WO2021141862A1

WO2021141862A1 - Methods of modulating sting pathway activation

Info

Publication number: WO2021141862A1
Application number: PCT/US2021/012094
Authority: WO
Inventors: David H. Raulet; Rutger D. Luteijn
Original assignee: The Regents Of The University Of California
Priority date: 2020-01-08
Filing date: 2021-01-04
Publication date: 2021-07-15

Abstract

Methods of modulating stimulator of interferon genes (STING) pathway activation are provided. Aspects of the methods include modulating Phosphatidylinositol 4-Kinase Beta (PI4KB) Golgi recruitment, e.g., via modulation of acyl-coenzyme A binding domain containing 3 (ACBD3) and/or oxysterol-binding protein (OSBP) activity, to modulate STING pathway activation. Also provided are compositions and kits for use in practicing the subject methods. The methods and compositions find use in a variety of applications, including therapeutic applications, such as methods of treating cancer or an inflammatory disease.

Description

METHODS OF MODULATING STING PATHWAY ACTIVATION

GOVERNMENT RIGHTS

This invention was made with Government support under contract AM 13041 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 62/958,598, filed January 8, 2020, the disclosure of which application is incorporated herein by reference.

INTRODUCTION

The innate immune system, once activated, is directly protective to the host and also initiates broader immune responses mediated by T cells, B cells and NK cells. The accumulation of DNA in the cytosol of infected, cancerous or mutant cells can trigger an innate immune response via the cGAS/STING pathway. The response is initiated by the binding of cytosolic DNA to the cytosolic enzyme cGAMP synthase (cGAS), leading to the synthesis of the second messenger 2’3’- cyclic GMP-AMP (2’3’-cGAMP). 2’3’-cGAMP activates the protein ‘stimulator of interferon genes’ (STING), which in turn activates the transcription factors IRF3 and NF-KB, and consequently the production of cytokines, including type I interferons, that support a broader immune response.

The cGAS/STING pathway senses cytosolic DNA originating from viruses and bacteria. STING is also activated by cytosolic self-DNA, which accumulates in cells in certain autoinflammatory disorders, including Aicardi-Goutieres Syndrome and systemic lupus erythematosus. Furthermore, cytosolic DNA accumulates in cells subjected to DNA damage, as occurs in tumor cells, resulting in activation of the cGAS/STING pathway and the initiation of an anti-tumor immune response.

The natural anti-tumor immune response can be weak. An amplified anti-tumor immune response can occur when STING agonists, such as cyclic dinucleotides (CDNs), are introduced into the tumor microenvironment, leading to immune activation and tumor regression.

SUMMARY

Methods of modulating stimulator of interferon genes (STING) pathway activation are provided. Aspects of the methods include modulating Phosphatidylinositol 4-Kinase Beta (PI4KB) recruitment to the Golgi, e.g., via modulation of acyl-coenzyme A binding domain containing 3 (ACBD3) and/or oxysterol-binding protein (OSBP) activity, to modulate STING pathway activation. Also provided are compositions and kits for use in practicing the subject methods. The methods and compositions find use in a variety of applications, including therapeutic applications, such as methods of treating cancer or an inflammatory disease.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 A to 3C provide experimental details as reported in the experimental section, below.

DEFINITIONS

The following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991 ) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

The term "administration" or “administering” as used herein with regard to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid, refers without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. By "administered together" it is not meant to be implied that two or more agents be administered as a single composition. Although administration as a single composition is contemplated by the present disclosure, such agents may be delivered to a single subject as separate administrations, which may be at the same or different time, and which may be by the same route or different routes of administration. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents; “affinity” can be expressed as a dissociation constant (Kd).

The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

The terms “cell uptake” and “cellular uptake” are used interchangeably herein and refer to the movement of a compound from the extracellular environment or matrix and into a cell, e.g., to the cytoplasm of a cell.

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

The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2.

"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The terms "subject", “individual” and "patient" are used interchangeably and refer to a member or members of any mammalian or non-mammalian species that may have a need for the pharmaceutical methods, compositions and treatments described herein. Subjects and patients thus include, without limitation, primate (including humans), canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)), avian, and other subjects. Humans and non-human animals having commercial importance (e.g., livestock and domesticated animals) are of particular interest.

"Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, particularly humans. Non-human animal models, particularly mammals, e.g., primate, murine, lagomorpha, etc. may be used for experimental investigations.

"Treating" or "treatment" of a condition or disease includes: (1) preventing at least one symptom of the conditions, i.e., causing a clinical symptom to not significantly develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. As used herein, the term “treating” is thus used to refer to both prevention of disease, and treatment of pre-existing conditions. For example, where the cyclic-di-nucleotide active agent is administered, the prevention of cellular proliferation can be accomplished by administration of the subject compounds prior to development of overt disease, e.g., to prevent the regrowth of tumors, prevent metastatic growth, etc. Alternatively, the compounds are used to treat ongoing disease, by stabilizing or improving the clinical symptoms of the patient.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

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

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

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

While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

Methods of Modulating STING Pathway Activation

As summarized above, methods of modulating STING pathway activation in a cell are provided. By modulating STING pathway activation is meant altering STING pathway activation, e.g., by increasing STING pathway activation or decreasing STING pathway activation, or by altering the specific outcomes of STING pathway activation with respect to which cytokines or mediators that are produced. As such, embodiments of the invention include methods of increasing STING pathway activation in a cell. The magnitude of increase in such embodiments relative to a suitable control (e.g., where a PI4KB Golgi recruitment modulating agent, such as described below, is not employed) may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. Other embodiments of the invention include methods of decreasing STING pathway activation in a cell. The magnitude of decrease in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more.

Aspects of the methods include modulating Phosphatidylinositol 4-Kinase Beta (PI4KB) recruitment to the Golgi in a cell (i.e., PI4KB Golgi recruitment), e.g., via modulation of acyl-coenzyme A binding domain containing 3 (ACBD3) and/or oxysterol-binding protein (OSBP) activity, to modulate STING pathway activation. Phosphatidylinositol 4-kinase beta (PI4KB) is a soluble protein shuttling between the cytoplasm and the nucleus, and can be recruited to the membranes of the Golgi system via protein-protein interactions, e.g. with small GTP binding proteins Arf1 and Rab11 , or a Golgi adaptor protein ACBD3. By modulating PI4KB recruitment to the Golgi is meant altering or changing recruitment of PI4KB to the Golgi apparatus. In some instances, the methods include increasing PI4KB Golgi recruitment in a cell. The magnitude of increase in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. Other embodiments of the invention include decreasing PI4KB Golgi recruitment in a cell. The magnitude of decrease in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more.

In those embodiments that increase PI4KB Golgi recruitment in a cell, e.g., where STING pathway activation is desired, the methods may include increasing ACBD3 activity and/or decreasing OSBP activity. As such, the methods may include increasing ACBD3 activity in the cell. The magnitude of increase in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. Alternatively, the methods may include decreasing OSBP activity in the cell. The magnitude of decrease in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. In some instances, the methods include both increasing ACBD3 activity in the cell and decreasing OSBP activity in the cell.

In those embodiments that decrease PI4KB Golgi recruitment in a cell, e.g., where inhibition of STING pathway activation is desired, the methods may include decreasing ACBD3 activity and/or increasing OSBP activity. As such, the methods may include decreasing ACBD3 activity in the cell. The magnitude of decrease in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. Alternatively, the methods may include increasing OSBP activity in the cell. The magnitude of increase in such embodiments relative to a suitable control may vary, where in some instances the magnitude is 2-fold or more, such as 5-fold or more, including 10-fold or more. In some instances, the methods include both decreasing ACBD3 activity in the cell and increasing OSBP activity in the cell.

In some instances, modulating STING pathway activation means increasing or enhancing the activity of a STING agonist, e.g., cyclic dinucleotide (CDN) or other STING agonist (e.g., amidobenzimidazole STING receptor agonists, such as described in Ramanjulu et al., "Design of amidobenzimidazole STING receptor agonists with systemic activity," Nature (2018) 564: 439-443), in a cell, in vitro or in vivo. When STING pathway activation is increased, one or more activities of a STING agonist, e.g., CDN or other STING agonist, of interest can also be increased or enhanced. In certain instances, activities of a STING agonist that are increased or enhanced include, but are not limited to, production of type I interferon (IFN), e.g., to provide an anti-tumor immune response or an immune response against a pathogen, and intercellular 2’3’-cGAMP signaling, e.g., between virus- infected cells and uninfected cells or between tumor cells and non-tumor cells. In certain cases, the parameter of interest, e.g., production of a type I interferon in a cell, is increased or enhanced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 2-fold or more, 3-fold or more, or even more, e.g., relative to a suitable control.

Modulating STING pathway activation is meant to encompass enhancing the treatment of a pathologic or disease condition in which a STING agonist, e.g., CDN, finds use, e.g., relative to treatment in the absence of a STING pathway activation modulating agent. Pathologic or disease conditions of interest are described herein and include, but are not limited to, cellular proliferative disease, cancer, autoimmune or inflammatory disease, viral infection (e.g., hepatitis virus), infections with intracellular bacteria and parasites. Enhancing the treatment of a pathologic or disease condition may include amelioration of the symptoms of a particular condition, arresting or reducing the development of the disease or its symptoms, and/or stabilizing or improving the clinical symptoms of the patient. Modulating STING pathway activation is also meant to encompass treatment of a pathologic or disease condition with reduced amounts of a STING agonist as compared to what would otherwise be required in the absence of STING pathway activation in accordance with the present invention. As such, modulating STING pathway activation is also meant to encompass treatment of a pathologic or disease condition with effective amount of a CDN that is reduced relative to the amount of the CDN that would otherwise be utilized as effective in the absence of the STING pathway activation modulating agent. Modulating STING pathway activation is also meant to encompass treatment of a pathologic or disease condition without administration of a STING agonist, such as a CDN, e.g., where there is intrinsic but weak activation of the pathway due to, e.g. dysregulated DNA replication, for example in cancer cells, or e.g., in certain infections, such that STING pathway modulation in accordance with embodiments of the invention enhances those responses sufficiently to induce an anti-tumor or anti-pathogen response without applying a STING agonist.

In certain instances, modulating the STING pathway activation means decreasing or inhibiting the activity of a CDN in a cell, in vitro or in vivo. When STING pathway activation is decreased, one or more activities of a CDN of interest can also be decreased or inhibited. Modulating the activity of a CDN in a cell is meant to encompass ameliorating undesirable side effects of a CDN therapy for a pathologic or disease condition, e.g., relative to CDN therapy in the absence of the PI4KB Golgi recruitment modulating agent. In certain instances, activities of a CDN that are decreased or inhibited include, but are not limited to, intercellular 2’3’-cGAMP signaling and cell toxicity. In certain cases, the parameter of interest is decreased or inhibited by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 2-fold or more, 3-fold or more, or even more, e.g., relative to a suitable control.

PI4KB Golgi recruitment-modulating agents

As summarized above, aspects of the subject methods include use of agents that modulate PI4KB Golgi recruitment, as described above. Aspects of the subject methods include contacting a cell with a PI4KB Golgi recruitment-modulating agent to modulate PI4KB Golgi recruitment in a cell thereby modulating STING pathway activation and, in some instances, the activity of the CDN of interest in the cell. A PI4KB Golgi recruitment - modulating agent is an agent that is capable of modulating the PI4KB Golgi recruitment either directly (e.g., via direct binding to produce an enhancing or inhibiting effect) or indirectly (e.g., via modulating expression of an activity of interest, e.g., ACDB3 and/or OSBP). Any convenient agent that is capable of modulating the activity of a target protein can be adapted for use in the subject methods. In some instances, the agent directly binds to a target protein, e.g., ACDB3, OSBP, etc., to modulate its activity. In certain instances, the agent acts indirectly, e.g., via modulating expression of the target protein.

PI4KB Golgi recruitment-modulating agents of interest include, but are not limited to, small molecule, nucleic acid, e.g., RNA or DNA, and peptide, e.g., protein, agents. PI4KB Golgi recruitment-modulating agents include small molecule compounds that selectively inhibit the activity of the target protein, e.g., ACDB3 or OSBP, of interest. PI4KB Golgi recruitment-modulating agents include small molecules that selectively enhance the activity of the target protein. Small molecule compounds that specifically and directly bind to the target protein are of interest. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. The compounds can include functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

For enhancing STING pathway activation, in some instances a small molecule inhibitor of OSBP is employed. Any convenient small molecule inhibitor of OSBP may be employed, where examples of such inhibitors include, but are not limited to: 25- hydroxycholesterol [250HC], T-00127-HEV2, AN-12-H5, itraconazole [ITZ], OSW-1 , Osw-1 analogs and conjugates, e.g., as described in published PCT application Publication No. WO2012159027A2, the disclosure of which is herein incorporated by reference, TTP-8307, and the like. For reducing STING pathway activation, in some instances a small molecule inhibitor of ACBD3 or PI4KB is employed. Any convenient small molecule inhibitor of ACBD3 may be employed. Any convenient small molecule inhibitor of PI4KB may be employed, where examples of such inhibitors include, but are not limited to: MI356, Compound 10, PIK93, UCB9608, those compounds described in published PCT application Publication Nos. WO2019141694A1 , WO2017055305A1 , WO2017097871 A1 , WO2015193167A1 (the disclosures of which are herein incorporated by reference, those compounds described in Chinese Patent No. CN102453712B and European Patent No. EP2319926B1 (the disclosures of which are herein incorporated by reference, those compounds described in Rutaganira et al., "Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIb," J. Med. Chem. (2016) 59: 1830-1839, and the like.

