicating the essential role of LCs to limit tumor growth. Moreover, if any therapy modulates reestablishing this derailed LCs population, this may become an important tool to treat this disease. In that context, our experiment data suggest that VPS72 is necessary’ for the development and maintenance of LCs (Fig. 18-20). We believe, if any intervention either pharmacological or genetic based topical activation of VPS72 is carried out, we can treat and reduce some burden of skin cancer.
[0339] VPS 72 in wound healing.
[0340] Skin wound healing is a complex process that involves replacing dead and missing cellular structures and tissue layers. During healing, LCs undergo migration and repopulation in skin and replaced by circulating LC precursors. In some cases, if there is any impairment in wound healing, this condition may lead to chronic would condition. Importantly, chronic wounds condition such as diabetic foot ulcers, venous ulcers were found with increased LC numbers around wound edge and reduced LCs numbers in the epidermis of healed wound, suggesting for important role of LCs in would healing. Moreover, if any therapy modulates reestablishing this derailed LCs population in wound healing, this may become an important tool to treat this condition. In that context, our experiment data suggest that VPS72 is necessary for the development and maintenance of LCs (Fig. 18-20). We believe, if any intervention either pharmacological or genetic based topical activation of VPS72 is carried out, we can treat and reduce some burden of chronic wound healing condition.
[0341] VPS72 in autoimmune disease (Skin lupus, Skin Scleroderma). The differentiation and distribution of macrophages are involved in regulating numerous diseases including chronic inflammatory condition, infectious disorders, and autoimmune disease disorder including systemic sclerosis, systemic lupus erythematosus and other organ-specific autoimmune diseases. For example, autoimmune disease including systemic sclerosis has skin and internal organ fibrosis mediated my M2 macrophage. Other accumulated evidence suggests that there is abundant role of activated macrophages in the blood and skin of patients with systemic sclerosis. Likewise, there is similar role of macrophage identified in systemic lupus erythematosus. After understanding these dynamics of TRM in autoimmune disease along with the required role of VPS72 in maintaining LCs (Fig. 18-20). we believe there may be pharmacological or genomic based intervention of VPS72 for macrophage population for reducing autoimmune diseases, by inhibiting the expression and/or activity of VPS72 in LCs. [0342] VPS72 controls my eloma.
[0343] Myeloma is a malignant disorder of plasma cells that build up in the bone marrow and form tumors foci in the skeleton of the body to initiate disease progression. In myeloma condition, macrophages are infiltrated and become major immune components for promoting tumor metastatic events. These myeloid macrophages are recruited from circulating monocytes, adapt into the tumor microenvironment, and differentiate into tumor associated macrophage. This established macrophage population become crucial in the myeloma microenvironment and activate various signaling pathways that require for protecting myeloma cells from undergoing apoptosis. After understanding these dynamics of macrophage in myeloma and the required role of VPS72 in maintaining resident macrophages (Fig. 18-20), we believe there may be VPS72 based pharmacological or genomic intervention for reducing infiltrating macrophage population in myeloma microenvironment.
[0344] VPS72 controls cancer through targeting resident macrophages in lung/ hepatic/renal cancer.
[0345] Current understanding on the fonnation and seeding of metastatic tumor cells into various organ suggest that TRM are the crucial immune cell that adapt to tumor microenvironment, get transformed to tumor associated macrophages (TAM) and become important element for tumorigenesis process in various organ including the lungs or liver or kidney tissue. Mostly M2 macrophage markers (CD206, CD 163) or pan-macrophage marker CD68 were used to distinguish these cells. Moreover, if any therapy modulates TRMs population, this may become an important tool for certain cancer therapies. After understanding these dynamics of macrophage and the required role of VPS72 in maintaining resident macrophages (Figure 18-20), we believe there may be pharmacological or genomic based intervention of inhibiting the expression and/or activity of VPS72 for reducing infiltrating macrophage population in cancer microenvironment.
[0346] VPS 72 controlling in aging.
[0347] A fully functioning immune system is essential for maintaining good health. However, the immune system undergoes deterioration with advancing age. and contributes towards increased susceptibility against infection, autoimmunity, and cancer. Additionally, macrophage immune cells can be an important player for maintaining tissue homeostasis and protecting tissue against infection. However, those macrophages mediated function are derailed with age. After understanding these dynamics of macrophage in aging with the required role of VPS72 in maintaining resident macrophages (Fig. 18-20), we believe there may be pharmacological or genomic based intervention of VPS72 for reducing infiltrating macrophage population in aging microenvironment
[0348] VPS72 in metabolic syndromes. Dysregulation of TRM function can have multiple consequences in many diseases, including cardiovascular and metabolic condition, obesity, cancer, amyloidosis, and infections. Reestablishment of functional macrophages may become an opportunity for treating those metabolic disease. Accumulating evidence suggest that post-natal microglial activation may become chronic inflammatory source to drive progressive neurodegeneration and induce diabetic retinopathy. Further, Kupffer cells play important roles in iron metabolism, cholesterol metabolism, and immune surveillance. In case, if liver KC become dysfunctional or diminished, this may lead to pathogenic situation by increased lipogenesis, released inflammatory cytokines, and activated stellate cells to generate fibrosis condition like non-alcoholic fatty liver disease (NAFLD). Hence, elimination and maintenance of tissue specific macrophage is crucially important. In that context, regulation of resident macrophage population may become an important tool to treat the symptoms of metabolic syndrome. After understanding these dynamics of macrophage and the required role of VPS72 in maintaining resident macrophages (Fig. 18-20), we believe there may be pharmacological or genomic-based intervention of VPS72 based macrophage population to treat metabolic syndrome. [0349] Synthesis and Design of VPS72|H2AZ Peptide Inhibitor
[0350] VPS72 is a specific H2A.Z deposition chaperone, later is a widely conserved histone variant; the crystal structure of human VPS72-H2A.Z- H2B complex have identified the specific binding sites of VPS72|H2A.Z. Here we predicted mouse VPS72 structure via https://swissmodel.expasy.org/ , and find that both human and mouse VPS72 structure aligned well (Fig. 21). Previous study identified critical binding sits of VPS72|H2A.Z-H2B dimer, including VPS72 F29A, Y30A, Y34A, F37A, D43A, E35A, and Y64A. With that information, we designed a peptide blocking align with VPS72 sequence (Fig. 21), with heighted in green. VPS72|H2A.Z was successfully synthesized with purity greater than 95%. [0351] Despite the incredible success of the latest cancer immunotherapies, some malignancies resist treatment. Emerging studies suggest that an intratumoral. immunosuppressive cellular niche may promote tumor immune escape, which could be a major obstacle to immunotherapy success. How7 regulatory T (Treg) cells mediate immunosuppression in the tumor microenvironment (TME) is not well understood, which is a major hurdle for improving immunotherapy. The transcription factor Forkhead Box P3 (FoxP3) is a marker for Treg cell lineage, stability, and immunosuppressive function.
Although progress has been made showing that certain transcriptional and epigenetic events are required for stable Foxp3 expression in Treg cells, and that the TME harbors significant microenvironmental stresses that promote successful Treg adaption to the TME, a significant gap remains in our understanding of how epigenetic networks control FoxP3 -mediated Treg survival and immunosuppressive activity within the stressful TME.
[0352] Recent studies suggest that the exchange of canonical histones to variant histones plays a key role in modulating chromatin structure during epigenetic regulation. The vacuolar protein sorting-associated protein 72 homolog (VPS72) is a histone chaperone that exchanges canonical histone H2A for variant histone H2A.Z. In vitro studies have shown that VPS72 is an oncogene that specifically and selectively regulates limited gene transcription through chromatin remodeling viaH2A.Z exchange. However, the in vivo biological function of VPS72, especially in immune cells, remains totally unknown, and this lack of understanding is a barrier to development of VPS72-mediated therapies for cancer. Our previous work indicates that intratumoral Tregs have elevated FoxP3 expression and are highly potent in immunosuppressive activity'. Importantly, it was found that increased FoxP3 expression is positively correlated with upregulated VPS72 and H2A.Z expression in tumorinfiltrating Tregs from tumor-bearing mice, implying a potential role for VPS72/H2A.Z in maintaining FoxP3 expression within the TME. Treg-specific VPS72 knockout (VPS72- TregKO) mice were generated, and it was found that VPS72 was required for peripheral Treg cell stability and immunosuppressive function even though targeted VPS72 deletion did not affect thymic Treg cell development. Consequently, VPS72-TregKO mice develop lethal, multi-organ autoimmune disease at 3-4 weeks of age. Treg-specific H2A.Z KO (H2A.Z- TregdKO) mice were then generated, which largely phenocopied VPS72-TregKO mice. These findings led to our central hypothesis that VPS 72 is a critical epigenetic factor that controls Treg stability and function through H2A.Z chromatin remodeling, and that TME- specific factors may selectively upregulate VPS72 to potentiate Treg immunosuppressive capacity and maintain Treg stability in the TME (Fig. 22). This hypothesis can be tested with three specific aims:
[0353] Aim 1. Define the roles of VPS72 and H2A.Z in peripheral Treg cell stability and function.
