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WO2025080949A1 - Méthodes et compositions pour améliorer l'immunothérapie - Google Patents

Méthodes et compositions pour améliorer l'immunothérapie Download PDF

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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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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

La présente divulgation concerne des compositions et des méthodes de modification d'une réponse de cellules immunitaires chez un sujet en ayant besoin, ladite méthode comprenant l'administration d'un agent inhibiteur de l'activité et/ou l'expression de VPS72 dans une cellule immunitaire du sujet.
PCT/US2024/050927 2023-10-12 2024-10-11 Méthodes et compositions pour améliorer l'immunothérapie Pending WO2025080949A1 (fr)

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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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