PI4KB Golgi recruitment-modulating agents are also found among biomolecules including proteins, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified using any convenient methods. In some cases, useful PI4KB Golgi recruitment -modulating agents exhibit an affinity (Kd) for a target protein, such as ACBD3 or OSBP, that is sufficient to provide for the desired modulation of PI4KB Golgi recruitment. The affinity of the PI4KB Golgi recruitment -modulating agent can be at least 1 -fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the agent for unrelated protein. In some cases, the affinity of a PI4KB Golgi recruitment - modulating agents to a target protein, e.g., ACBD3 or OSBP, can be, for example, from about 100 nanomolar (nM) to about 1 nM, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or from about 10 nanomolar (nM) to about 0.1 nM. In some embodiments, the affinity between the agent and a target protein is characterized by a K_d (dissociation constant) of 10 ⁶ M or less, such as 10⁷ M or less, including 10⁸ M or less, e.g., 10⁹ M or less, 10¹⁰ M or less, 10¹¹ M or less, 10¹² M or less, 10¹³ M or less, 10¹⁴ M or less, including 10¹⁵ M or less.

PI4KB Golgi recruitment-modulating agents include antibodies that specifically bind to a target protein. In some cases, the antibody specifically binds an epitope of the target protein that provides for inhibition of the function of the target protein. In certain cases, the antibody specifically binds a distinct epitope of the target protein that provides for the desired modulation of STING pathway activation in a cell. Antibodies that can be used as PI4KB Golgi recruitment-modulating agents in connection with the present disclosure can encompass, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)₂ antibody fragments, Fv antibody fragments (e.g., V_H or V_L), single chain Fv antibody fragments and dsFv antibody fragments.

Furthermore, the antibody molecules can be fully human antibodies, humanized antibodies, or chimeric antibodies. The antibodies that can be used in connection with the present disclosure can include any antibody variable region, mature or unprocessed, linked to any immunoglobulin constant region. Minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain 75% or more, e.g., 80% or more, 90% or more, 95% or more, or 99% or more of the sequence. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether an amino acid change results in a functional peptide can be determined by assaying the specific activity of the polypeptide derivative.

"Antibody fragments" comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

Antibodies that can be used in connection with the present disclosure thus can encompass monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single chain Fv antibody fragments and dsFv antibody fragments. Furthermore, the antibody molecules can be fully human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, the antibody molecules are monoclonal, fully human antibodies. The antibodies that can be used in connection with the present disclosure can include any antibody variable region, mature or unprocessed, linked to any immunoglobulin constant region. If a light chain variable region is linked to a constant region, it can be a kappa chain constant region. If a heavy chain variable region is linked to a constant region, it can be a human gamma 1 , gamma 2, gamma 3 or gamma 4 constant region, more preferably, gamma 1 , gamma 2 or gamma 4 and even more preferably gamma 1 or gamma 4. In some cases, the PI4KB Golgi recruitment -modulating agent is an antibody. In certain cases, the PI4KB Golgi recruitment -modulating agent is an antibody fragment or binding derivative thereof. The antibody fragment or binding derivative thereof can be selected from a Fab fragment, a F(ab')₂ fragment, a scFv, a diabody and a triabody.

Depending on the particular embodiments being practiced, a variety of different types of PI4KB Golgi recruitment-modulating agents may be employed. In some instances, the agent modulates the activity of a target protein following expression, such that the agent is one that changes the activity of the protein encoded by the target gene following expression of the protein from the target gene. In other embodiments, the PI4KB Golgi recruitment- modulating agent modulates expression of the RNA and/or protein from the gene encoding the target protein, such that it changes the expression of the RNA or protein from the target gene in some manner. In these instances, the agent may change expression of the RNA or protein in a number of different ways. As would be readily understood by one of ordinary skill in the art, one can reduce expression (protein production) of an endogenous gene at the DNA, RNA, or protein level. For example, expression can be reduced by reducing the total amount of wild type protein made by the endogenous locus, and this can be accomplished either by changing the nature of the protein produced (e.g., via gene mutation to generate a loss of function allele such as a null allele or an allele that encodes a protein reduced function) or by reducing the overall levels of protein produced without changing the nature of the protein itself.

In certain embodiments, the PI4KB Golgi recruitment-modulating agent is one that reduces, including inhibits, expression of a functional target protein, e.g., ACBD3 or OSBP. Inhibition of protein expression may be accomplished using any convenient means, and one of ordinary skill in the art will be aware of multiple suitable methods. For example, in order to reduce/inhibit expression, one can reduce protein levels post-translationally; one can block production of protein by blocking/reducing translation of mRNA (e.g., using an RNAi agent such as an shRNA or siRNA that targets the mRNA of an endogenous gene); one can reduce mRNA levels post-transcriptionally (e.g., using an RNAi agent such as an shRNA or siRNA that targets the mRNA of an endogenous gene); one can reduce mRNA levels by blocking transcription (e.g., using gene editing tools to either alter a promoter and/or enhancer sequence or to modulate transcription, or by using modified gene editing tools, e.g., CRISPRi, that can modify transcription without cutting the target DNA). Additionally, one can alter the nature of the protein made from an endogenous locus by inducing (e.g., using gene editing technology) a loss of function mutation, which can range from an allele with reduced wild type activity to a dead protein or no protein (e.g., catalytically inactive mutant, a frameshift allele, a gene knockout, etc.). Moreover, one can reduce mRNA levels via gene editing methods that result in low net transcript levels (e.g., frameshift mutations can trigger nonsense mediated mRNA decay).

Any convenient inhibitor of expression can be utilized as an antagonist in the subject methods. Such antagonists can act to inhibit expression at a transcriptional, translational, or post-translational level. In some embodiments, the inhibitors are nucleic-acid based, including, without limitation, DNA, RNA, chimeric RNA/DNA, protein nucleic acid, and other nucleic acid derivatives. In some embodiments, the expression inhibitors encompass RNA molecules capable of inhibiting receptor production when introduced into a receptorexpressing cell (termed RNAi), including short hairpin double-stranded RNA (shRNA). In some instances, the expression inhibitors are small interfering RNA (siRNA). In some instances, the expression inhibitors are small interfering microRNA. It will be understood that any sequence capable of reducing the cell surface expression of a receptor, or reducing the expression of a receptor ligand, can be used in practicing the methods of the present disclosure.

Examples of agents that inhibit expression of an endogenous gene (.g., as described herein) include but are not limited to: (a) an RNAi agent such as an shRNA or siRNA that specifically targets mRNA encoded by the endogenous gene; (b) a genome editing agent (e.g., a Zinc finger nuclease, a TALEN, a CRISPR/Cas genome editing agent such as Cas9, Cpf1 , CasX, CasY, and the like) that cleaves the target cell’s genomic DNA at a locus encoding the endogenous gene (e.g., ACBD3 or OSBP) - thus inducing a genome editing event (e.g., null allele, partial loss of function allele) at the locus of the endogenous gene; (c) a modified genome editing agent such as a nuclease dead zinc finger, TALE, or CRISPR/Cas nuclease fused to a transcriptional repressor protein that modulates (e.g. reduces) transcription at the locus encoding the target protein encoding endogenous gene (e.g., ACBD3 or OSBP) (see, e.g., Qi et al„ Cell. 2013 Feb 28;152(5):1173-83’; Gilbert et al, Cell. 2014 Oct 23;159(3):647-61 ; Larson et al., Nat Protoc. 2013 Nov;8(11):2180-96).

Antisense molecules can be used to down-regulate expression of a target gene in the cell. The anti-sense reagent may be antisense oligodeoxynucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted protein, and inhibits expression of the targeted protein. Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may include multiple different sequences.

Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner etal. (1993), supra, and Milligan etal., supra.) Oligonucleotides may be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman etal. (1995), Nucl. Acids Res. 23:4434-42). In addition, the transcription level of a protein can be regulated by gene silencing using RNAi agents, e.g., double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). RNAi, such as double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), has been extensively documented in the nematode C. elegans (Fire, A., et al, Nature, 391 , 806-811 , 1998) and routinely used to “knock down” genes in various systems. RNAi agents may be dsRNA or a transcriptional template of the interfering ribonucleic acid that can be used to produce dsRNA in a cell. A number of options can be utilized to deliver the dsRNA into a cell or population of cells such as in a cell culture, tissue, organ or embryo. For instance, RNA can be directly introduced intracellularly. Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma 107: 430-439). Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate. A number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.

In some instances, methods include enhancing expression of target protein, e.g., ACBD3 or OSBP, to increase the activity thereof. Expression of a target protein may be enhanced using any convenient protocol. In some instances, a vector is employed to introduce a nucleic acid coding sequence for the target protein into the cell, whereby the target protein is expressed in the cell. Accordingly, one aspect of the invention is a nucleic acid vector that includes a coding sequence for a target protein, e.g., ACBD3 or OSBP. Application of the subject vector to a subject, e.g. using any convenient method such as a gene therapy method, may result in expression of one or more coding sequences of interest in cells of the subject, to produce a biologically active product that may modulate a biological activity of the cell and thereby modulating STING pathway activation. In some cases, the vector is a nucleic acid vector comprising a coding sequence for ACBD3. In some cases, the vector is a nucleic acid vector comprising a coding sequence for OSBP.

In some instances, the vector comprises a coding sequence for the target protein, e.g., ACBD3 for enhancing STING pathway activation or OSBP for reducing STING pathway activation, suitable for use in gene therapy. Gene therapy vectors of interest include any kind of particle that comprises a polynucleotide fragment encoding the target protein, operably linked to a regulatory element such as a promoter, which allows the expression of a functional target protein in the targeted cells.

The target protein sequence used in the gene therapy vector may be derived from the same species as the subject. Any convenient target protein sequences, or fragments or functional equivalents thereof, may be utilized in the subject vectors, including sequences from any convenient animal, such as a primate, ungulate, cat, dog, or other domestic pet or domesticated mammal, rabbit, pig, horse, sheep, cow, or a human. For example, gene therapy in humans may be carried out using the human target protein sequence, e.g., human ACBD3 or human OSBP coding sequence.