[0354] 1.1. Determine the role of VPS72 in (1) Treg immunosuppressive activity', depolarization, and reprogramming; and (2) test if Foxp3 overexpression rescues defective Treg immunosuppressive activity in VPS72KO mice.
[0355] 1.2. Determine the role of H2A.Z in Treg stability and immunosuppressive function by examining whether the two H2A.Z isoforms (H2A.Z1 and H2A.Z2) regulate Treg stability and function individually or together.
[0356] 1.3. Determine the mechanisms by which VPS72 and H2A.Z regulate FoxP3 expression by using multi-omics analysis, RNA-seq, scRNA/scATAC-seq, and CUT&RUN- seq, to delineate global VPS72-mediated H2A.Z chromatin exchanges and identify FoxP3- related targets and signaling pathways.
[0357] Aim 2. Evaluate the role of VPS72 and EI2A.Z in immunosuppression during antitumor immunity.
[0358] 2. 1. Analyze the efficacy of antitumor immunity in inducible VPS72-TregiKO and H2A.Z-TregiKO mice.
[0359] 2.2. Examine the role of VPS72 and H2A.Z in Treg antitumor immunity against spontaneous melanoma.
[0360] 2.3. Evaluate the synergy of Treg VPS72 suppression with checkpoint blockade (anti-PD-1) in antitumor therapy.
[0361] Aim 3. Identify TME factors that upregulate VPS72 expression for Treg adaptation. It can be determined whether TME cytokines (such as TGF-(31), metabolites (low glucose & amino acids), and hypoxia induce VPS72-mediated FI2A.Z exchange in Tregs and dissect the underlying molecular mechanisms. [0362] In view of these aims, new insights can be gleaned into the role of VPS 72- mediated H2A.Z exchange in Treg stability and function, which will not only provide new insight into the biology of Tregs, but also facilitate the development of Treg-based intervention strategies for cancer.
[0363] Tumors have long been recognized as having the distinctive properties of uncontrolled growth, tissue invasion, and metastasis, but their ability to evade immune destruction has recently attracted attention as a potential target for new therapeutics. In many cases, immune evasion does not occur because tumor cells lack sufficient antigenicity to activate a CD8+ T cells; in fact, tumors that retain high levels of tumor-infiltrating CD8+ T cells are typically associated with improved clinical outcomes. Rather, numerous tumors concurrently display an accumulation of FoxP3+ Tregs within the tumor microenvironment (TME), which is correlated with poor prognoses for patients with many solid tumors. These findings suggest that malignant tumors may overcome their inherent antigenicity in part by promoting the accumulation and suppressive function of tumor-infiltrating Tregs. This is supported by preliminary’ observations and previous studies, which indicate that tumorinfiltrating Tregs display increased immunosuppressive capacity that correlates with increased FoxP3 expression relative to Tregs in peripheral lymphoid organs in mice and circulating blood in humans. The factors within the TME and the underlying molecular mechanisms that upregulate FoxP3 expression remain unknown.
[0364] VPS72 overexpression has been identified in different types of cancers, such as hepatocarcinoma and melanoma (TCGA GDAC team) and is recognized as an oncogene. VPS72 is on the top 20 list of highly expressed genes in skin cutaneous melanoma (TCGA- GDAC database). Interestingly, high H2A.Z expression w as positively correlated with high expression of VPS72 in different types of the aforementioned cancers (TCGA database), and high expression of VPS72 in hepatocarcinoma was associated with poor survival. Overall, the VPS72-mediated H2A.Z epigenetic axis (Fig 23) is involved in the biological processes of different cancers and has been shown to modulate local chromatin structure to activate or repress target genes and regulate cellular and metabolic processes, mitochondrial function, cell cycle progression, and autophagy regulation. However, the function of VPS72 and H2A.Z in the immune system, especially in Treg biology and Tregs in TME, remain totally unknown.
[0365] FoxP3 upregulation alongside VPS72 and H2A.Z overexpression maintains Treg stability and adaptation to the TME. The TME is a heterogeneous milieu where different types of cells interact, including tumor cells, immune cells, and stromal cells. In addition to directly promoting tumor cell growth. TME factors are also critical for reprogramming the immune response that allows for tumor immune escape. For example, cytokines and chemokines produced by both infiltrating immune cells and tumor cells can induce and attract immune suppressive Tregs. The intratumoral Tregs then have the capability to adapt themselves to the harsh TME generated by the vigorous growth of malignant cells. Preliminary studies and data from recently published in Science Advance found that both Treg frequencies and FoxP3 expression levels were significantly higher in Tregs from B16 melanoma tissues than in Tregs from the peripheral lymphoid organs (Fig. 24 A-C), and this higher expression was required for maintaining Treg stability and adaptation to the TME. The surface expression of CD25, PD-1, CTLA-4 and GITR in Treg cells was also analyzed (see Fig. 24D). Interestingly, our preliminary studies showed significantly higher VPS72 and H2A.Z expression in tumor-infiltrating Tregs than in Tregs from spleen of the same tumorbearing mouse or Tregs from skin of wild ty pe (WT) mice (Fig. 25 A-C). In contrast, VPS72 expression measured by mean fluorescence intensity (MFI) was comparable in CD4+ and CD8+ T cells from spleen and tumors of those same mice (Fig. 25 D). Consistent with our preliminary data in mice, analysis of published single-cell data revealed a dramatic increase in VPS72 expression in tumor infiltrating Tregs from human melanoma and liver cancers relative to Tregs from human peripheral blood mononuclear cells (PBMC) (Fig. 26). Therefore, our preliminary discoveries imply that TME-specific factors induce both VPS72 and FoxP3 upregulation and may control Treg stability and adaptation.
[0366] VPS72-mediated H2A.Z chromatin remodeling is required for Treg stability and immune suppressive function. To investigate the role of VPS72 and H2A.Z in the immune system, a T cell-specific VPS72 knockout (KO) mouse model (VPS72-TKO) was generated (see Fig. 11 A and Fig. 27 A and B). Cell-specific VPS72 deletion did not affect thymic conventional T cell and Treg cell development, but it did result in dramatically reduced peripheral Tregs (Fig. 28 A-C). Additionally, it was found that VPS72 is required for Treg homeostasis in the periphery in a cell-intrinsic manner (Fig. 29). To further investigate the specific role of VPS72 in Treg development, Treg-specific VPS72 KO (VPS72-TregKO) mice were generated. Loss of VPS72 did not affect thymic Treg development but resulted in significantly reduced peripheral Treg cell frequency and FoxP3 expression (Fig. 30 A-B), with Fig. 30C showing a representative image of Teff cells with their respective absolute numbers. The VPS72-TregKO mice spontaneously developed a severe and lethal multi-organ autoimmune disorder starting at 3-4 weeks of age and died very quickly (Fig. 13). Fig. 31 A- C illustrates data showing that VPS72 is required for TGF-0-induced Treg (iTreg differentiation and FoxP3 expression). Interestingly, T cell-specific H2A.Z1/Z2 double knockout (H2A.Z-TdK0) mice were generated, and as expected, it was found that Tregs from H2A.Z-TdKO mice phenocopied the Tregs in VPS72-TKO mice (Fig. 32), suggesting a functional role for VPS72 as an H2A.Z chaperone in Treg regulation. CUT&RUN-seq (cleavage under targets and release using a nuclease sequencing) has recently been used to analyze histone-DNA interactions. We performed H2A.Z CUT&RUN-seq in splenic Treg cells and found H2A.Z enriched peaks at FoxP3 and TGF-J31 promoter regions, suggesting that VPS72-mediated H2A.Z remodeling may regulate Treg stability and function, at least through regulating FoxP3 and TGF-pi expression (Fig. 33, including SEQ ID NO: 7 for NFAT; SEQ ID NO: 8 for RUNX1; SEQ ID NO: 9 for MYB). Thus, preliminary data suggest that VPS72 is required for peripheral Treg maintenance and function, likely through H2A.Z chromatin remodeling. In Aim 1, the role of VPS72 and H2A.Z in Treg stability and function can be determined and the mechanisms by which VPS72 and H2A.Z regulate Treg homeostasis and function can be uncovered.
[0367] VPS72-mediated H2A.Z chromatin remodeling controls Treg adaptation in the TME. Depletion of FoxP3+ Tregs has been shown to be beneficial in anti-tumor immunotherapy in experimental animals bearing tumors, as show n by using CD4 or CD25 monoclonal antibodies. Since the VPS72-TregKO mice die at an early age due to autoimmunity, a tamoxifen (TAM)-inducible Treg cell -specific VPS 72 deletion model was developed (VPS72-TregiKO) to evaluate the efficacy of VPS72 inhibition in promoting the ability of Treg cells to suppress B16 melanoma. It was found that VPS72 deficiency in Tregs after tumor formation significantly blocked tumor development and resulted in increased anti-tumor immunity (Fig. 34). Strikingly, it was found that VPS72-deficient Tregs failed to maintain high levels of FoxP3 in the TME. Inhibition of VPS72 can block liver cancer cell proliferation and migration and promote tumor cell apoptosis. Because VPS72 is now considered an oncogene that is highly expressed in some cancers and its high expression predicts poor tumor prognosis, VPS72 inhibition may hold great therapeutic potential as an anti-tumor therapeutic target in both Treg and tumor cells. In Aim 2, the preclinical efficacy of Treg-specific VPS72 suppression in inducible VPS72-TregiKO and H2A.Z-TregiKO mice can be analyzed for antitumor immune therapy using multiple syngeneic tumor models, including B16 melanoma, EG7 lymphoma, and MC38 colon cancer, as well as a spontaneous melanoma model.
[0368] Low glucose upregulates VPS72 expression in Tregs. TME-specific factors are critical for reprogramming the immune response that allows for tumor immune escape. Intratumoral Tregs can adapt to the harsh TME that is generated by vigorous growth of malignant cells, including metabolic changes (low glucose and amino acids and high lactate), hypoxia, high CO2 concentrations, and low pH (acidity). However, the exact mechanisms by which TME factors potentiate Treg immunosuppressive activity and control Treg adaptation to the TME remain to be identified. Low glucose can increase VPS72 expression (Fig. 35). Given that VPS72 regulates FoxP3, TME factors may increase the expression of FoxP3 by upregulating VPS72.
[0369] VPS72-H2A.Z chromatin remodeling contributes to cancer development. T cell-specific H2A.Z-TdKO mice were generated, which phenocopied the VPS72-TKO mice by showing defective peripheral Tregs. Furthermore, VPS72 deficiency in Tregs enhances anti-tumor immunity by decreasing FoxP3 expression and TME-specific factors regulate VPS72 and H2A.Z expression in Tregs. Accordingly, VPS72-mediated H2A.Z remodeling and how it controls Treg stability and function in the TME can be determined.
[0370] Described herein is evidence that VPS72 and H2A.Z regulate peripheral Treg stability and function in tumor immunity. VPS72-H2A.Z has a role in Treg and Treg immunosuppressive function in the TME, and anti-tumor immune therapies that target VPS72 can be developed. Novel mouse models with spatial/temporal-specific VPS72 and H2A.Z KO can be generated to dissect VPS72/H2A.Z function in Tregs; and multi-omics analysis can be employed, including scRNA-seq/scATAC-seq/CUT&RUN-seq and imaging mass cytometry (IMC).
[0371] Scientific rigor, transparency, and statistical analysis: Mice can be matched for biological variables including sex, age, and weight in all experiments. Both male and female mice can be used. All findings can be repeated to confimi reproducibility and rigor. Transparency can be maintained in reporting methodological details and experimental results so that other investigators may reproduce and extend our findings. Rigorous statistical analyses can be applied to each Aim. Whenever possible, altered genotypes can be confirmed by measuring protein and mRNA expression. Where appropriate, examples of primary (typically FACS) data can be shown with the gating scheme and quantified across mice/experiments. For quantification, statistical analysis can be performed with GraphPad Prism 9, and analyses can be stated in the figure legends and methods. Comparisons can be based on at least 3 biological replicates. For animal studies, a sample size of 8-10 mice per group has >85% power to detect a significant difference of P<0.05 with an effect size of 1.7.
[0372] Tumor-infiltrating Tregs have elevated expression of FoxP3 in mice. To explore the role of Tregs in tumorigenesis, mouse tumor models can be used, including but not limited to: B16 melanoma, EG7 lymphoma, MC38 colon cancer, and LLC1 small cell lung carcinoma. Both Treg frequency and FoxP3 expression levels were significantly- increased in Tregs from B16 melanoma tissues relative to Tregs from peripheral lymphoid organs (Fig. 24 A-C & data not shown). Consistent with the elevated FoxP3 expression, Treg surface markers including CD25, PD-1, CTLA-4, and GITR were all significantly increased in tumor Tregs (Fig. 24 D). Similar results were obtained when EG7 lymphoma, MC38 colon cancer, and LLC1 small cell lung carcinoma tumor models were analyzed.
[0373] VPS72 and H2A.Z are upregulated in mouse tumor Tregs. Given that VPS72 and H2A.Z are upregulated in tumor tissue, Treg cells expressing VPS72 and H2A.Z were analyzed. It was found that the VPS 72 expression in tumor Tregs was significantly higher than that in Tregs from spleens of the same tumor-bearing mice (Fig. 25 A). Interestingly and unexpectedly, H2A.Z1 and H2A.Z2 mRNA expression was also upregulated (Fig. 25 B). Furthermore, VPS72 protein levels by FACS analysis showed higher expression in tumor Tregs than in Tregs from spleen of tumor-bearing or wild ty pe skin (Fig. 25 C). However, VPS72 expression in CD4+ and CD8+ T cells was unaltered (Fig. 25 D). These data suggest that TME-specific factors may simultaneously upregulate FoxP3, VPS72, and H2A.Z expression in tumor Tregs.
[0374] VPS72 is upregulated in tumor-infiltrating Tregs in human melanoma and liver cancer. VPS72 was also more highly expressed in tumor Tregs than in peripheral blood Tregs from human patients. Publicly available single-cell RNA-seq data for CD4+FoxP3+ Tregs in liver cancer (GSE98638), melanoma (GSE120575), and CD4+FoxP3+ Tregs were analyzed from peripheral blood (GSE175602) and found that VPS72 was highly expressed in the liver Tregs, moderately expressed in melanoma Tregs, and only slightly expressed in Tregs from peripheral blood (Fig. 26). suggesting a potential function of VPS72 in human cancer Treg stability.
[0375] VPS72 is required for peripheral Treg maintenance but not for thymic T and Treg cell development. To investigate the function of VPS72 in the immune system, VPS721oxp mice were generated by crossing VPS72 targeted allele mice (MGI:5008033) with Flp recombinase expressing (Jax 016226) mice. VPS72fl/fl mice were then crossed with CD4-Cre mice (Jax 004194) to generate T cell-specific VPS72 KO mice (VPS72-TKO) (Fig. 11 A). VPS72 contains 6 exons, and exons 2-4 are flanked by loxP sites. VPS72 protein deletion in thymic CD4 T cells (Fig. 27 A) from VPS72-TKO mice was confirmed byWestern blot. The immune phenotype of VPS72-TKO mice was also characterized. VPS72 deletion had no effect on the frequency or absolute number of conventional CD4, CD8, and Treg cells in the thymus (Fig. 28 A & B, which shows the same data as Fig. 12 A-D but with a different Treg gating strategy), indicating that VPS72 is dispensable for thymic development of both conventional T cells and Tregs. However, the frequency and absolute number of CD4+ and CD8+ T cells and Tregs in the spleen were significantly lower in VPS72-TKO mice than in WT mice (Fig. 28 A & B, which shows the same data as Fig. 12 E- H but with a different Treg gating strategy). The MFI of FoxP3 expression in splenic Tregs was significantly reduced (Fig. 28 C). To determine whether VPS72 regulates Treg cell development and homeostasis in a cell-intrinsic manner, bone marrow (BM) chimeras were generated by reconstituting lethally irradiated CD45.1+CD45.2+ SJL/B6 recipients with a 1:1 mixture of CD45.1+ SJL and CD45.2+ WT or VPS72-TKO BM cells (Fig. 12 K). Analysis of mixed chimera mice after 8 weeks revealed that VPS72-TKO donor BM cells poorly reconstituted Tregs in spleen and lymph nodes but generated comparable reconstitution in thymus (Fig. 29), which confirmed a cell-intrinsic defect of Treg homeostasis in the absence of VPS72. Fig. 29 shows the same data as 12N (for the spleen), but with a different Treg gating strategy.
[0376] Treg-specific VPS72 KO mice (VPS72-TregKO) develop a lethal, multi-organ autoimmune disease. Treg-specific VPS72 KO (VPS72-TregKO) mice were generated by breeding VPS721oxp mice with FoxP3-Cre-YFP knock-in mice (Jax: 016959) (Fig. 11 A). VPS72 protein deletion in splenic CD4+CD25+ cells (Fig. 27 B) from VPS72-TregKO mice was confirmed by Western blot. Consistently, VPS72-TregKO mice developed normally in the first two postnatal weeks. Starting at 3-4 weeks of age, VPS72-TregKO mice had a runted size (Fig. 13 A) and significantly reduced body weight (Fig. 13 B) relative to age/sex- matched control mice (heterozygote and WT). Also, most VPS72-TregKO mice died before 6 weeks of age (Fig. 13 C). Histological examination of multiple organs revealed significant leukocyte infiltration in most tissues, including the liver, lung, and heart (Fig. 13 E). Thus, loss of VPS72 in Tregs causes a severe and lethal multi-organ autoimmune disorder, suggesting that VPS72 is a key regulator that is essential for Treg immunosuppressive function.