Human ACBD3 mRNA coding sequence has been assigned NCBI ref seq number NM_022735 and has the following sequence:

1 agaggtcagc aggaagtcga tacgtggctg ccgtctgtcc ccgctgagga ggtgcagcag

61 ccggagatgg cggcggtgct gaacgcagag cgactcgagg tgtccgtcga cggcctcacg

121 ctcagcccgg acccggagga gcggcctggg gcggagggcg ccccgctgct gccgccaccg

181 ctgccaccgc cctcgccacc tggatccggt cgcggcccgg gcgcctcagg ggagcagccc

241 gagcccgggg aggcggcggc tgggggcgcg gcggaggagg cgcggcggct ggagcagcgc

301 tggggtttcg gcctggagga gttgtacggc ctggcactgc gcttcttcaa agaaaaagat

361 ggcaaagcat ttcatccaac ttatgaagaa aaattgaagc ttgtggcact gcataagcaa

421 gttcttatgg gcccatataa tccagacact tgtcctgagg ttggattctt tgatgtgttg

481 gggaatgaca ggaggagaga atgggcagcc ctgggaaaca tgtctaaaga ggatgccatg

541 gtggagtttg tcaagctctt aaataggtgt tgccatctct tttcaacata tgttgcgtcc

601 cacaaaatag agaaggaaga gcaagaaaaa aaaaggaagg aggaagagga gcgaaggcgg

661 cgtgaagagg aagaaagaga acgtctgcaa aaggaggaag agaaacgtag gagagaagaa

721 gaggaaaggc ttcgacggga ggaagaggaa aggagacgga tagaagaaga aaggcttcgg

781 ttggagcagc aaaagcagca gataatggca gctttaaact cccagactgc cgtgcagttc

841 cagcagtatg cagcccaaca gtatccaggg aactacgaac agcagcaaat tctcatccgc

901 cagttgcagg agcaacacta tcagcagtac atgcagcagt tgtatcaagt ccagcttgca

961 cagcaacagg cagcattaca gaaacaacag gaagtagtag tggctgggtc ttccttgcct

1021 acatcatcaa aagtgaatgc aactgtacca agtaatatga tgtcagttaa tggacaggcc

1081 aaaacacaca ctgacagctc cgaaaaagaa ctggaaccag aagctgcaga agaagccctg

1141 gagaatggac caaaagaatc tcttccagta atagcagctc catccatgtg gacacgacct

1201 cagatcaaag acttcaaaga gaagattcag caggatgcag attccgtgat tacagtgggc

1261 cgaggagaag tggtcactgt tcgagtaccc acccatgaag aaggatcata tctcttttgg

1321 gaatttgcca cagacaatta tgacattggg tttggggtgt attttgaatg gacagactct

1381 ccaaacactg ctgtcagcgt gcatgtcagt gagtccagcg atgacgacga ggaggaagaa

1441 gaaaacatcg gttgtgaaga gaaagccaaa aagaatgcca acaagccttt gctggatgag

1501 attgtgcctg tgtaccgacg ggactgtcat gaggaggtgt atgctggcag ccatcaatat

1561 ccagggagag gagtctatct cctcaagttt gacaactcct actctttgtg gcggtcaaaa

1621 tcagtctact acagagtcta ttatactaga taaaaatgtt gttacaaagt ctggagtcta

1681 gggttgggca gaagatgaca tttaatttgg aaatttcttt ttacttttgt ggagcattag

1741 agtcacagtt taccttattg atattggtct gatggtttgt gaactcttgc tgggaatcaa

1801 aatttccttg agactcttta gcattcatac tttggggtta aaggagattc ctcagactca

1861 tccagccctt gggtgctgac cagcagagtc actagtggat gctgaagtta catgagctac

1921 atgttaaata tttaaagtct ccaaaataaa acaccccaac gttgacctta cccggctgat 1981 ggttagcccc ttgctgcctg ctccatgtgt cttatgagag cccgtagtta cagtgtcctc 2041 taatttgaaa tccataagtt aacaagtcta tatcaggtgc agctggcttt gattaaaggc 2101 catttttaaa acttaaaaac tcaacacctc acagattata atagaaaaag aaatggcctc 2161 agtttgatct cgttcagaat gacccagatt gtttctgctt tgggtgcagc tgtttagttc 2221 agagttatat tacagagaat tattttctga gataatctta aactagaatg ttcaaaacta 2281 attgataatt gaagtatcaa gatacgtaga acacctcaga gatttttctt caggaacttc 2341 cacaaacttt gaatccttgt atctttattt ggtattcata ctactagtag caaaatacag 2401 gttttttgtt ttgttttgtt ttgttttggc ttcatagagt atctcaaatt gaaacttttc 2461 tgcacaaaga ataaaattaa ggattttata aactcaaatt ggcacctact gaattaaaat 2521 acataaaatc atttaaatat aattcagcat atgggaagta acattgcact aatatggaaa 2581 tcactgccag agacagtcta ttttctttta atttgttact acttagtcac aaaccccaca 2641 ttattccagt ttggaattac ttattaagga gaattggaaa tacatatgcc catgcttaaa 2701 ttttatagct ttaatttgtg ttatttcttt attgacggga agaggtacat ctttttttcc 2761 ttactgaaaa caaatatgga ttaattgcct caaatttgta taagtgattg gctagtgatt 2821 cttgttttca gaagggagag tggtatagat agaaaatgac aaagatggca atatacactt 2881 aatgttgtta ttgtatgttg ttactgaagt acttagattt ttaaaatttc aaatcctaaa 2941 tcacttcttg taggagggtt ttcattaact gcagtatata cagttcacta catatgggtt 3001 gtttgagttt tttgtgtgct gtatttcttt ctgtttttta atacctggtt ttgtacatat 3061 ctaactctgt tctcttttgg ttgttcagaa actggatttt ttttttctta agcagtgctt 3121 aatttgtgtt ttttaatttt gattcagaag tagtcccagc tcataggtgt tcatactgtt 3181 acatccagaa catttgtcag gctctctgtc agctttcatg tacatatggt atagaaacca 3241 tggagttagg cacttcctgg attttttttt tatgagaaaa atactgtatt taaaatgtaa 3301 aataaacttt taaaaagcag gcactaatat atatttcttc cagcctttga ttacaaattt 3361 gtccttgcac atgttaagat gaattatctc ctaaaaatat cattgttctt gggagcagtg 3421 tatgttactt tacatagcag cggttcctgt catgtgttca tgtcagaata tttttggttt 3481 taaactttct tattgccttt ggctgttgat tagtacagta caagtgcgat ttcaaaaaga 3541 tcttgaaagt aatatattta atcaattaaa atgtttatct gtaa (SEQ ID NO:01)

Human ACBD3 has a sequence encoded by the above coding sequence of:

MAAVLNAERLEVSVDGLTLSPDPEERPGAEGAPLLPPPLPPPSP PGSGRGPGASGEQPEPGEAAAGGAAEEARRLEQRWGFGLEELYGLALRFFKEKDGKAF HPTYEEKLKLVALHKQVLMGPYNPDTCPEVGFFDVLGNDRRREWAALGNMSKEDAMVE FVKLLNRCCHLFSTYVASHKIEKEEQEKKRKEEEERRRREEEERERLQKEEEKRRREE EERLRREEEERRRIEEERLRLEQQKQQIMAALNSQTAVQFQQYAAQQYPGNYEQQQIL IRQLQEQHYQQYMQQLYQVQLAQQQAALQKQQEVWAGSSLPTSSKVNATVPSNMMSV NGQAKTHTDSSEKELEPEAAEEALENGPKESLPVIAAPSMWTRPQIKDFKEKIQQDAD SVITVGRGEVVTVRVPTHEEGSYLFWEFATDNYDIGFGVYFEWTDSPNTAVSVHVSES SDDDEEEEENIGCEEKAKKNANKPLLDEIVPVYRRDCHEEVYAGSHQYPGRGVYLLKF DNSYSLWRSKSVYYRVYYTR (SEQ ID NO:02)

Human OSBP mRNA coding sequence has been assigned NCBI ref seq number NM_002556 and has the following sequence:

1 ggggacgctg cgcggcggtg gctgatgcgg tagccgtgtg gggcgctccg ggcggcgacg 61 gcggctctcg taggcggttc cggtcttgta tctccaggcg gcggcggctc atggcggcga 121 cggagctgag aggagtggtg gggccaggcc cggcagccat tgcagcactt ggcggcggcg 181 gcgccggtcc cccagtggtg ggaggaggcg gcggccgcgg agatgcgggg ccaggctccg 241 gggccgcgtc agggacggtg gtcgcggcgg cggcgggagg cccgggcccg ggggccgggg 301 gagtggcggc ggctggcccg gcccctgcgc cgccgactgg gggctcgggc ggctcgggcg 361 ctgggggttc gggctcggct cgagagggct ggctcttcaa atggaccaat tatatcaaag 421 gctaccagcg gcgatggttc gtgctgagca acgggctcct gagctactac agatcaaagg 481 cagaaatgag acatacctgc cgtggtacca tcaacctcgc cacagccaac atcaccgtgg 541 aggactcctg caacttcatc atttccaatg ggggtgctca gacctaccat ctgaaagcta 601 gttcagaagt tgagcggcag cgctgggtga cggccctgga actggccaag gccaaagctg 661 tgaagatgct ggcagagtca gatgaatcag gagatgaaga gtctgtctca caaactgaca 721 agactgagct gcagaatacc cttcggaccc tctctagcaa agtagaggac ttgagcacgt 781 gcaatgactt gatagctaag catggcacag ctctgcagcg ttctctcagt gagctggagt 841 ccctgaagtt gcctgctgag agcaatgaaa agatcaaaca ggtcaacgaa cgagccacac 901 tctttaggat aacatccaat gccatgatca acgcctgcag agatttcctc atgttagccc 961 agacccatag taaaaaatgg caaaagtcac tacagtatga aagagaccag cgtatccgac