[0377] VPS72 is required for maintaining peripheral Tregs and FoxP3 expression in Tregs from VPS72-TregKO mice. We next analyzed Treg cells in the thymus, spleen, and lymph nodes of 3-4 w eek old mice. Significant changes were not uncovered in thymic Tregs, but the percentage and absolute number of splenic CD4+YFP+ Tregs (Fig. 30 A) and CD4+FoxP3+ Tregs (Fig. 30 B, which has different flow cytometry data than Fig. 14G) were dramatically reduced in VPS72-TregKO mice, leading to the activation of CD4 and CD8 T cells (Fig. 30 C; see also Fig. 14 A-B for the spleen). Expression of FoxP3 (MFI) was also significantly reduced in Tregs from KO mice (Fig. 30 B). Thus. VPS72 is required in vivo for both peripheral Treg stability and FoxP3 expression in Tregs.
[0378] VPS72 is required for TGF-P-induced Treg (iTreg) differentiation and FoxP3 expression. VPS72 is required for generating iTreg in vitro. CD4 naive cells were sorted from VPS72-TregKO mice to generate iTreg in vitro and found significantly lower Treg differentiation (Fig. 31 A-B) with dramatically decreased expression of FoxP3 (Fig 31 C). Thus, VPS72 is also required for in vitro iTreg differentiation and FoxP3 expression.
[0379] H2A.Z regulates peripheral Treg stability. To confirm the functional specificity of VPS72 as a histone chaperone that exchanges H2A.Z. H2A.Zlfl/fl.H2A.Z2fl/fl mice with CD4-Cre mice were crossed to generate H2A.Z1.Z2 double KO mice (H2A.Z- TdKO). As shown in Fig 32 A, we confirmed H2A.Z deletion in thymocytes and splenic T cells by Western blot (H2A.Z antibody recognizes both H2A.Z1 and Z2 isoforms). Loss of H2A.Z did not affect thymic conventional CD4 and CD8 cells and Treg cells, but significantly reduced splenic Treg. CD4, and CD8 T cells (Fig. 32 B-C), which phenotypically copies VPS72-TKO mice (Fig. 28). These results support our notion that VPS72-H2A.Z modification is required for peripheral Treg cell stability.
[0380] VPS72 suppression in Treg cells enhances the anti-tumor response against pre- established B 16 melanoma. As VPS72-TregKO mice die at an early age, an inducible deletion Cre-ERT2 mouse model was used. FoxP3eGFP-Cre-ERT2 mice w ere crossed to VPS72-floxed mice to allows for inducible deletion of VPS72 in Tregs (VPS72-TregiKO) following TAM treatment. To investigate the role of VPS72 in the context of cancer, WT and age and sex-matched inducible VPS72-KO mice were subcutaneously injected with B16 melanoma cells (0.3M/mice) and monitored for tumor progression. When the tumor reached 100 mm3, mice were injected with TAM for 5 consecutive days. It was found that Treg VPS72 deficiency resulted in significantly reduced tumor size and weight (Fig. 34A & B). The tumor-infiltrating CD8 T cell frequency was significantly increased in Bl 6 melanomabearing VPS72-TregiKO mice relative to WT mice (Fig 34 C), resulting in increased Granzyme B (Fig 34 D) and IFN-y (Fig. 34 E) production. To determine whether the increased CD8 T cell activation in tumors w as due to reduced Treg functioning, tumor CD4+FoxP3+ T cells were analyzed. Interestingly, both Treg frequency and FoxP3 expression were significantly reduced in tumor cells from VPS72-TregiKO mice compared to WT (Fig. 34 F&G). These findings indicate that Treg-specific VPS72 suppression enhances the anti-tumor immune response in mice. [0381] Mapping H2A.Z binding peaks across the genome in splenic Tregs. To investigate VPS72-H2A.Z remodeling, CUT&RUN sequencing was used to identify genes that are directly bound to H2A.Z in splenic CD4+CD25+ Tregs from C57BL/6 mice. Most H2A.Z binding sites were found at intergenic, intron, promoters flanking the transcription start site (TSS) (promoter-TSS) regions (Fig. 33 A). These peaks were mapped back to 1437 genes. Consistently, H2A.Z binding peaks were highly enriched in the GO terms of metabolic process, transcription factors (TF), and apoptosis process (Fig. 33 B). KEGG pathway analysis also indicated that the enriched peaks were found mostly in apoptosis, TCR, MAPK, and TGF-P signaling pathways (Fig. 33 C). Motif enrichment analysis showed significant enrichment of target motifs for the TFs related to FoxP3 and Tgfpi expression, such as RUNX1 and NFAT for FoxP3 and MYBfor Tgipi (Fig 33 D). Unexpectedly, it was found that H2A.Z binding peaks in the promoter regions of both FoxP3 and Tgfpi (Fig. 33 E). These data indicate that VPS72-H2A.Z mediated chromatin modification may regulate Tregs by directly targeting FoxP3 or indirectly targeting genes (like Tgfpi) related to FoxP3 expression in Tregs.
[0382] Loss of VPS72 and H2A.Z results in reduced peripheral Treg frequency and FoxP3 expression in Tregs with elevated autoimmunity, indicating an essential role for VPS72-H2A.Z-mediated chromatin remodeling in Treg maintenance and immunosuppressive function. The present disclosure serves delineate the cellular mechanisms underlying the effect of VPS72-H2A.Z deficiency in Treg suppressive functions in vitro and in mice and then define the role of H2A.Z1 and H2A.Z2 in maintaining Treg stability and function. Finally, the mechanisms by which VPS72 and H2A.Z regulate FoxP3 expression can be identified.
[0383] Treg cells attain their immunosuppressive ability through the expression of inhibitory cell surface receptors (e.g., CTLA-4, PD-1, and G1TR) and the generation of regulatory cytokines such as IL-10. Therefore, to determine how VPS72 maintains Treg function, we will analyze the surface markers on Tregs related to Treg immunosuppressive function, such as CD25, ICOS, CTLA-4, Helios, PD-1, CD44, and Nrpl, as well as IL-10 production in Tregs from the spleen and lymph nodes in VPS72-TregiKO and WT mice. If VPS72 maintains Treg immunosuppressive activity by regulating expression of the aforementioned surface markers/cytokines, decreased levels of those markers in VPS72- deficient Tregs can be anticipated. Treg-specific VPS72 deletion results in decreased Treg frequency and FoxP3 expression in the peripheral organs and causes early onset of autoinflammation (Fig. 13 & 30), suggesting that VPS72 is required for Treg homeostasis and immunosuppressive activity. To confirm these findings, in vitro and in vivo immunosuppressive assays can be performed. In vitro immunosuppression assay: CD4+CD25+YFP+ Tregs sorted from VPS72-TregKO and WT mice will be co-cultured with sorted naive CD4+CD25-CD441oCD62Lhi T cells labelled with carboxyfluorescein succinimidyl ester (CFSE) in different ratios in 96-well U-bottom plates for 3 days. Immunosuppressive Treg function will be analyzed by the proliferation of activated CD4+ T cells as determined by CFSE dilution. In addition, apoptosis and proliferation in Tregs from spleen and lymph nodes can be assessed by annexin V and Ki67 staining, respectively. In vivo immunosuppression assay: Sorted naive CD4+ T cells from congenic CD45.1 B6.SJL mice can be adoptively transferred alone or in combination with sorted YFP+ Treg cells (CD45.2+) from WT or VPS72-TregKO mice into recipient immunodeficient Ragl-/- mice. Adoptive transfer of naive CD4 T cells into Ragl-/- mice results in colitis, which can be protected against by Tregs. If VPS72-deficency impairs Treg immunosuppressive functions, we anticipate that mice receiving transfer of VPS72-defi cient Tregs will be less protected from developing disease than those receiving WT Tregs. Histological analysis can be performed to assess immune profiles and cytokine production in the colon. In addition, the CD45.2+CD4+ T cells isolated from inflamed colon tissues can be gated to analyze FoxP3 expression to determine the role of VPS72 on Treg stability in vivo.
[0384] FoxP3 is a lineage-specific TF required for Treg stability’, particularly in inflammatory conditions. Tregs can lose FoxP3 expression and convert into pathogenic effector T cells under certain inflammatory conditions, suggesting that inflammatory environmental factors may destabilize FoxP3 expression. To determine whether VPS72- deficiency results in Treg depolarization and/or reprogramming, WT and VPS72-deficient Tregs (both natural and in vitro polarized) can be co-cultured with different cytokines known to depolarize Tregs (IL-6, IFN-y, IL-4 and TNF-a. Whether VPS72-deficient Tregs produce Thl, Th2, or Thl7 cytokines can be analyzed and their FoxP3 expression can be examined by intracellular staining. We expect that loss of VPS72 will lead to Treg depolarization and production of Thl, Th2, or Th 17 cytokines, which is further enhanced by co-cultivation with inflammatory’ cytokines.