1021 tggaagaaac cctcgagcag ctggcgaagc agcataatca cctggagagg gccttccgag

1081 gagccacggt gctgccggca aacactcctg gcaatgtggg ttctggtaaa gatcagtgct

1141 gctctggcaa aggggacatg agcgatgaag atgatgagaa tgaatttttt gatgcacctg

1201 agatcatcac catgcctgaa aatttgggcc acaaacgtac tggcagcaat atcagtggag

1261 ccagcagtga catcagcctt gatgaacagt acaagcatca gctggaggag accaaaaagg

1321 aaaagagaac cagaatacca tacaagccaa actatagcct caatttatgg agcatcatga

1381 agaactgcat tggaaaagaa ctctctaaga tccccatgcc ggtaaacttt aatgagccct

1441 tgtccatgct tcagcgcctt actgaagatc tggaatacca tgagctgtta gaccgagctg

1501 caaaatgtga gaattctcta gaacagctct gttatgttgc agctttcacc gtgtcctcct

1561 actccactac tgtcttccgc accagtaagc cattcaaccc actgcttggg gagacctttg

1621 agctggaccg attagaggag aatgggtacc gatccctctg tgaacaggtg agtcatcatc

1681 cccctgctgc tgcgcaccat gctgagtcca aaaatggctg gacattgcgt caggaaatca

1741 aaatcaccag caagtttcga ggcaaatacc tctccattat gcccctcggt accattcatt

1801 gtattttcca tgcaactggg caccactaca cttggaagaa agttaccaca actgtacaca

1861 acattattgt gggcaagttg tggatagatc agtctggcga aattgatatt gtgaatcaca

1921 agacaggaga caagtgtaat cttaaatttg ttccttatag ctacttctct cgggatgtag

1981 caagaaaggt gacgggggaa gtgacagatc catcaggaaa agtccacttt gctcttctgg

2041 ggacgtggga tgagaaaatg gaatgtttca aagtacagcc agtcattggg gaaaatgggg

2101 gtgatgctcg acagagaggc catgaagcag aggaaagcag ggtcatgctg tggaaaagga

2161 atcctttacc gaagaatgca gaaaacatgt actacttctc agagcttgct ctgactctca

2221 atgcttggga aagtggcact gcccccacag acagccggtt acgacctgac cagagactga

2281 tggaaaatgg acgctgggat gaagcaaatg cggagaagca gcgcctggag gaaaaacaaa

2341 gactttccag aaagaagaga gaagcggaag ctatgaaagc cacagaggat ggcacaccat

2401 atgatcccta taaggcactg tggtttgagc ccctgttacc aaggagttaa

2461 cccatattta taggggagaa tactgggagt gtaaagaaaa acaggactgg agctcatgcc

2521 cggacatttt ctgaaacggc agtaacaaaa aagaggagca tataatggag aagaggacag

2581 aggatgtgtg ggaaagctgg aagttgtgac tctcttacca agtgctttcc tcaagtttgt

2641 ctctcttgac caatcatttt ttccaaatca atcaccagaa ggagacacca gtggtggtga

2701 ttggctggtt gccttagctg attgaaactg aaatcgacat aggagatact atgtctgtaa

2761 gggagaggtt tagtggccga aggttagtgt tattccacat ccacgaagtc aagtattctt

2821 ggctcttctt tctccctctg cccccattta agacaatgtc actcaccctc cccttgtagt

2881 atcctgctca gccaagatct gcctaagatt atgattcagg ggcattttgg tgctgttaaa

2941 agcaagagcc gaatttagaa gttcccttaa aaagaaacac ataagactta ccaatggagg

3001 cattgtgaat ggttgcagtg gagcttaggt ataacatcat caagtgtgtt cacacgcggg

3061 gtcggtttaa tggagtgtcc acgcggagat aactgcgata ttggaacacc tgtgagagag

3121 attgttctat agggctggaa tattcagagt tacattcttg gaagtttctg tttttacttg

3181 catcaaacac ccctctgttg ttctccatca tctttaatag caactggaga ccactttggt

3241 cattggtaag ggggtgcatt ctcctcacaa aggggtttta tggacttcct caggcggaga

3301 gcttctgaga acacaggcag gatggaaaaa gactactagc cacttttgct ttcccaaccc

3361 cccttaatgc catccttcat tgtctttctg gcttctcttc ttctggcaca gtaccatttt

3421 gggtctgtgc cccagtgtgg agcaaaacat tgcctgtccc attctgatat acttcagaat

3481 ttgagagcag aagttaatgt ggaacaaaag ttttcaccat ctctcaagcc ccaaggactg

3541 gagccacctc tggaataatg tgttaaatat ctgtatatta tatatatgta gagaaaaatc

3601 taattttttg gtttgatttc cttcccatta agaatcttgt tttttgatac ccttgtctcc

3661 ctgagactct ttcttggaca cgtgttgatg tggatggcac tcctgctctc catcttttat

3721 gggcagagag atttcaaaac ctgagcagga tcatgtctgg cagttactaa acagggactt

3781 tgtccatggt ttgtatcatg gggtgcctac tgctgtctaa actctgctct cctaattagc

3841 tgactcttga aaatgaaatc ctaacctctt tcctgcctct tttctttctc ttttcaggaa

3901 gagtgtgggt agaaagtccg gagggcaact tccaatttaa ttctgctctg tccggcgcat

3961 aggctgaatt ccaaaagaac tttcttccta ggatgtgggg cttaaagcac cgtcatgtgg

4021 ggtggatcag ataattgctc ctgtggggcc ctggcttcct tctctgtttg tgcagtattt

4081 gtttttgttg tcttgttgag aaatatcccc tgtgcaaccc acccttggtt gaggatcaca

4141 aattgaggtg ccttccttgt atgctttttg ttagtatttt ttgtttttgt tccttaactg

4201 ctgagcctca gccagcgtgg gtcctggggg aaataccatt ccaaaggaag ccatccctat

4261 ggggaaattg gttttactgg gtaaattaat acttttattt cttcccctat cccttccttc

4321 ctagttgtga taaacatggg agaggagtga ctttgactgc tggggaattt cctcctaaga

4381 accttattcc caaaataagc cagggattat gtcaaggctt ctttcaagga ggctcataaa

4441 agcagtgacc tcatccgggg ctttggatag agtgagaatt ccccaactaa attggacttc

4501 tctgctttat ccccttctct tccttcacca tctgcactac atttctggct gatcccaatc

4561 agattcccgc taatggaaga agtttagaat ctttcaggtg gaataaagtc acatgaaaac 4621 aaaacacaac tatatatatt ttcagttttt ttgccttatt gatttttttc caaaaaaaaa 4681 aaactactaa attaaataat tttttaaaag tea (SEQ ID NO:03)

Human OSBP has a sequence encoded by the above coding sequence of:

MAATELRGVVGPGPAAIAALGGGGAGPPWGGGGGRGDAGPGSG AASGTWAAAAGGPGPGAGGVAAAGPAPAPPTGGSGGSGAGGSGSAREGWLFKWTNYI KGYQRRWFVLSNGLLSYYRSKAEMRHTCRGTINLATANITVEDSCNFIISNGGAQTYH LKASSEVERQRWVTALELAKAKAVKMLAESDESGDEESVSQTDKTELQNTLRTLSSKV EDLSTCNDLIAKHGTALQRSLSELESLKLPAESNEKIKQVNERATLFRITSNAMINAC RDFLMLAQTHSKKWQKSLQYERDQRIRLEETLEQLAKQHNHLERAFRGATVLPANTPG NVGSGKDQCCSGKGDMSDEDDENEFFDAPEIITMPENLGHKRTGSNISGASSDISLDE QYKHQLEETKKEKRTRIPYKPNYSLNLWSIMKNCIGKELSKIPMPVNFNEPLSMLQRL TEDLEYHELLDRAAKCENSLEQLCYVAAFTVSSYSTTVFRTSKPFNPLLGETFELDRL EENGYRSLCEQVSHHPPAAAHHAESKNGWTLRQEIKITSKFRGKYLSIMPLGTIHCIF HATGHHYTWKKVTTTVHNIIVGKLWIDQSGEIDIVNHKTGDKCNLKFVPYSYFSRDVA RKVTGEVTDPSGKVHFALLGTWDEKMECFKVQPVIGENGGDARQRGHEAEESRVMLWK RNPLPKNAENMYYFSELALTLNAWESGTAPTDSRLRPDQRLMENGRWDEANAEKQRLE EKQRLSRKKREAEAMKATEDGTPYDPYKALWFERKKDPVTKELTHIYRGEYWECKEKQ DWSSCPDIF (SEQ ID NO:04)

As used herein, "functional equivalent" refers to a nucleic acid molecule that encodes a polypeptide that has target protein activity. The functional equivalent may display 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared to a parent target protein sequence. Functional equivalents may be artificial or naturally-occurring. For example, naturally-occurring variants of the target protein sequence in a population fall within the scope of functional equivalent. Target protein sequences derived from other species also fall within the scope of the term "functional equivalent. In one embodiment, the functional equivalent is a nucleic acid with a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the parent sequence. In a further embodiment, the functional equivalent is a polypeptide with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to a parent sequence. In the case of functional equivalents, sequence identity should be calculated along the entire length of the nucleic acid. Functional equivalents may contain one or more, e.g. 2, 3, 4, 5, 10, 15, 20, 30 or more, nucleotide insertions, deletions and/or substitutions when compared to a parent sequence. The term "functional equivalent" also encompasses nucleic acid sequences that encode a target protein polypeptide with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity to the parent amino acid sequence, but that show little homology to the parent nucleic acid sequence because of the degeneracy of the genetic code.

As used herein, the term "active fragment" refers to a nucleic acid molecule that encodes a polypeptide that has target protein activity or polypeptide that has target protein activity, but which is a fragment of the nucleic acid as set forth in the parent polynucleotide sequence or the amino acid sequence as set forth in parent polypeptide sequence. An active fragment may be of any size provided that target protein activity is retained. A fragment will have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% identity to the parent sequence along the length of the alignment between the shorter fragment and longer parent sequence.

Fusion proteins including these fragments can be comprised in the nucleic acid vectors needed to carry out the invention. For example, an additional 5, 10, 20, 30, 40, 50 or even 100 amino acid residues from the polypeptide sequence, or from a homologous sequence, may be included at either or both the C terminal and/or N terminus without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit biological activity. Sequence identity may be calculated by any one of the various methods in the art, including for example BLAST (Altschul S F, Gish W, Miller W, Myers E W, Lipman D J (1990). "Basic local alignment search tool". J Mol Biol 215 (3): 403-410) and PASTA (Lipman, D J; Pearson, W R (1985). "Rapid and sensitive protein similarity searches". Science 227 (4693): 1435-41 ; http://fasta.bioch.virginia.edu/fasta www2/fasta Iist2.shtml) and variations on these alignment programs.

The vector may further include one or more regulatory sequences. Any convenient regulatory sequences or promoter sequences may be utilized in the subject vectors, e.g., as described herein. In some embodiments, the regulatory sequence that is operatively linked to the coding sequence is the cytomegalovirus promoter (CMV), although any other convenient regulatory sequences may be utilized.

Any convenient viruses may be utilized in delivering the vector of interest to the subject. Viruses of interest include, but are not limited to a retrovirus, an adenovirus, an adeno-associated virus (AAV), a herpes simplex virus and a lentivirus. Viral gene therapy vectors are well known in the art, see e.g., Heilbronn & Weger (2010) Handb Exp Pharmacal. 197:143-70. Vectors of interest include integrative and non-integrative vectors such as those based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, pox viruses, alphaviruses, and herpes viruses.

In some cases, non-integrative viral vectors, such as AAV, may be utilized. In one aspect, non-integrative vectors do not cause any permanent genetic modification. The vectors may be targeted to adult tissues to avoid having the subjects under the effect of constitutive telomerase expression from early stages of development. In some instances, non-integrative vectors effectively incorporate a safety mechanism to avoid over-proliferation of target protein expressing cells. The cells may lose the vector (and, as a consequence, the target protein expression) if they start proliferating quickly.

Non-integrative vectors of interest include those based on adenoviruses (AdV) such as gutless adenoviruses, adeno-associated viruses (AAV), integrase deficient lentiviruses, pox viruses, alphaviruses, and herpes viruses. In certain embodiments, the non-integrative vector used in the invention is an adeno-associated virus-based non-integrative vector, similar to natural adeno-associated virus particles. Examples of adeno-associated virus- based non integrative vectors include vectors based on any AAV serotype, i.e. AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAVI I and pseudotyped AAV. Vectors of interest include those capable of transducing a broad range of tissues at high efficiency, with poor immunogenicity and an excellent safety profile. In some cases, the vectors transduce post-mitotic cells and can sustain long-term gene expression (up to several years) both in small and large animal models of age-related disorders.

Examples of agents that increase or activate expression of an endogenous gene (e.g., as described herein) may also be employed, where such agents include, but are not limited to, CRISPR activation (CRISPRa) agents. When the agent is a CRISPR/Cas editing agent, the agent can include both the protein and guide RNA component. The guide nucleic acid (e.g., guide RNA) can be introduced into the cell as an RNA or as a DNA encoding the RNA (e.g., encoded by a DNA vector - on a plasmid, virus, and the like). The CRISPR/Cas protein can be introduced into the cell as a protein or as a nucleic acid (mRNA or DNA) encoding the protein. Programmable gene editing agents and their guide nucleic acids include, but are not limited to, CRISPR/Cas RNa-guided proteins such as Cas9, CasX,

CasY, and Cpf1 , Zinc finger proteins such as Zinc finger nucleases, TALE proteins such as TALENs, CRISPR/Cas guide RNAs, and the like.

STING Agonists

In embodiments of the invention where STING pathway activation is enhanced, the method may further include contacting a cell with a STING agonist, e.g., a CDN or other STING agonist of interest. Where the STING agonist is a CDN, the CDN of interest can be a CDN that is contacted with a cell in vitro or administered to a subject in vivo. As such, aspects of the subject methods include contacting a target cell with the CDN of interest. A variety of CDNs find use in the subject methods in conjunction with the PI4KB Golgi recruitment modulating agents (e.g., as described herein).

In some cases, the CDN is naturally occurring. Naturally occurring CDNs of interest include those involved in intercellular signaling, such as 2’3’-cGAMP. In certain instances, the CDN is one that is implicated in a disease or condition associated with aberrant signaling, such as an autoimmune/inflammatory disease (e.g., as described herein). In some cases, the CDN is involved in intercellular signaling between tumor cells and non-tumor cells where amplification of the signal can provide for anti-tumor immunity. In some cases, the CDN is involved in intercellular signaling between virus-infected and uninfected cells where amplification of the signal can provide for anti-viral immunity. In some cases, the CDN of interest is a CDN that is produced endogenously in a cell sample in vitro or in vivo by a cell of a subject. In some instances, the endogenously produced CDN is 2’3’-cGAMP. As such, aspects of the subject methods include increasing intercellular 2’3’-cGAMP signaling between cells in vivo, such as between virus-infected and uninfected cells for amplification of anti-viral immunity. In some cases, the endogenous production of a CDN of interest can be triggered or enhanced in a CDN producing cell by administration of an CDN production promoting agent, see e.g., Vance et al. in U.S. Publication No. 2014/0329889.

In certain instances, the CDN is non-naturally occurring. In some cases, the CDN is a CDN drug that finds use in cancer therapeutic applications. A variety of CDNs that are agonists of Stimulator of Interferon Genes (STING) find use in cancer immunotherapy, including synthetic CDNs that are analogues of a naturally occurring CDN such as 2’3’- cGAMP. An amplified anti-tumor immune response can occur when a CDN STING agonist is delivered to a tumor microenvironment, leading to immune activation and tumor regression.

As used herein, “cyclic dinucleotide” or “CDN” refers to a compound containing two nucleosides (i.e., a first and second nucleoside), wherein the 2’ or 3’ carbon of each nucleoside is linked to the 5’ carbon of the other nucleoside via a phosphodiester internucleoside linkage. Therefore, a 2’-5’ phosphodiester linkage containing CDN refers to a CDN where the 2’ carbon of at least one of the nucleosides is linked to the 5’ carbon of the other nucleoside. As discussed herein, 2’-5’ phosphodiester linkage containing CDNs can be used in practicing the subject methods to increase production of a type I interferon in a cell or subject. In certain embodiments, the CDN has two 2’-5’ phosphodiester linkages. In some embodiments, the CDN has a 2’-5’ phosphodiester linkage and a 3’-5’ phosphodiester linkage. In certain embodiments, the CDN has two 3’-5’ phosphodiester linkages.

Cyclic-di-nucleotides include those specifically described herein as well as isoforms (e.g., tautomers) of those specifically described herein that can be used in practicing the subject methods. A “cyclic-di-nucleotide” also includes all of the stereoisomeric forms of the cyclic-di-nucleotides described herein.

The term “nucleoside” refers to a composition containing a nitrogenous base covalently attached to a sugar (e.g., ribose or deoxyribose) or an analog thereof. Examples of nucleosides include, but are not limited to, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nitrogenous base” refers to a nitrogen-containing heterocycle having the chemical properties of a nucleobase. Nitrogenous bases of interest include, but are not limited to, pyrimidines (e.g., cytosine, thymine, and uracil) and purines (e.g., adenine and guanine), as well as substituted pyrimidine derivatives and substituted purine derivatives, pyrimidine analogs and purine analogs, and tautomers thereof.

In some embodiments, the nucleoside contains a deoxyribose sugar. Analogs of nucleosides include, but are not limited to dexoyadenosine analogues (e.g., Didanosine and Vidarabine); deoxycytidine analogues (e.g., Cytarabine, Ematricitabine, Lamivudine, and Zalcitabine); deoxyguanosine analogues (Abacavir and Entecavir); (deoxy-) thymidine analogues (e.g., Stavudine, Telbivudine, and Zidovudine); and deoxyuridine alaogues (e.g., Idoxuridine and Trifluridine).

The CDN can include a guanosine nucleoside. In some cases, the CDN contains two guanosine nucleosides. The CDN can include an adenosine nucleoside. In some embodiments, the CDN contains two adenosine nucleosides. In certain cases, the CDN contains an adenosine nucleoside and a guanosine nucleoside.

While not being bound by any particular theory of operation, CDNs can increase type-1 IFN production in a cell. In certain embodiments, the CDN increases type-1 IFN production through a mechanism that involves stimulator of interferon genes (STING). CDNs can be obtained using any suitable method. For example, CDNs may be made by chemical synthesis using nucleoside derivatives as starting material. CDNs can also be produced via in vitro synthesis, using recombinant purified cGAMP synthase (cGAS) or other recombinant purified CDN synthases such as the bacterial cGAMP synthase from V. cholerae (DncV) or mutant versions of any recombinant purified CDN synthases. Moreover, the structures of such cyclic-di-nucleotides can be confirmed using any convenient methods, such as NMR analysis.

Any convenient CDNs may be utilized in the subject methods, compositions and kits. CDN’s of interest include, but are not limited to, those described by Vance et al. in U.S. Publication No. 2014/0329889; Dubensky et al. in U.S. Publication No. 2015/0056224; Dubensky et al. in U.S. Publication No. 2014/0205653; Dubensky et al. in U.S. Patent No. 9549944; Altman et al. in WO2017027645; and Altman et al. in WO2017027646, US20060040887, US20080286296, US20120041057, US20160362441 , US20180002369, US20180064745, US2018186828, US2018237468, US2018273578, US20190055277, US2019183917, US2019185509, US2019185510, US2019248828, US9718848, W02005030186, WO2014179335, WO2015074145, WO2017011444, WO2017075477, WO2017123657, WO2017123669, WO2017161349 WO2018009466, US20180092937, WO2018065360, WO2018045204, WO2018098203, WO2018009648, WO2018009652, WO2018100558, WO2018138684, WO2018138685, WO2018156625, WO2018198076, WO2018198084, WO2018208667, WO2019023459, WO2019043634, WO2019046496, WO2019046498, WO2019046500, WO2019046511 , WO2019051488, WO2019051489, WO2019074887, WO2019079261 , WO2019092660, WO2019118839, WO2019125974, WO2019175776, WO2019180683, WO2019185477, WO2019185476, WO2019193533, WO2019193542, WO2019193543, and WO2019211799, the disclosures of which are herein incorporated by reference in their entirety. In some cases, the CDN that finds use in the subject methods is one that is described by Vance et al. in U.S. Publication No. 2014/0329889. In certain embodiments, the cyclic-di-nucleotide has one of the following formulae (I) and (II):

(I) (II) wherein X and Y are each independently a nitrogenous base or an analog thereof, or a salt thereof. In certain embodiments of formulae (l)-(ll), X and Y are each independently selected from the following:

CDNs described herein can also be described by the following nomenclature: cyclic[Xi(a-5’)pX₂(b-5')p], wherein Xi and X2 are first and second nucleosides, “a” is the designation of the carbon of the first nucleoside (e.g., 2’ or 3’ position) that is linked to the 5’ carbon of the second nucleoside via a phosphodiester bond and “b” is the designation of the carbon of the second nucleoside (e.g., 2’ or 3’ position) that is linked to the 5’ carbon of the first nucleoside by a phosphodiester bond. In some cases, at least one of “a” and “b” is 2’ in the formula. For instance, based on this nomenclature, cyclic[G(2’-5’)pA(3’-5')p] has the following formula:

or a salt thereof.

In certain embodiments, the CDN contains a 2’-5’ phosphodiester bond. In particular embodiments, the CDN further contains a 3’-5’ phosphodiester bond (e.g., cyclic[Xi(2’- 5’)pX₂(3’-5')p] or cyclic[Xi(3’-5’)pX₂(2’-5')p]). In some instances, the CDN contains two 2’-5’ phosphodiester bonds (cyclic[Xi(2’-5’)pX₂(2’-5')p]). In some instances, the CDN contains two 3’-5’ phosphodiester bonds (cyclic[Xi(3’-5’)pX₂(3’-5')p]).

In certain embodiments, the cyclic-di-nucleotide is: cyclic[A(2’-5’)pA2’-5')p]; cyclic[T(2’-5’)pT(2’-5')p]; cyclic[G(2’-5’)pG(2’-5')p]; cyclic[C(2’-5’)pC(2’-5')p]; or cyclic[U(2’- 5’)pU(2’-5')p]. In certain embodiments, the cyclic-di-nucleotide is: cyclic[A(2’-5’)pA(3’-5')p]; cyclic[T(2’-5’)pT(3’-5')p]; cyclic[G(2’-5’)pG(3’-5')p]; cyclic[C(2’-5’)pC(3’-5')p]; cyclic[U(2’- 5’)pU(3’-5')p]; cyclic[A(2’-5’)pT(3’-5')p]; cyclic[T(2’-5’)pA(3’-5')p]; cyclic[A(2’-5’)pG(3’-5')p]; cyclic[G(2’-5’)pA(3’-5')p]; cyclic[A(2’-5’)pC (3’-5')p]; cyclic[C(2’-5’)pA(3’-5')p]; cyclic[A(2’- 5’)pU(3’-5')p]; cyclic[U(2’-5’)pA(3’-5')p]; cyclic[T(2’-5’)pG(3’-5')p]; cyclic[G(2’-5’)pT(3’-5')p]; cyclic[T2’-5’)pC(3’-5')p]; cyclic[C(2’-5’)pT(3’-5')p]; cyclic[T(2’-5’)pU(3’-5')p]; cyclic[U(2’-

5’)pT(3’-5')p]; cyclic[G(2’-5’)pC(3’-5')p]; cyclic[C2’-5’)pG(3’-5')p]; cyclic[G(2’-5’)pU(3’-5')p]; cyclic[U(2’-5’)pG(3’-5')p]; cyclic[C(2’-5’)pU(3’-5')p]; or cyclic[U(2’-5’)pC(3’-5')p].

In certain embodiments, the cyclic-di-nucleotide has the following formula (cyclic[

or a salt thereof. In certain embodiments, the cyclic-di-nucleotide has the following formula (cyclic[

or a salt thereof. In other embodiments, the cyclic-di-nucleotide has the following formula cyclic[

or a salt thereof.

In other embodiments, the cyclic-di-nucleotide has the following formula cyclic[

or a salt thereof.

In yet other embodiments, the cyclic-di-nucleotide has the following formula cyclic[G(2’5’)pG(3’5’)p] :

or a salt thereof.

In certain embodiments, the cyclic-di-nucleotide has the following formula cyclic[

or a salt thereof.

or a salt thereof.

In certain embodiments, the cyclic-di-nucleotide has one of the following formulae:

wherein R is any amino acid side chain, and X and Y are as defined above for formula (I)- (II), or a salt thereof.

CDN’s of interest include, but are not limited to, those described by Dubensky etal. in U.S. Publication No. 2015/0056224. In certain embodiments, the CDN has the structure:

(a)

covalently linked to

wherein:

R3 is a covalent bond to the 5' carbon of (b),

R4 is a covalent bond to the 2' or 3' carbon of (b),

R1 is a purine linked through its N9 nitrogen to the ribose ring of (a),

R5 is a purine linked through its N9 nitrogen to the ribose ring of (b), each of Xi and X₂are independently O or S,

R2 is H or an optionally substituted straight chain alkyl of from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9 carbons, an optionally substituted alkynyl of from 1-9 carbons, or an optionally substituted aryl, wherein substitution(s), when present, may be independently selected from the group consisting of Ci-₆ alkyl straight or branched chain, benzyl, halogen, trihalomethyl, Ci-₆alkoxy, — N0₂, — NH₂, — OH, =0, — COOR' where R' is H or lower alkyl, — CH₂OH, and — CONH₂, and the 2 ' or 3' carbon of (b) which is not in a covalent bond with (a) is — O — R6, wherein R6 is H or an optionally substituted straight chain alkyl of from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9 carbons, an optionally substituted alkynyl of from 1-9 carbons, or an optionally substituted aryl, wherein substitution(s), when present, may be independently selected from the group consisting of Ci-₆ alkyl straight or branched chain, benzyl, halogen, trihalomethyl, Ci-₆ alkoxy, — N0₂, — NH₂, — OH, =0, — COOR' where R' is H or lower alkyl, — CH₂OH, and — CONH₂, or a prodrug or pharmaceutically acceptable salt thereof.

In certain cases, the CDN has one of the following formula: c-[G(2',5')pG(3',5')p], c- [A(2',5')pA(3',5')p], c-[G(2',5')pA(3',5')p] or c-[G(2',5')pA(3',5')p] where each p refers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage. In certain instances, the CDN is a compound of the formula:

where Ri and R₂ are each H, or a pharmaceutically acceptable salt thereof.

In certain cases, the CDN is a bisphosphorothioate analog of a naturally occurring CDN such as c-di-AMP. In some cases, the CDN is ADU-S100 or 2’3’-c-di-AM(PS)₂(Rp,Rp), also known as dithio-(Rp, Rp)-[cyclic[A(2’,5’)pA(3’,5’)p]] or (ML RR-S2 CDA), as described by Corrales et al. (“Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity” Cell Reports 11 , 1018-1030, May 19, 2015).

Embodiments of the invention include administration of other, non-CDN STING agonists. Any convenient non-CDN STING Agonist may be employed, wherein examples of such STING agonists include, but are not limited to E7766, PMID: 30405246, etc., as well as those described in Published United States Patent Application Publication Nos. US20190359608, US20190337918, US20190300513, US20190359608, W02007070598, WO2017004499, WO2017011622, WO2017011920, WO2017175147, WO2017175156, WO2018234805, WO2018234807, WO2018234808, WO2018013887, WO2018013908, WO2019027858, WO2019195063, WO2019069269, WO2019069270, WO2019069275, WO2019165032, WO2019219820, WO2019243823, WO2019243825, the disclosures of which are herein incorporated by reference.

Methods

As summarized above, aspects of the methods include modulating, e.g., increasing or decreasing, PI4KB Golgi recruitment in a cell to modulate, e.g., enhance or reduce,

STING pathway activation. By increasing or decreasing PI4KB Golgi recruitment, STING pathway activation can be modulated in the cell can be modulated to provide for a desired biological effect. PI4KB Golgi recruitment can be modulated by contacting the cell, in vitro or in vivo, with a PI4KB Golgi recruitment-modulating agent (e.g., as described herein).

In some cases, the PI4KB Golgi recruitment -modulating agent enhances or increases PI4KB Golgi recruitment. Examples of such agents include small molecule inhibitors of OSPB, e.g., as described above. In some cases, the PI4KB Golgi recruitment- modulating agent decreases PI4KB Golgi recruitment. Examples of such agents include PI4KB small molecule inhibitors, e.g., as described above.

In certain instances, the PI4KB Golgi recruitment-modulating agent is a small molecule and the effective amount which is contacted with the cell or cellular sample, or administered to a subject, will generally contain between from about 1 mg to about 1000 mg of the agent, in some cases, 25 mg or more, such as 50 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, 500 mg or more, 600 mg or more, 800 mg or more, or 1000 mg or more.

Any convenient activities of STING pathway activation can be targeted for modulation according to the subject methods in a variety of applications. The pathway leads, for example, to activation of IRF3 transcription factor, which induces IFN and certain other cytokines and mediators. Accordingly, this pathway and elements thereof may be modulated in embodiments of the invention. The pathway leads, for example, to activation of NF-kB, which induces other cytokines and mediators. Accordingly, this pathway and elements thereof may be modulated in embodiments of the invention. This pathway leads, for example to activation of STAT6 which induces yet other cytokines and mediators. Accordingly, this pathway and elements thereof may be modulated in embodiments of the invention. Embodiments of the invention may be employed to modulate the response to preferentially activate one or another of these “axes”, which may be employed to change the type of immune response induced, as desired.