[0385] Given the diminished FoxP3 levels in VPS72-TregKO cells, the defective Treg immunosuppressive capability’ could be at least partially rescued by overexpression of FoxP3. A rescue experiment with FoxP3 overexpression can be performed. Briefly, sorted YFP+ Tregs from WT or VPS72-TregKO mice can be stimulated for 48h in the presence of CD3/CD28 dynabeads with 2000U hIL-2. After 48h of stimulation, cells will be transfected with FoxP3-overexpressing retrovirus and cultured for 72h. After rest, YFP+GFP+ virus- infected Tregs will be sorted. Naive CD4+CD25- T cells will also be sorted and labelled with APC CFSE cell proliferation dye to use as responder T cells. These labelled naive T cells will be cultured with anti-CD3 and anti-CD28 (2 pg/ml) for 72h together with an increasing ratio of sorted GFP+YFP+ Tregs. The immunosuppressive function of Treg cells can be assessed by flow cytometry based on CFSE dilution.
[0386] Defining the role of H2A.Z in maintaining Treg stabil ity and function. H2A.Z is encoded by two different genes, H2AFZ and H2AFV, which encode two isoforms, H2A.Z1 and H2A.Z2. They can regulate both distinct and overlapping sets of genes in a context- dependent manner. Furthennore. H2A.Z1 and H2A.Z2 can replace each other at TSSs. We generated T cell-specific CD4Cre.H2A.Zl/H2A.Z2 double KO (H2A.Z-TdKO) mice and observed comparable levels of T cells and Tregs in the thymus and significantly reduced levels of peripheral Treg and T cells, suggesting that H2A.Z is not required for thymic T cell and Treg development but is required for peripheral Treg and T cell homeostasis (Fig. 32), which phenotypically resembles VPS72-TKO mice (Fig. 28). To further define H2A.Z function in Treg cells, Treg-specific H2A.Zl-TregKO, H2A.Z2-TregKO, and H2A.Z1.Z2- TregdKO mice can be generated and then dissected if H2A.Z1, H2A.Z2, or both are required for Treg cell development and function, evaluating (1) the frequency and number of Treg cells in the thymus and peripheral organs; (2) Treg immunosuppressive function and reprogramming; and (3) Treg apoptosis and proliferation. This will uncover independent and/or redundant roles for both H2A.Z isoforms in VPS72-mediated Treg stability and function. Because VPS72 acts as a chaperone for H2AZ, similar Treg phenotypes are likely to exist in VPS72.TregKO mice.
[0387] Identifying the mechanisms by which VPS72 and H2A.Z regulate FoxP3 expression. Global gene expression analysis with the latest next generation sequencing tools is useful for gaining insight into cell-specific molecular mechanisms. Thus, RNA-Seq can be performed to identify' the genes regulated by the VPS72-mediated H2A.Z chromatin remodeling in Tregs from VPS72-TregKO, H2A.Z-TregKO, and WT mice. VPS72 is an H2A.Z chaperone that does not directly bind to DNA. CUT&RUN-seq can be used to analyze DNA-protein interactions and has many advantages over ChlP-seq, including generating fewer false-positive binding sites, requiring fewer input cells, and having higher signal-to- noise ratios and higher resolution. H2A.Z CUT&RUN can be performed using 50,000 splenic CD3+CD4+CD25+ Tregs from C57BL/6 mice and identified over 1400 genes and TF binding motifs, including genes related to apoptosis, MAPK, and Tgf- pathways (Fig. 33). Unexpectedly, H2A.Z binding peaks were mapped to the FoxP3 and Tgf-pi regions. Even more unexpectedly, binding motifs for two FoxP3 TFs, RUNX1 and NF AT, were significantly enriched within the FoxP3 promoter and CNS2 regions, while the binding motif for MYB, a Tgf-01 TF, was significantly enriched in the Tgf-pi promoter region. As showed in Fig. 36, these data support that VPS72-H2A.Z mediated chromatin remodeling may regulate FoxP3 by directly interacting with RUNX1 and NF AT or indirectly controlling FoxP3 expression by targeting TGF-pi through MYB. CUT&RUN-seq can be performed in Tregs from VPS72-TregKO, H2A.Z-TregKO, and WT mice to help validate H2A.Z interactions with RUNX1, NF AT, or MYB. The following experiments can be performed: [0388] 1) RNA-Seq profiling to identify the genes regulated by VPS72 and H2A.Z.
CD4+YFP+ Tregs sorted from VPS72-TregKO. H2A.Z-TregKO. and WT mice will be used for bulk RNA-Seq. Differentially expressed genes can be identified.
[0389] 2) Genome- wide profiling of H2A.Z chromatin localization to validate the gene regions enriched by H2A.Z. As shown in Fig. 33, H2A.Z CUT&RUN analysis was successfully performed. A CUT&RUN analysis with H2A.Z can be conducted using sorted YFP+CD4+ Treg cells and iTreg cells from WT and VPS72-TregKO mice, as well as H2A.Z-TregKO mice as a negative control. To identify the genomic distribution of H2A.Z binding peaks, the peaks can be annotated with the priority order (promoter > exon > intron > intergenic) using ChlPseeker (version 1.26.2) when a single peak spans more than two genomic features. For clustering analysis, RPKM values can be calculated using a 5 kb sliding window^ to compare genome-wide ChlP-seq enrichment among samples or promoter regions (TSS ±1 kb) can be used to compare promoter ChlP-seq enrichment among samples. Hierarchical clustering will be performed in R by stats package and hclust function ith RPKM values via Pearson's correlations. We will perform peak calling using model-based analysis of ChlP-seq (MACS) and define the decreasing and increasing number of H2A.Z peaks. The accumulation of H2A.Z peaks in promoter regions may be associated with genes required for Treg function.
[0390] 3) Multi-omics analysis to determine VPS72 target genes. To identify the target genes regulated by VPS72/HZA.Z chromatin modification, an integrated CUT&RUN- seq and RNA-seq analysis can be implemented to determine the genome-wide chromatin occupancy of H2A.Z in VPS72-TregKO and WT mice. From GO and Ingenuity Pathway Analysis (IP A), specific genes and gene clusters can be selected and validated. A correlation analysis of genome-wide co-occupancy can be performed. Differentially expressed genes within identified open chromatin regions and TF binding sites will be considered as potential VPS72/H2A.Z direct targets for Treg stability and function. The TFs that have binding motifs in chromatin regions regulated by VPS72/H2A.Z can be identified as functionally related TFs. Combined with RNA-seq analysis, the downstream target genes of those TFs can be evaluated in combination with related IPA and KEGG analysis. These target genes can be confirmed at the protein level by flow cy tometry' or Western blot, or at the mRNA level by RT-PCR.
[0391] 4) To assess whether VPS72-H2A.Z cooperates with putative TFs in Tregs. If analysis from (3) confirms that RUNX1 and NF AT localize to FoxP3 promoter and CNS2 regions and MYB localizes to the Tgtpi promoter region, an immunoprecipitation can be performed using Tregs from VPS72-TregKO and WT mice to test if H2A.Z interacts with MYB, RUNX1 and NF AT. Cell lysates can be incubated with anti-H2A.Z antibody and protein interactions can be analyzed by immunoblotting with anti -MYB, anti-RUNXl, and anti-NFAT antibodies with HRP-mediated visualization using the Western Lighting Plus ECL detection kit.
[0392] 5) To test whether MYB. RUNX1, and NF AT directly regulate H2A.Z dependent FoxP3 expression, CUT&RUN-PCR can be performed to see if MYB, RUNX1, and NF AT can bind to the TGF-pi or FoxP3 promoters in Tregs from VPS72-TregKO and WT mice, using H2A.Z-TregKO as a negative control. CUT&RUN DNA obtained from anti- MYB, RUNX1 and NF AT pulldown will be purified and quantified by real-time PCR.