In some cases, STING pathway activation provides an anti-tumor immune response via production of type I interferon (IFN) in a cell. As such, aspects of this disclosure include methods of increasing the production of a type I interferon (IFN) in a cell, e.g., in vitro or in vivo. By increasing type-1 interferon production is meant that the subject methods increase type-1 interferon production in a cell, as compared to a suitable control. The magnitude of the increase in type-1 interferon production in a cell relative to what can be achieved with the CDN alone may vary, and in some instances is 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, or 10- fold or greater, as compared to a suitable control. In those embodiments where, prior to practice of the subject methods, interferon production is not-detectable, the increase may result in detectable amounts of interferon production.

In some instances, the subject methods provide for increasing the production of a type I interferon (IFN)-stimulated gene in a cell, e.g., in vitro or in vivo. By increasing interferon-stimulated gene production is meant that the subject methods increase production of an interferon-stimulated gene or gene product in a cell, as compared to a suitable control. IFN-stimulated genes of interest include, but are not limited to, CXCL10, IRF7, IFIT3, ISG15 and RANTES. The magnitude of the increase in production in a cell relative to what can be achieved with the CDN alone may vary, and in some instances is 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5- fold or greater, or 10-fold or greater, as compared to a suitable control.

Type-I interferon production can be measured using any convenient method including, but not limited to, vesicular stomatitis virus (VSV) challenge bioassay, enzyme- linked immunosorbent assay (ELISA) replicon based bioassays or by using a reporter gene (e.g., luciferase) cloned under regulation of a type I interferon signaling pathway. See, e.g., Meager J. Immunol. Methods 261 :21-36 (2002); Vrolijk et al. C.J. Virol. Methods 110:201- 209 (2003); and Francois et al. Antimicrob Agents Chemother 49(9) :3770-3775 (2005).

The methods may be used to increase the production of any convenient type I interferon including, but not limited to: IFN-a (alpha), IFN-b (beta), IFN-k (kappa), IFN-d (delta), IFN-e (epsilon), IFN-t (tau), IFN-w (omega), and IFN-z (zeta, also known as limitin).

In some embodiments, the method is for increasing the production of IFN-a. In some embodiments, the method is for increasing the production of IFN-b.

The methods may also be employed to modulate product of other cytokines downstream of the pathway. For example, STING pathway activation increases the expression of costimulatory molecules and other cell surface molecules which induce cell to cell signals that enhance or modulate immune responses.

In practicing certain embodiments of the methods provided herein, an effective amount of the active agent (such as described above), is provided in the target cell or cells. As used herein "effective amount" or "efficacious amount" means the amount that, when contacted with the cell, e.g., by being introduced into the cell in vitro, by being administered to a subject, etc., is sufficient to result in the desired outcome, e.g., increased levels of type I interferon in the cell. The "effective amount" will vary depending on cell and/or the organism and/or active agent and or the nature of the desired outcome and/or the disease and its severity and the age, weight, etc., of the subject to be treated.

In some instances, an effective amount of PI4KB Golgi recruitment-modulating agent is provided to the cells to result in a change in STING pathway activation in the cells. In some cases, an effective amount of PI4KB Golgi recruitment-modulating agent is the amount to result in a 10% increase or more in the amount of STING pathway activation product observed (directly or indirectly) in the cell, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, or 10-fold or greater, relative to a negative control, e.g., a cell not contacted with the PI4KB Golgi recruitment-modulating agent. The amount of the STING pathway activation product observed may be measured by any suitable method, directly or indirectly. For example, the amount of type I interferon produced by the cell may be assessed after contact with the active agent(s), e.g., 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or more after contact with the active agent(s).

Contact of the cell with the agent(s) may occur using any convenient protocol. The protocol may provide for in vitro or in vivo contact of the agent(s) with the target cell, depending on the location of the target cell. For example, where the target cell is an isolated cell, e.g., a cell in vitro (i.e., in culture), or a cell ex vivo (“ex vivo" being cells or organs are modified outside of the body, where such cells or organs are typically returned to a living body), the agent may be introduced directly to the cell under cell culture conditions permissive of viability of the target cell. The choice of method is generally dependent on the type of cell being contacted and the nature of the active agent, and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. As another example, where the target cell or cells are part of a multicellular organism, the active agent may be administered to the organism or subject in a manner such that the agent is able to contact the target cell(s), e.g., via an in vivo protocol. By "in vivo,” it is meant the agent is administered to a living body of an animal.

The active agent(s) can be employed to increase the production of type I interferon in vivo. In these in vivo embodiments, the active agent(s) can be administered directly to the individual. The agent(s) may be administered by any suitable methods for the administration of peptides, small molecules or nucleic acids to a subject. The STING agonists and/or PI4KB Golgi recruitment-modulating agents can be incorporated into a variety of formulations. More particularly, the agent(s) of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents. Pharmaceutical compositions that can be used in practicing the subject methods are described herein.

In some instances, an effective amount of the STING agonist is administered to the subject in conjunction with the PI4KB Golgi recruitment-modulating agent. By an “effective amount” or a “therapeutically effective amount” of the agent it is meant an amount that is required to reduce the severity, the duration and/or the symptoms of the disease. In some embodiments, the effective amount of a pharmaceutical composition containing a STING agonist active agent for use in conjunction with the PI4KB Golgi recruitment-modulating agent, as provided herein, is between 0.025 mg/kg and 1000 mg/kg body weight of a human subject. In certain embodiments, the pharmaceutical composition is administered to a human subject at an amount of 1000 mg/kg body weight or less, 950 mg/kg body weight or less, 900 mg/kg body weight or less, 850 mg/kg body weight or less, 800 mg/kg body weight or less, 750 mg/kg body weight or less, 700 mg/kg body weight or less, 650 mg/kg body weight or less, 600 mg/kg body weight or less, 550 mg/kg body weight or less, 500 mg/kg body weight or less, 450 mg/kg body weight or less, 400 mg/kg body weight or less, 350 mg/kg body weight or less, 300 mg/kg body weight or less, 250 mg/kg body weight or less, 200 mg/kg body weight or less, 150 mg/kg body weight or less, 100 mg/kg body weight or less, 95 mg/kg body weight or less, 90 mg/kg body weight or less, 85 mg/kg body weight or less, 80 mg/kg body weight or less, 75 mg/kg body weight or less, 70 mg/kg body weight or less, or 65 mg/kg body weight or less. In some embodiments, the STING agonist is employed in mitotic or post-mitotic cells in vitro or ex vivo, i.e., to produce modified cells that can be reintroduced into an individual. Mitotic and post-mitotic cells of interest in these embodiments include any eukaryotic cell, e.g., pluripotent stem cells, for example, ES cells, iPS cells, and embryonic germ cells; somatic cells, for example, hematopoietic cells, fibroblasts, neurons, muscle cells, bone cells, vascular endothelial cells, gut cells, and the like, and their lineage-restricted progenitors and precursors; and neoplastic, or cancer, cells, i.e., cells demonstrating one or more properties associated with cancer cells, e.g., hyperproliferation, contact inhibition, the ability to invade other tissue, etc. In certain embodiments, the eukaryotic cells are cancer cells. In certain embodiments, the eukaryotic cells are hematopoietic cells, e.g., macrophages, NK cells, etc. Cells may be from any mammalian species, e.g., murine, rodent, canine, feline, equine, bovine, ovine, primate, human, etc. Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e., splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. In some cases, the primary cell lines are maintained for fewer than 10 passages in vitro.

If the cells are primary cells, they may be harvested from an individual by any convenient method. For example, blood cells, e.g., leukocytes, e.g., macrophages, may be harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. may be harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution will generally be a balanced salt solution, e.g., normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused. In such cases, the cells may be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells. The CDN active agent(s) may be produced by eukaryotic cells or by prokaryotic cells, it may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art. Combination Therapy

For use in the subject methods, the STING agonist described herein may be administered in combination with the PI4KB Golgi recruitment-modulating agent (e.g., as described herein). “In combination with” refers to uses where, for example, the first compound (e.g., the STING agonist, such as a CDN active agent) is administered during the entire course of administration of the second compound (e.g., PI4KB Golgi recruitment- modulating agent); where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g., where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, “in combination” can also refer to regimen involving administration of two or more compounds. “In combination with” as used herein also refers to administration of two or more compounds that may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

Any convenient additional active agents, e.g., agents that find use in a combination therapeutic application with a STING agonist of interest, can also be utilized in conjunction with the STING agonist and PI4KB Golgi recruitment-modulating agent in the subject methods. In some cases, the additional active agent is a chemotherapeutic agent or other cancer therapy, an antiviral agent, a cGAS activity modulating agent, agents that increase cytosolic DNA (e.g., agents targeting RNaseH2 or SAMHD1), ionizing radiation, etc. In some embodiments, the additional cancer therapy comprises radiation therapy, surgery, chemotherapy, or an immunotherapy (for example, without limitation, an immunomodulator, an immune checkpoint inhibitor, a cellular immunotherapy, or a cancer vaccine). In some embodiments, the one or more additional cancer therapies comprise an inactivated tumor cell that expresses and secretes one or more cytokines or one or more heat shock proteins. In some embodiments, the cytokine is selected from the group consisting of GM-CSF, CCL20, CCL3, IL-12p70, and FLT-3 ligand. In some embodiments the heat shock protein is a gp96- Ig protein. In some cases, the additional active agent is an immune checkpoint inhibitor (e.g., CTLA-4, PD-1 , TIM-3, Vista, BTLA, LAG-3, KIR, or TIGIT pathway antagonists, including, without limitation, PD-1 pathway blocking agents such as anti-PD-1 antibodies PDR001 , nivolumab, pembrolizumab, SHR-1210, REGN2810 (cemiplimab), or pidilizumab, or PD-1 inhibitor AMP-224; PD-L1 inhibitors such as anti-PD-L1 antibodies BMS-936559, MPDL3280A(atezolizumab), MEDI4736 (durvalumab), or avelumab; anti-CTLA-4 antibodies such as ipilimumab, tremelimumab, IBI310, and AGEN1884; Vista inhibitors including anti- Vista antibodies; B7-H3 inhibitors including anti-B7-H3 antibodies; and CD70 inhibitors including anti-CD70 antibodies); Co-stimulatory checkpoint receptor agonist (e.g., CD40 agonists, including an anti-CD40 antibody; CD137 agonists, including an anti-CD137 antibody; GITR agonists, including an anti-GITR antibody; 0X40 agonists, including an anti- 0X40 antibody); an immune activating cytokine (e.g. IL-2, IL-12, IL-15); a TLR agonist (e.g., CpG or monophosphoryl lipid A); a RIG-1 agonist (e.g. 5’pp-dsRNA or 3p-hpRNA); a vaccine selected to stimulate an immune response to one or more cancer antigens, for example an inactivated or attenuated bacteria which induce innate immunity and is engineered to express cancer antigens (e.g., inactivated or attenuated Listeria monocytogenes); a therapeutic antibody that induces antibody-dependent cellular cytotoxicity; an immunomodulatory cell line; an antigen selected for the purpose of inducing an immune response, an agent which mediate innate immune activation (i) via Toll-like Receptors (TLRs) including, without limitation, TLR agonist (e.g., CpG or monophosphoryl lipid A), (ii) via (NOD)-like receptors (NLRs), (iii) via Retinoic acid inducible gene-based (RIG)-l-like receptors (RLRs), (iv) via C-type lectin receptors (CLRs), or (v) via pathogen-associated molecular patterns ("PAMPs"); a chemotherapeutic agent, etc.

In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 pathway antagonist, a PD-1 pathway antagonist, a TIM-3 pathway antagonist, a Vista pathway antagonist, a BTLA pathway antagonist, a LAG-3 pathway antagonist, and a TIGIT pathway antagonist. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti- TIM-3 antibody, an anti-Vista antibody, an anti-BTLA antibody, an anti-B7-H3 antibody, an anti-CD70 antibody, an anti-KIR antibody or an anti-LAG-3 antibody. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, cemiplimab, SHR-1210, PDR001 , MEDI0680, AMP-224, ipilimumab, tremelimumab, IBI310, AGEN1884, BMS-936559, atezolizumab, durvalumab, and avelumab.

Examples of chemotherapeutic agents for use in combination therapy include, but are not limited to, an indoleamine 2,3-dioxygenase (ID01) inhibitor (e.g., epacadostat and navoximod), daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, lomustine (CCNU), carmustine, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, methylcyclohexylnitrosurea, nitrogen mustards (e.g. mechlorethamine, melphalan, cyclophosphamide, chlorambucil, uramustine, ifosfamide, bendamustine), prednimustine, estramustine phosphate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5- FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxanes (e.g., taxol, docetaxel, cabazitaxel, larotaxel), vincristine, vinblastine, anhydrovinblastine, etoposide (VP- 16), trimetrexate, irinotecan, topotecan, gemcitabine, decitabine, teniposide, cisplatin, carboplatin, diethylstilbestrol (DES), abiraterone acetate, altretamine, auristatin, bexarotene, bicalutamide, cachectin, cemadotin, cryptophycin, dolastatin, abemaciclib, acalabrutinib, afatinib, alectinib, axitinib, binimetinib, bosutinib, brigatinib, cabozantinib, ceritinib, cobimetinib, copanlisib, crizotinib, dabrafenib, dasatinib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, midostaurin, neratinib, nilotinib, osimertinib, palbociclib, pazopanib, ponatinib, regorafenib, ribociclib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vatalanib, vemurafenib, finasteride, flutamide, enasidenib, lenalidomide, liarozole, lonidamine, niraparib, olaparib, enzalutamide, mivobulin isethionate, abexinostat, belinostat, chidamide, entinostat, givinostat, panobinostat, quisinostat, resminostat, romidepsin, vorinostat, rhizoxin, rucaparib, sertenef, streptozocin, nilutamide, onapristone, sotrastaurin, tasonermin, tretinoin, venetoclax, vindesine sulfate, vinflunine, 3',4'-didehydro-4'-deoxy-8'-norvin-caleukoblastine, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, and N,N- dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1 -Lproline-t-butylamide. Examples of additional agents for use in combination therapy of neoplastic disease include, but are not limited to, thalidomide, marimastat, COL-3, BMS-275291 , squalamine, 2-ME, SU6668, neovastat, Medi-522, EMD121974, CAI, celecoxib, interleukin-12, IM862, TNP470, avastin, gleevec, herceptin, and mixtures thereof.

Examples of TLR agonists for use in combination therapy include, but are not limited to, Pam2Cys, Pam3Cys, Complete Freund’s Adjuvant (CFA), monocyte activating lipopeptide-2 (MALP2), lipopeptide derived from Mycoplasma salivarium (FSL-1), Haemophilus inf luenzae type b outer membrane protein complex (Hib-OMPC), Poly l:C, Poly AU, Hiltonol® (poly-ICLC), monophosphoryl lipid A, lipopolysaccharide (LPS), bacterial flagellin, sialyl-Tn, imiquimod, resiquimod, lefitolimod, tilsotolimod, loxoribine, and CpG oligodeoxynucleotides (e.g., agatolimod, and unmethylated CpG dinucleotide).

Additional antiviral agents can also be delivered in conjunction with a CDN of interest in the treatment methods of this disclosure. For example, compounds that inhibit inosine monophosphate dehydrogenase (IMPDH) may have the potential to exert direct antiviral activity, and such compounds can be administered in a combination therapy, as described herein. Drugs that are effective inhibitors of hepatitis C NS3 protease may be administered in combination with the CDN, as described herein. Hepatitis C NS3 protease inhibitors inhibit viral replication. Other agents such as inhibitors of HCV NS3 helicase are also attractive drugs for combinational therapy and are contemplated for use in combination therapies described herein. Ribozymes such as HeptazymeTM and phosphorothioate oligonucleotides which are complementary to HCV protein sequences and which inhibit the expression of viral core proteins are also suitable for use in combination therapies described herein.

Examples of additional agents for use in combination therapy of multiple sclerosis include, but are not limited to; glatiramer; corticosteroids; muscle relaxants, such as Tizanidine (Zanaflex) and baclofen (Lioresal); medications to reduce fatigue, such as amantadine (Symmetrel) or modafinil (Provigil); and other medications that may also be used for depression, pain and bladder or bowel control problems that can be associated with MS.

Because of the adjuvant properties of the CDNs of the present disclosure, their use in the subject methods may also combined with other therapeutic modalities including other vaccines, adjuvants, antigen, antibodies, and immune modulators.

Examples of antibodies for use in combination therapy include, but are not limited to, muromonab-CD3, infliximab, omalizumab, daclizumab, rituximab, ibritumomab, tositumomab, cetuximab, trastuzumab, brentuximab vedotin, alemtuzumab, vitaxin, bevacizumab, and abciximab.

In some instances, the active agent is a CDN transporter modulatory agent, e.g., as described in PCT application serial nol. PCT/US2019/32663 published as WO 2019/222500; the disclosure of which is herein incorporated by reference (see also Luteijn et al., "

SLC19A1 transports immunoreactive cyclic dinucleotides," Nature (2019) 573:434-438). Membrane transporters that can be targeted to modulate cellular uptake of a CDN of interest according to the subject methods include membrane folate transporters that are capable of transporting CDNs of interest into cells. Membrane folate transporters include a class of transporters which can actively transport molecules including folate, folate derivatives and/or antifolates, see e.g., Matherly et al. (“Membrane transport of folates”, Vitam. Horm. 2003;66:403-56). Membrane folate transporters of interest include, but are not limited to, the SLC19 family of transporters, reduced folate carrier (RFC), the SLC46 family of transporters including the proton-coupled folate transporters (PCFT). RFC transporter is ubiquitously expressed and can transport folate in mammalian cells and tissues. In some instances of the method, the transporter is a member of the SLC19 family of transporters. In some instances of the method, the transporter is RFC transporter. In certain cases, the RFC transporter is RFC1 , also known as solute carrier family 19 (folate transporter), member 1 , also known as SLC19A1 , RFC, CHMD, FOLT, IFC1 , REFC or IFC-1 . In some instances of the method, the transporter is a member of the SLC46 family of transporters. In certain cases, the transporter is solute carrier family 46, member 1 , also known as SLC46A1 , PCFT, G21 or HCP1 . In certain cases, the transporter is solute carrier family 46, member 3, also known as SLC46A3 or FKSG16. Exemplary transporters of interest include those described by Hou and Matherly (“Biology of the Major Facilitative Folate Transporters SLC19A1 and SLC46A1”, Curr Top Membr. 2014; 73: 175-204), Zhao and Goldman (Folate and Thiamine Transporters mediated by Facilitative Carriers (SLC19A1-3 and SLC46A1) and Folate Receptors) Mol. Aspects Med. 2013; 34) and Hamblett et al. (“SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm”, Cancer Res. 2015 Dec 15;75(24):5329-40). A CDN transporter-modulating agent is an agent that modulates the transport of the CDN across the membrane of a cell thereby modulating the activity of the CDN of interest in the cell. A CDN transporter-modulating agent is an agent that is capable of modulating the action of a target membrane transporter either directly (e.g., via direct binding to produce an enhancing or inhibiting effect) or indirectly (e.g., via modulating expression of a membrane transporter). Any convenient agents that are capable of modulating the activity of a target membrane transporter can be adapted for use in the subject methods. In some instances, the agent directly binds to the target membrane transporter to modulate its activity. In certain instances, the agent acts indirectly, e.g., via modulating expression of the target membrane transporter.

This disclosure provides a pharmaceutical composition that contains any of the PIK4B Golgi recruitment modulating agents (e.g., as described herein) and/or any of the STING agonists (e.g., as described herein) and a pharmaceutically acceptable carrier. The pharmaceutical composition can include a PIK4B Golgi recruitment modulating agent as the only active agent. The pharmaceutical composition can include both a PIK4B Golgi recruitment modulating agent and a STING agonist, e.g., CDN. The subject pharmaceutical compositions find use in the kits and methods described herein.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized foreign pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the agent of interest is administered. Such pharmaceutical carriers can be, for example, sterile liquids, such as dimethyl sulfoxide (DMSO) or saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The inhibitors can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, hereby incorporated by reference herein in its entirety. Such compositions will contain a therapeutically effective amount of the mitochondrial transport protein (e.g., a Miro protein, a TRAK protein, or Khc) inhibitor, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The pharmaceutical composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides or proteins, ions (e.g., sodium, potassium, calcium, magnesium, manganese) and lipids.

Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use may be sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

The pharmaceutical composition can be formulated for intravenous, oral, via implant, transmucosal, transdermal, intramuscular, intrathecal, or subcutaneous administration. In some cases, the pharmaceutical composition is formulated for intravenous administration.

In other cases, the pharmaceutical composition is formulated for subcutaneous administration. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGAs). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone. Osteopontin or nucleic acids of the invention can also be administered attached to particles using a gene gun.

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

The pharmaceutical composition containing an active agent can be formulated to cross the blood brain barrier (BBB). One strategy for drug delivery through the blood brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin. A BBB disrupting agent can be co-administered with the therapeutic compositions when the compositions are administered by intravascular injection. Other strategies to go through the BBB may entail the use of endogenous transport systems, including caveoil-1 mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. Active transport moieties may also be conjugated to the therapeutic compounds for use in the invention to facilitate transport across the endothelial wall of the blood vessel. Alternatively, drug delivery of the pharmaceutical composition behind the BBB may be by local delivery, for example by intrathecal delivery, e.g., through an Ommaya reservoir (see, e.g., US Patent Nos. 5,222,982 and 5385582, incorporated herein by reference); by bolus injection, e.g., by a syringe, e.g., intravitreally or intracranially; by continuous infusion, e.g., by cannulation, e.g., with convection (see, e.g., US Application No. 20070254842, incorporated here by reference); or by implanting a device upon which the inhibitor pharmaceutical composition has been reversibly affixed (see e.g., US Application Nos. 20080081064 and 20090196903, incorporated herein by reference).

In certain embodiments, the pharmaceutical composition containing the active agent is formulated in a delivery vehicle, e.g., to enhance passive cytosolic transport. Any convenient protocol may be employed to facilitate delivery of the CDN active agent across the plasma membrane of a cell and into the cytosol.

In some instances, the STING agonist, e.g., CDN, and/or PIK4B Golgi recruitment modulating active agent may be encapsulated in a delivery vehicle comprising liposomes in the pharmaceutical composition. Methods of using liposomes for drug delivery and other therapeutic uses are known in the art. See, e.g., US Pat. Nos. 8329213, US6465008, US5013556, US Application No. 20070110798, and Andrews et al., Mol Pharm 2012 9:1118, which are incorporated herein by reference. Liposomes may be modified to render their surface more hydrophilic by adding polyethylene glycol ("pegylated") to the bilayer, which increases their circulation time in the bloodstream. These are known as "stealth" liposomes and are especially useful as carriers for hydrophilic (water soluble) molecules.

In certain embodiments, nano- or microparticles made from biodegradable materials such as poly(lactic acid), poly(y-glutamic acid), poly(glycolic acid), polylactic-co-glycolic acid, polyethylenimine, or alginate microparticles, and cationic microparticles, including dedrimers, such as cyclodextrins, may be employed as delivery vehicles for the active agents to promote cellular uptake. See, e.g., US Pat. No. 8187571 , Krishnamachari et al., Adv Drug Deliv Rev 2009 61 :205, Garzon et al., 2005 Vaccine 23:1384, incorporated herein by reference. In certain embodiments, the delivery vehicle for delivering the active agents can also be targeting delivery vehicles, e.g., a liposome containing one or more targeting moieties or biodistribution modifiers on the surface of the liposome. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target. The specific binding agent can be any molecule that specifically binds to a protein, peptide, biomacromolecule, cell, tissue, etc. that is being targeted (e.g., protein, peptide, biomacromolecule, cell, tissue, etc. wherein the active agent exerts its desired effect). Depending on the nature of the target site, the specific binding agent can be, but is not limited to, an antibody against an epitope of a peptidic analyte, or any recognition molecule, such as a member of a specific binding pair. For example, suitable specific binding pairs include, but are not limited to: a member of a receptor/ligand pair; a ligand-binding portion of a receptor; a member of an antibody/antigen pair; an antigen-binding fragment of an antibody; a hapten; a member of a lectin/carbohydrate pair; a member of an enzyme/substrate pair; biotin/avidin; biotin/streptavidin; digoxin/antidigoxin; a member of a peptide aptamer binding pair; and the like. In certain embodiments, the specific binding moiety includes an antibody. In some cases, the specific binding moiety is a fragment of an antibody which retains specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The specific binding moiety may also include Fab', Fv, F(ab')2, and or other antibody fragments that retain specific binding to antigen. In certain embodiments, the targeting moiety is a binding agent that specifically interacts with a molecule expressed on a tumor cell or an immune cell (e.g., CD4, CD8, CD69, CD62L, and the like), such that the targeting delivery vehicle containing the cyclic-di-nucleotide or STING active agents is delivered to the site of a tumor or to specific immune cells.

Where desired, any combinations of the above listed delivery vehicles may be used advantageously to enhance delivery of the active agents to the target cells.

Components of the pharmaceutical composition can be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ample of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In some embodiments, the pharmaceutical composition is supplied as a dry sterilized lyophilized powder that is capable of being reconstituted to the appropriate concentration for administration to a subject. In some embodiments, the pharmaceutical composition is supplied as a water free concentrate. In some embodiments, the pharmaceutical composition is supplied as a dry sterile lyophilized powder at a unit dosage of at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, or at least 75 mg.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, xanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

In some embodiments, the pharmaceutical composition is formulated as a salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In certain embodiments, the pharmaceutical composition contains a prodrug derivative of any of the CDN active agents provided herein. Such prodrugs can be subsequently converted to an active form of the CDN in the body of the subject administered the pharmaceutical composition.