[0393] Loss or decrease of VPS72 can likely decrease Treg expression of surface markers related to immunosuppressive Treg function, such as ICOS, CTLA-4, PD-1, and CD44 and lead to reduced IL- 10 production and increased Treg immune suppressive function in vitro and in vivo. In addition, in vitro overexpression of FoxP3 in VPS72-deficient Tregs may partially rescue immunosuppressive function. Given that VPS72 regulates chromatin structure through H2A.Z exchange, specific H2A.Z1 or H2A.Z2 deletion in Tregs can impair Treg immunosuppressive function. However, H2A.Z1 and H2A.Z2 may be functionally redundant; thus, deletion of both H2A.Z1 and Z2 may be required to mediate Treg functional deficiency. By using bulk RNA-Seq and H2A.Z CUT&RUN-seq data to validate if VPS72/H2A.Z directly regulates FoxP3 expression through interacting with RUNX1 and NFAT, VPS72-regulated genes can be identified that control Treg cell maintenance, stability7, and function (Fig. 36 A). However, if TGF-|H expression is downregulated in VPS72- TregKO Tregs and it can be confirmed that H2A.Z maps onto the TGF-(31 region with MYB TF. then VPS72/H2A.Z can also indirectly regulate FoxP3 expression through a MYB-TGF- pi-Smad2/3/4 pathway (Fig. 36 B). Also, through integrative multi-omics analysis, new VPS72/H2A.Z direct targets can be identified that regulate Treg cell maintenance, stability, and function. Finally, H2A.Z may be also deposited by other chaperone proteins and that VPS72 may directly target non-histone proteins; thus, gene expression profiles from VPS72- TregKO and H2A.Z-TregKO mice may be slightly different. It may be beneficial to focus on the genes regulated in both VPS72 and H2A.Z KO mice. In addition to FoxP3, other signaling molecules can be identified that are involved in Treg homeostasis and immune suppressive function, under direct and/or indirect control of VPS72/H2A.Z mediated chromatin modification.
[0394] Evaluating the role of VPS72 and H2A.Z in antitumor immunity. Given that VPS72/H2A.Z genetic deletion in Tregs impairs Treg immunosuppressive activity and enhances conventional T cell immunity, a rationale is provided for targeting VPS72 to stimulate antitumor immunity. Furthermore, VPS72 and FoxP3 expression is upregulated in tumor Treg cells (Figs. 24 & 25), and VPS72 is an oncogene. Accordingly, suppressing VPS72 results in increased apoptosis and cell cycle arrest in cancer cells, which further supports the present disclosure that VPS72 is a potential target for antitumor immune therapy. Indeed, the inducible VPS72-TregiKO mouse model can be used to analyze the degree to which Treg VPS72 suppression enhances the anti-tumor response against pre-established B16 melanoma (Fig. 34). Thus, it can follow that VPS72/H2A.Z mediated chromatin modification stabilizes Treg populations within xenograft and spontaneous tumors, such as melanoma, resulting in tumor immunosuppression and enhanced tumor growth.
[0395] Analyzing the efficacy of the anti-tumor immunity in VPS72-TregiKO and H2A.Z-TregiKO mice. To rule out potential bias from using specific tumor types, multiple syngeneic tumor models can be used, including MC-38 colon adenocarcinoma, EG7 lymphoma, and LLC1 Lewis lung carcinoma to determine the role of VPS72 and H2AZ in anti-tumor immunity in VPS72-TregiKO and H2A.Z-TregiKO mice. As mentioned in Fig. 34, age- and sex-matched inducible VPS72-TregiKO, H2A.Z-TregiKO, and WT mice can be subcutaneously injected with tumor cells, and tumor growth and progression can be monitored. After 7-10 days of tumor injection, the mice can be injected with TAM for 5 consecutive days. To test if VPS72 deletion in Tregs promotes antitumor immunity, tumorinfiltrating lymphocytes can be isolated, and the numbers and activation of T cells (CD4 and CD8) can be measured. We can also examine the immune responses in the peripheral lymphoid organs by analyzing expression of granzyme B and IFN-y in CD8+ T cells. It is possible for targeted VPS72/H2AZ suppression to result in an elevated antitumor immune response and that VPS72/H2AZ-TregiKO mice can have better tumor rejection than WT mice. To confirm that Treg tumor infiltration can be reduced by VPS72 deletion, FoxP3 MFI and levels of suppressive markers (PD-1, CTLA-4, GITR, and CD 103) can be measured by flow cytometiy in CD4+CD25+FoxP3+ Tregs. Because immune suppression in the TME is also promoted through myeloid-derived suppressive cells (MDSCs), whether MDSCs are reduced in the tumor can be more closely analyzed.
[0396] scRNA-seq and scATAC-Seq analysis. To examine immuno-profiles in the TME of VPS72-TregiKO, H2A.Z-TregiKO, and WT mice, scRNA-seq and scATAC-Seq can be used to determine the transcriptome and chromatin landscape of the various immune cells, especially potential subpopulations of Tregs regulated by VPS72/H2A.Z. Single-cell suspensions of the tumors (n=3 per group) can be generated and CD45+ immune cells can be sorted in a cell sorting core to generate sc-RNA-Seq libraries using a 10X Genomics Chromium Controller, for example. The libraries can be sequenced by Illumina HiSeq4000. The FindAllMarkers function in Seurat can be implemented to identify differentially expressed genes between clusters with a Bonferroni adjustment <0.05 as a statistical significance threshold. To infer the functionality’ of genes within each cluster, a pathway analysis using IPA (Qiagen Bioinformatics) can be carried out. An integrative analysis of scATAC-seq and scRNA-seq data can be performed, especially7 in Treg cells, using the coupled nonnegative matrix factorizations (coupled NMF) method. The VPS72-regulated Treg subset-specific gene signatures can be obtained from scRNA-seq and regulatory’ elements (chromatin accessibility regions and TF motifs) from scATAC-seq analysis.
[0397] Imaging Mass Cytometry7 (IMC) or CyTOF. To determine the cellular interactions among different immune cells and between immune and tumor cells, tumor tissue sections can be analyzed using imaging mass cytometry (IMC) with 45 immune related markers. The multiplex metal-labeled staining allows for the examination of tumor immune populations within architecturally preserved tumor tissues from WT and KO mice. IMC raw images can be transformed into single-cell data using a combination of Ilastik and CellProfiler softw are. Data from all regions of interest can be imported into histoCAT software to create a global t-SNE map of all samples, which can be segregated using PhenoGraph clustering. Neighborhood analysis to infer cell-cell interactions can be performed using imcRtools.
[0398] Examining VPS72 and H2A.Z suppression in Tregs during antitumor immunity7 against spontaneous melanoma. Despite being a useful xenograft melanoma model, non-orthotopic growth may not be ideal. To address this concern, the effects of Treg-specific VPS72 ablation can be analyzed in the newly developed orthotopic melanoma model of BrafV 600EPTEN mice, which develop clinically relevant spontaneous melanoma (Fig. 37). Approximately lcm2 sections of skin graft from BrafV600EPTEN mice can be transplanted into WT and VPS72-TregiKO or H2A.Z-TregKO mice. Seven days after transplantation, the skin graft can be treated by topical application of 4-hydroxy -tamoxifen (TMX). Melanoma development in the graft can be monitored weekly for the first 5 weeks and daily thereafter. To characterize the dynamics of tumor development, recipient mice can be grouped and euthanized during early (day 30), middle (day 45), and late phase (day 60 or after) tumor grow th. The immune response in peripheral lymphoid organs (spleen and lymph nodes) and inside tumors can be characterized to ascertain the degree to which (1) Treg-specific VPS72/H2A.Z suppression delays onset of tumor development; (ii) Treg-specific VPS72 suppression results in reduced tumor growth; and (iii) Treg-specific VPS72/EI2A.Z suppression enhances antitumor immune responses in recipient mice. Overall, Treg function is likely to be highly impaired (Tregs can be isolated and their and function checked in vitro), and tumor growth can be inhibited as a result of increased numbers of activated CD8 cells. Collectively, it appears as though targeting VPS72/EI2A.Z in Tregs can slow tumor growth by inhibiting the Treg immunosuppressive function and enhancing endogenous antitumor CD8 T cell responses. In BrafV600EPTEN mice, metastasis can be detected in lymph nodes and lungs during the later stages of the disease. Recipient WT and VPS72-TregiKO or H2AZ-TregiKO mice can be euthanized 60 days after TMX treatment. Then metastases in draining lymph nodes and lung can be analyzed to further investigate VPS72 deficiency in melanoma metastasis.
[0399] Evaluating the synergy of Treg-specific VPS72 suppression with checkpoint blockade (anti-PD-1) in antitumor therapy. VPS72 depletion, which decreases Treg suppressive function in VPS72-TregiKO mice, combined with anti-PD-1 treatment, which enhances Teff cell function, will have a beneficial synergistic effect on antitumor immunity. To ascertain the degree to which this depletion has an impact, 8- to 10-week-old VPS72- TregiKO and WT mice can be injected with B16 melanoma cancer cells. Tumor size can be measured, and anti-PD-1 treatment can begin when tumors reach 100 mm3 (usually 7-10 days after tumor cell injection). Mice can be injected with TAM and different doses of anti- PD-1 every7 3 days (0-300 pg). Teff cell activation and Treg FoxP3 level and immunosuppressive function can also be analyzed as described herein. Thus, genetic VPS72/H2A.Z suppression can enhance the therapeutic efficacy of anti-PD-1 tumor growth suppression in mice. Genetic deletion of VPS72 can enhance antitumor immunity in subjects. In the combined therapy setting, Treg-specific VPS72 deletion will dramatically enhance the antitumor efficacy of immune check point blockade wi th anti-PD-1.
[0400] Identifying TME-specific factors that promote VPS72-H2A.Z exchange to control Treg adaptation. Both Treg frequency and FoxP3 expression levels required for Treg stability' and adaptation to the TME are significantly higher in Tregs from B16 melanoma tissue than in Tregs from peripheral lymphoid organs (Fig. 24). Unexpectedly, a significant increase in VPS72 and H2A.Z expression in tumor-Tregs was detected (Fig. 25).
Furthermore, dramatically higher VPS72 expression in tumor-Tregs from human melanoma and liver cancers was uncovered, as compared with Tregs from human PBMC (Fig. 26). Accordingly, VPS72-mediated H2A.Z remodeling likely regulates Treg stability and function through targeting FoxP3 expression, and it follows that TME-specific factors induce VPS72 expression, which controls Treg stability and adaptation through targeting FoxP3. TME- specific factors can be advantageous for reprogramming the immune response during tumor immune escape. Intratumoral Tregs have the capability to adapt to the harsh elements promoted by the fast-growing malignant tumor cells within the TME. such as cytokines (e.g., TGF-P), metabolic changes (low glucose and amino acids / high lactate), hypoxia, high CO2 concentrations, and low pH. To explore this, it can be analyzed whether TME metabolic factors can reshape Tregs by inducing VPS72 expression. Unexpectedly, Tregs cultured in low glucose medium had significantly enhanced VPS72 expression (Fig. 35). indicating that tumor-infiltrating Tregs can maintain their immunosuppressive function through VPS72 upregulation in response to low glucose. Thus, other TME factors may affect Treg VPS72 expression. The molecular mechanisms underlying how these TME factors potentiate Treg immunosuppressive function by inducing VPS72 can be further elucidated. Unexpectedly, it was uncovered that the TGF-P-related TF Smad, the hypoxia-related TFs HIFla and Spl, and the glucose sensing TF FOxO3a are located in the VPS72 promoter region. Thus, it is possible for TME-specific factors to upregulate VPS72 through these TFs (Fig. 38). In vitro polarized Tregs can be used for further analysis and natural Tregs (CD4+CD25+YFP+) sorted from mice can be used for further validation.
[0401] Tumor conditioned medium induces VPS72 expression in Tregs. Conditioned media from Bl 6, LLC1, and EG7 cells can enhance Treg stability and immunosuppressive function through upregulation of lineage TF FoxP3 deubiquitinases, and TGF-pi, hypoxia, and glucose are examples of key TME-related factors. Mouse CD4+CD25+YFP+ Tregs in conditioned medium from Bl 6, LLC1, and EG7 cells can be cultivated to provide insight on tumor cells’ production of soluble factors that induce VPS72 expression. Thus, it is possible for different tumor conditioned media to increase VPS72 expression and downregulate FoxP3 expression.
[0402] It is possible that TGF-P produced by tumor cells induces VPS72 expression in Tregs. Tumor cells and infdtrating immune cells produce a variety of cytokines (e.g., TGF- pi) that promote immune evasion by inducing Treg activity and maintaining Treg stability. TGF- suppression alters tumor-conditioned media-induced USP21 and USP22 expression, which can be analyzed using a TGF-P inhibitor (TGFpi). Two further experiments can be performed: (1) Tregs cultured in tumor-conditioned medium with or without TGFpi LY3200882 at 25 pg/ml; and (2) Tregs cultured in regular medium with or without 20ng/mL TGF-P or with TGFpi for 8 hours. VPS72 expression can be determined by qPCR and FACS analysis. This can indicate how the TME cytokine TGF- P induces VPS72 expression in Tregs. TGF- P induces gene transcription through either SMAD activation or non-SMAD pathways. Genomic analysis identifies multiple conserved SMAD4 binding sites in VPS72 promoter regions (Fig. 39). Together with the fact that tumor TME-induced VPS72 expression and TGF- p induced Tregs (iTreg) are defective in VPS72KO mice (Figs. 25 & 31), it follows that TGF- P induces VPS72 gene expression through activation of SMAD4 TF. A ChIP assay or CUT & RUN can be used to determine the binding of SMAD4 onto VPS72 promoters in Tregs upon TGF- P or tumor-conditioned media cultivation. It is possible for both TGF- p and conditioned media from B16 and LLC1 but not EG7 cells to induce SMAD binding to the VPS72 promoter. If the SMAD protein binds to a VPS72 promoter, Tregs sorted from CD4.Smad4KO mice can be used to confirm whether TGF- P induces VPS72 expression in a SMAD-dependent manner.
[0403] Hypoxia may be used to induce VPS72 expression in Tregs. Hypoxia often arises in the TME through the uncontrolled oncogene-driven proliferation of cancer cells. Tregs can be cultured under hypoxic (1% oxygen) or normoxic conditions for 12 hours. VPS72 expression can be measured with qPCR and western blotting. Given that multiple hypoxia-related TFs, including HIFla and SP1, are located in the VPS72 promoter region, it follows that hypoxia in the TME is involved in VPS72 induction to maintain FoxP3 stability. The molecular mechanism can be dissected and ChIP analysis can be performed to assess binding of HIFla or SP1 at the VPS72 promoter. Once direct binding of HIFla or SP1 to the VPS72 promoter is detected, it can show' an extent to which loss of HIFla in Tregs abolishes hypoxia-induced VPS72 expression.
[0404] Elucidating the effects of tumor metabolites on VPS72 expression and immunosuppressive functions. In addition to cytokines and hypoxia, metabolic changes in the TME such as low glucose and amino acids, high lactate, elevated C02, and low pH, can potentially have a role in VPS72-mediated induction of Treg immunosuppressive activity. Example factors to assess include, but are not limited to: (i) VPS72 expression induction in low glucose medium. FoxP3 levels are maintained under glucose-deprived conditions and glucose-deprived conditions promote VPS72 expression (Fig. 35). When glucose availability is insufficient to generate enough ATP, AMPK is activated, which consequently promotes downstream AMPK TFs, including FOXO3. Unexpectedly, a FOXO3 binding site is located in the VPS72 promoter region (Fig. 38). Thus, the molecular mechanisms can be dissected by performing a ChIP analy sis to detect FOXO3 binding at the VPS72 promoter. It follows that treating Tregs with AMPK-specific inhibitors will abolish, at least partially, the increased VPS72 expression seen in Tregs in low glucose medium, (ii) Low amino acid levels involved in VPS72 induction in Tregs. Similarly, Tregs can be cultured in low amino acid medium and whether the expression of VPS72 is altered can be analyzed. It follows that VPS72 expression is induced in low amino acid conditions. The mTOR (mammalian target of rapamycin) pathway is often inhibited in cells cultivated in medium lacking sufficient amino acids. Rapamycin treatment is a potential treatment option for recapitulating the effect of low amino acids to induce Treg VPS72 expression.
[0405] Expected Outcomes and Alternative Approaches. TME-specific factors (cytokines, hypoxia, and metabolic changes) can be more particularly identified that reshape Tregs through selective induction of VPS72-mediated H2A.Z remodeling. Previously, the molecular mechanisms underlying how the TME promotes Treg function resulting in tumor immune evasion were underappreciated. Potential alternatives: (i) In addition to TGF-P, other cytokines such as IL- 10 and many inflammatory cytokines (IL-1 P, IL-6 & TNF-a, etc.) produced by tumor cells or by infiltrating immune cells can potentially be involved in regulating VPS72 expression in Tregs. To assess this, a panel of inflammatory and antiinflammatory cytokines using in vitro culture to identify all cytokines that induce Treg VPS72 expression can be used. Once the cytokines are identified, the underlying molecular pathways can be dissected, (ii) Hypoxia can also induce gene expression through HIF- independent mechanisms, such as the AMPK-FOXO3 pathway. Thus, FOXO3 can be activated by both hypoxia and low glucose, which increase VPS72 expression, (iii) The Smad, HIFla, and AMPK-FOXO3 pathways can play a role in VPS72 induction in Tregs under TGF-P, hypoxia, and metabolic stress. Unbiased bulk RNA-Seq and scRNA-seq analysis discussed herein can be used to identify additional pathways involved in VPS- 72/H2A.Z-induced Treg stability and function in the TME, revealing aspects of the molecular mechanisms, (v) Moreover, human Tregs can be used to validate results from TME-induced VPS72 expression in mouse Tregs. To achieve this, CD4 T cells from PBMCs can be isolated and polarized into iTregs. The effects of TME-specific factors, including cytokines (TGF-0, hypoxia, and metabolites (low glucose and amino acids) on human Treg VPS72 expression can be determined by qPCR and western blotting. In addition to polarized Tregs, data can be validated using Tregs (CD4+CD25+FR4+ or CD4+CD25+CD1271ow) directly sorted from PBMCs.

Claims

CLAIMS We claim:
1. A method of altering an immune cell response in a subject in need thereof, said method comprising administering an inhibitory7 agent that inhibits VPS72 activity7 and/or expression in an immune cell of the subject.
2. The method of claim 1, wherein inhibiting VPS72 activity7 and/or expression by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% alters an immune cell response.
3. The method of any one of claims 1-2, wherein the immune response altered comprises hematopoiesis.
4. The method of claim 3, wherein altering hematopoiesis modifies specific hematopoietic stem cell lineages.
5. The method of claim 4, wherein modifying the specific hematopoietic stem cell lineage is a therapeutic treatment for a hematological disease.
6. The method of any one of claims 1 -2, wherein inhibiting VPS72 activity and/or expression reduces tissue resident macrophage responses by reducing tissue resident macrophage numbers.
7. The method of claim 6, wherein reducing tissue resident macrophage numbers include numbers of Langerhan cells, a tissue resident macrophage of skin, as a therapeutic treatment for a skin disease or wound healing.
8. The method of claim 7, wherein the skin disease is Langerhans cell histiocytosis.
9. The method of claim 6, wherein reducing tissue resident macrophage number is a therapeutic treatment for autoimmune disease.
10. The method of claim 9, wherein the autoimmune disease is systemic sclerosis, systemic lupus erythematosus, or other organ specific autoimmune disease.
11. The method of claim 6, wherein reducing the numbers of tissue resident macrophage, known as tumor associated macrophages, is a treatment for cancer.
12. The method of claim 11, wherein the cancer treated is myeloma, lung, liver, or kidney.
13. The method of claim 6, wherein reducing tissue resident macrophage number is a therapeutic treatment for metabolic syndrome.
14. The method of claim 13, wherein treatment of metabolic syndrome treats symptoms of metabolic syndrome comprising non-alcoholic fatty liver disease (NAFLD), heart disease, diabetic retinopathy, stroke risk, obesity, hypertension, or a neurodegenerative disease associated with metabolic syndrome.
15. The method of claim 6, wherein reducing tissue resident macrophages number is a therapeutic treatment for age-related macrophage dysfunction (ARMD).
16. The method of claim 15, wherein treating ARMD increases immunity to infections, improves vaccine response, or reduces susceptibility to autoimmune disease or cancer.
17. The method of any one of claims 1-2, wherein inhibiting VPS72 activity and/or expression reduces Treg cell functional responses.
18. The method of claim 17, wherein reducing Treg cell function responses is a therapeutic treatment for cancer or an autoimmune disease.
19. The method of claim 17, wherein reducing Treg cell function responses promotes endogenous antitumor responses.
20. The method of claim 18, wherein the endogenous antitumor responses are CD8 T cell responses.
21. The method of claim 17, wherein inhibiting VPS72 activity and/or expression reduces Treg expression of surface markers related to immunosuppressive function.
22. The method of any one of claims 1-21 , wherein a peptide inhibitor blocks VPS72 from binding to H2A.Z-H2B dimer by competitively inhibiting the binding of VPS72 with H2A.Z-H2B.
23. The method of claim 22, wherein amino acids in the peptide inhibitor correspond to VPS72 amino acids at positions F29, Y30, Y34, F37, D43, E35, and Y64.
24. The method of any one of claims 1-23, wherein inhibiting the VPS72 activity comprises contacting a cell with an isolated or a VPS72 peptide inhibitor having an amino acid sequence which is at least 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 2.
25. The method of any one of claims 1-21, wherein inhibiting the VPS72 expression comprises contacting a cell with an isolated antisense oligonucleotide operable to bind to a mRNA, or a portion thereof which encodes VPS72, having an nucleic acid sequence which is at least 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% identical to an nucleic acid sequence of SEQ ID NO: 2.
26. The method of any one of claims 22-25, wherein the isolated antisense oligonucleotide or peptide inhibitor further comprises a cell penetrating peptide.
27. The method of any one of claims 22-26, wherein the isolated antisense oligonucleotide or peptide inhibitor further comprises a nuclear locating signal.
28. The method of any one of claims 22-27, wherein the isolated antisense oligonucleotide or peptide inhibitor further comprises a cell-specific targeting moiety.
29. The method of claim 28, wherein the cell-specific targeting moiety comprises a cellspecific receptor antigen, an antibody, or antibody fragment.
30. The method of any one of claims 26-28, wherein the isolated antisense oligonucleotide or peptide inhibitor further comprises a linker peptide or chemical conjugation.
31. The method of any one of claims 22-30, wherein a therapeutically effective concentration of the isolated antisense oligonucleotide or peptide inhibitor, or pharmaceutical salt thereof, is formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
32. The method of claim 31, wherein the pharmaceutical composition includes a chemotherapeutic drug or an immunotherapeutic drug.
33. The method of any one of claims 22-31, wherein the isolated antisense oligonucleotide or peptide inhibitor, or pharmaceutical salt thereof, or pharmaceutical composition is linked with, or covalently or non-covalently bonded with or encapsulated by, a carrier selected from the group consisting of organic nanoparticles or microparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, extracellular vesicles, exosomes, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, polyplexes: polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, roN-e-caprolactone (PCL), starch hydroxyethyl (HES, MEA), poly glycolate (PGA), poly (lactic-co-glycolide) , polylactide (PLA), poly (d, 1-lactide-co- glycolide) (PLGA), polyethylene glycol (PEG), (2-Hydroxypropyl) methacry lamide (poly (HPMA) or PHPMA); and dextran; dendrimers, selected from the group consisting of: polyether-hydroxylamine (PEHAM), poly amidoamine (PAMAM), polyesteramine, polypropylene imine and poly glycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA) nanofibers, of polylactide (PLA), polyethylene oxide and poly-s-caprolactone (PCL); or inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD.
34. An isolated polypeptide or oligonucleotide for use in the manufacture of a medicament for altering an immune cell response, which is a therapeutic treatment for cancer, metabolic syndrome, inflammatory' disease, autoimmune disease, or age-related macrophage dysfunction.
35. A method of altering an immune cell response in a subject in need thereof, said method comprising administering an agent that regulates FOXP3 expression via VPS72 activity and/or expression and/or H2A.Z activity and/or expression in an immune cell of the subject.
36. The method of claim 35, wherein the regulation of FOXP3 expression is indirect through a MYB-TGF-pi-Smad2/3/4 pathway.
PCT/US2024/050927 2023-10-12 2024-10-11 Methods and compositions for improving immunotherapy Pending WO2025080949A1 (en)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN TIEJUN, TU YINUO, LV DONGNUO, LIN KUNPENG, TANG HUI, HUANG WENJIE: "Vacuolar protein sorting-associated protein 72 homolog (VPS72) binding to lysine acetyltransferase 5 (KAT5) promotes the proliferation, invasion and migration of hepatocellular carcinoma through regulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway", BIOENGINEERED, LANDES BIOSCIENCE, US, vol. 13, no. 4, 1 April 2022 (2022-04-01), US , pages 9197 - 9210, XP093305772, ISSN: 2165-5979, DOI: 10.1080/21655979.2022.2056692 *
GUI YONGHUI, LIU XUENI, WANG CHAO, YANG PENG: "Bioinformatics Analysis of Prognostic Significance of VPS72 and Correlations with Immune Infiltrates in Hepatocellular Carcinoma", RESEARCH SQUARE, 22 February 2021 (2021-02-22), XP093305774, Retrieved from the Internet <URL:https://assets-eu.researchsquare.com/files/rs-265387/v1/1db64768-1a0d-4805-93b5-1b436dc21132.pdf?c=1631880714> DOI: 10.21203/rs.3.rs-265387/v1 *
LIU FURONG, LIAO ZHIBIN, QIN LU, ZHANG ZE, ZHANG QIAOFENG, HAN SHENQI, ZENG WEIFENG, ZHANG HONGWEI, LIU YACHONG, SONG JIA, CHEN WE: "Targeting VPS72 inhibits ACTL6A/MYC axis activity in HCC progression", HEPATOLOGY, 71ST ANNU MEET AM ASSOC STUDY LIVER DIS (AASLD) · 2020-11-13 / 2020-11-17 · VIRTUAL, N/A · ABST 215, vol. 78, no. 5, 1 November 2023 (2023-11-01), pages 1384 - 1401, XP093305771, ISSN: 0270-9139, DOI: 10.1097/HEP.0000000000000268 *
SUBEDI KALPANA, JIE WANG, LI ZHOU, QING-SHENG MI : "Vacuolar Protein Sorting-Associated Protein 72 (VPS72) is Required for Hematopoietic Stem Cell Development", J. IMMUNOL., vol. 212, no. 1, 1 May 2024 (2024-05-01), pages 0065_4931, XP093305779 *

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