Kits

Kits including a PIK4B Golgi recruitment -modulating agent (e.g., as described herein) and a STING agonist, e.g., CDN active agent, (e.g., as described herein) are provided. In some cases, the kit includes a unit dose of the subject active agents e.g., in an oral or injectable dose.

In the subject kits, the one or more components are present in the same or different containers, as may be convenient or desirable. In addition to the containers containing the components of the kits (e.g., unit doses) instructions can be included describing the use and attendant benefits of the STING agonist, e.g., CDN, and PIK4B Golgi recruitment-modulating agent in treating a pathological condition of interest. Instructions may be provided in a variety of different formats. In certain embodiments, the instructions may include complete protocols for practicing the subject methods or means for obtaining the same (e.g., a website URL directing the user to a webpage which provides the instructions), where these instructions may be printed on a substrate, where substrate may be one or more of: a package insert, the packaging, reagent containers and the like. Utility

This disclosure provides methods, compositions and kits that find use in a variety of applications. The subject methods find use in a variety of applications where it is desirable to either inhibit or enhance STING pathway activation in a target cell. Therapeutic applications of interest include, but are not limited to, cancer immunotherapy, antiviral applications, treatment of autoimmune or inflammatory disease, and other in applications similar to those described herein. In some cases, this disclosure provides for methods and applications involving STING pathway activation in immune cells in vivo, leading to greater activation of the immune response.

Specific applications of interest include those in which a subject is treated for a disease condition that would benefit from an increase in type I interferon by providing the subject with a therapeutically effective amount of a STING agonist, e.g., CDN active agent.

In some instances, it may be desirable to increase a type I interferon or STING mediated response in a healthy individual, e.g., for the prevention of a disease or condition. The subject methods can be applied to enhance STING pathway activation for improved cancer immunotherapy. In anti-cancer therapy, the subject methods can also be applied locally or systemically to inhibit or block uptake of a STING agonist, e.g., CDN active agent, by target cells where it is desirable to alleviate the possible toxic effects of such a STING agonist, e.g., a CDN administered for anti-cancer treatment.

The present invention also provides a method of inhibiting type I interferon production mediated by the cGAS-STING pathway. In some embodiments, subjects suitable for treatment with a method described herein include individuals having an immunological or inflammatory disease or disorder including, but not limited to a cancer, an autoimmune disease or disorder, an allergic reaction, a chronic infectious disease and an immunodeficiency disease or disorder. The subject methods can be applied with the purpose of inhibiting CDN uptake and signaling in autoimmune/inflammatory diseases linked to aberrant CDN signaling, In some embodiments, the disease or disorder can be a type I interferonopathy (e.g., Aicardi-Goutieres Syndrome, Sjogren's syndrome, Singleton-Merten Syndrome, proteasome-associated autoinflammatory syndrome, SAVI (STING-associated vasculopathy with onset in infancy), CANDLE syndrome, chilblain lupus erythematosus, systemic lupus erythematosus, spondyloenchondrodysplasia), rheumatoid arthritis, juvenile rheumatoid arthritis, idiopathic thrombocytopenic purpura, autoimmune myocarditis, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, psoriasis, Type 1 diabetes, or Type 2 diabetes. In other embodiments, the disease or disorder can be an inflammatory disorder (e.g., atherosclerosis, dermatomyositis, SIRS, sepsis, septic shock, atherosclerosis, celiac disease, interstitial cystitis, transplant rejection, rheumatoid arthritis, juvenile rheumatoid arthritis, inflammatory bowel disease (ulcerative colitis, Crohn’s disease), age-related macular degeneration, IgA nephropathy, glomerulonephritis, vasculitis, polymyositis, or Wegener’s disease). In some instances, the methods find use in inhibiting STING pathway activation in inflammatory diseases. Inappropriate activation of the STING pathway may underlie certain inflammatory diseases, including inflammatory bowel diseases, arthritis and possibly lupus. Inhibitors of PI4KB are effective in ameliorating the pathological manifestations of such diseases.

In some embodiments, subjects suitable for treatment with a method of the present invention where STING pathway activation is enhanced include individuals having a cellular proliferative disease, such as a neoplastic disease (e.g., cancer). Cellular proliferative disease is characterized by the undesired propagation of cells, including, but not limited to, neoplastic disease conditions, e.g., cancer. Examples of cellular proliferative disease include, but are not limited to, abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying, for example, rheumatoid arthritis, psoriasis, diabetic retinopathy, other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration, corneal graft rejection, neurovascular glaucoma and Oster Webber syndrome, psoriasis, restenosis, fungal, parasitic and viral infections such cytomegaloviral infections. Subjects to be treated according to the methods of the invention include any individual having any of the above-mentioned disorders.

In some instances, the methods find use in enhancing the effects of STING agonists as cancer immunotherapies. Examples of such instances include (i) co-administering STING agonists and an OSBP inhibitor into tumors; (ii) administering low doses of STING agonist systemically and OSBP inhibitors intratumorally; (iii) administering STING agonists intratumorally and OSBP inhibitors systemically; (iv) administering low doses of STING agonists and OSBP inhibitors systemically.

In some instances, the methods find use in enhancing the immuno-therapeutic effects of chemo- and radiotherapies. In addition to killing tumor cells, chemo and radio therapies are known to activate the cGAS-STING pathway and can thereby promote antitumor immune responses. Combining OSBP inhibitors with chemo or radiotherapy greatly amplifies the anti-tumor immune responses that accompany radio and chemotherapy.

In some instances, the methods find use in protecting specific tissues from STING pathway activation. STING pathway agonists such as CDNs, at high concentrations, can cause local tissue damage. In the context of systemic treatments with STING agonists (or systemic effects that result when STING agonists migrate out of a locally treated tumor), critical tissues are protected if they are locally treated with a PI4KB inhibitor.

The subject methods can be applied with the purpose of increasing intercellular 2’3’- cGAMP signaling between virus-infected and uninfected cells for amplification of anti-viral immunity. In some embodiments, subjects suitable for treatment with a subject method include individuals who have been clinically diagnosed as infected with a virus. In some embodiments, the virus is a hepatitis virus (e.g., HAV, HBV, HCV, delta, etc.), particularly HCV, are suitable for treatment with the methods of the instant invention. Individuals who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti- HCV antibody in their serum. Such individuals include naive individuals (e.g., individuals not previously treated for HCV, particularly those who have not previously received IFN-a-based or ribavirin-based therapy) and individuals who have failed prior treatment for HCV.

In certain embodiments, subjects suitable for treatment with a method of the present invention include individuals having multiple sclerosis. Multiple sclerosis refers to an autoimmune neurodegenerative disease, which is marked by inflammation within the central nervous system with lymphocyte attack against myelin produced by oligodendrocytes, plaque formation and demyelization with destruction of the myelin sheath of axons in the brain and spinal cord, leading to significant neurological disability over time. Typically, at onset an otherwise healthy person presents with the acute or sub-acute onset of neurological symptomatology (attack) manifested by unilateral loss of vision, vertigo, ataxia, dyscoordination, gait difficulties, sensory impairment characterized by paresthesia, dysesthesia, sensory loss, urinary disturbances until incontinence, diplopia, dysarthria or various degrees of motor weakness until paralysis. The symptoms may be painless, remain for several days to a few weeks, and then partially or completely resolve. After a period of remission, a second attack will occur. During this period after the first attack, the patient is defined to suffer from probable MS. Probable MS patients may remain undiagnosed for years. When the second attack occurs the diagnosis of clinically definite MS (CDMS) is made (Poser criteria 1983; C. M. Poser et al., Ann. Neurol. 1983; 13, 227).

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Using a genetic screen, ACBD3 was identified as a gene involved in STING pathway activation via exogenously provided cyclic dinucleotides (See FIGS. 1A and 1 B). ACBD3 recruits PI4KB to the Golgi, to stimulate PI4P accumulation in the Golgi (See FIGS 2A and 2B). ACBD3 depletion in cells results in impaired STING activation in response to certain cyclic dinucleotides. A negative regulator of PI4KB recruitment to the Golgi is the protein OSBP. Modulation of ACBD3 and OSBP functions can be used to modulate STING pathway function as a means to either enhance activation of the pathway in the context of immunotherapy, or inhibit pathway activation in the case of pathology resulting from inappropriate STING pathway activation. As a proof of principle, molecules that inhibit OSBP have been described (itraconazole and OSW-1), including one (itraconazole) that is FDA approved for treating fungal diseases albeit by a different mechanism. We have shown that these inhibitors, which are predicted to increase PI4P accumulation in the Golgi, greatly enhance STING pathway activation induced by exogenously supplied cyclic dinucleotides. See FIGS 3A to 3C. Conversely, we find that genetic depletion of PI4KB depresses STING signaling in cells induced by STING agonists, indicating the utility of PI4KB inhibitors for treating inflammatory diseases that result from inappropriate STING pathway activation. Further details are provided in Appendix A of priority provisional application serial no. the disclosure of which is herein incorporated by references.

Findings include:

ACBD3 knockdown:

• Decreased response to c-di-AMP * synthetic and bacteria! CDNs

• Slightly increased response to cGAMP

• differential STING regulation depending on CDNs

• ACBD3 does not affect CDN uptake

• ACBD3 and STING colocaiize and interact upon stimulation with CDNs

ACBD3 increases Pi4P in Golgi <-> OSBP shuttles PI4P out of Go!gi * OS8P inhibitors increase sensitivity to both CDNs -> boost in vivo response to injected CDNs

Notwithstanding the appended claims, the disclosure is also defined by the following clauses:

1 . A method of modulating stimulator of interferon genes (STING) pathway activation in a cell, the method comprising: modulating Phosphatidylinositol 4-Kinase Beta (PI4KB) Golgi recruitment to modulate STING pathway activation in the cell.

2. The method according to clause 1 , wherein the method comprises increasing PI4KB Golgi recruitment to enhance STING pathway activation in the cell.

3. The method according to clause 2, wherein the method comprises inhibiting Oxysterol-binding protein (OSBP) activity in the cell. 4. The method according to clauses 2 or 3, wherein the method comprises increasing acyl-coenzyme A binding domain containing 3 (ACBD3) activity in the cell.

5. The method according to any of clauses 2 to 4, wherein the method further comprises contacting the cell with a STING agonist.

6. The method according to clause 5, wherein the STING agonist comprises a cyclic dinucleotide (CDN).

7. The method according to clause 6, wherein the method results in increasing STING- dependent type I interferon production in the cell.

8. The method according to clause 7, wherein the type I interferon is interferon alpha or interferon beta.

9. The method according to clause 1 , wherein the method comprises decreasing PI4KB Golgi recruitment to reduce STING pathway activation in the cell.

10. The method according to clause 9, wherein the method comprises increasing OSBP activity in the cell.

11 . The method according to clauses 9 or 10, wherein the method comprises inhibiting ACBD3 activity in the cell.

12. The method according to any one of clauses 1 to 11 , wherein the cell is in vitro.

13. The method according to any one of clauses 1 to 11 , wherein the cell is in vivo.

14. A method of enhancing STING pathway activation in a cell, the method comprising: contacting the cell with an agent that increases PI4KB Golgi recruitment to enhance

STING pathway activation in the cell.

15. The method according to clause 14, wherein the agent inhibits OSBP activity in the cell.

16. The method according to clauses 14 or 15, wherein the agent increases ACBD3 activity in the cell.

17. The method according to any of clauses 14 to 16, wherein the agent is selected from the group consisting of a small molecule, a nucleic acid and a polypeptide.

18. The method according to any of clauses 14 to 17, wherein the method further comprises contacting the cell with a STING agonist.

19. The method according to clause 18, wherein the sting agonist comprises a CDN.

20. The method according to clause 19, wherein the CDN comprises a 2'-5' phosphodiester linkage.

21 . The method according to clause 20, wherein the CDN comprises two 2’-5’ phosphodiester linkages.

22. The method according to clause 20, wherein the CDN comprises a 2’-5’ phosphodiester linkage and a 3’-5’ phosphodiester linkage.

23. The method according to clause 22, wherein the CDN has the formula:

wherein X and Y are each independently:

or a salt thereof. 24. The method according to clause 19, wherein the CDN comprises a 2'-5' thiophosphate linkage.

25. The method according to clause 24, wherein the CDN comprises two 2’-5’ thiophosphate linkages.

26. The method according to clause 19, wherein the CDN is cyclic[G(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

27. The method according to clause 19, wherein the CDN is cyclic[A(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage. 28. The method according to clause 19, wherein the CDN is cyclic[G(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

29. The method according to clause 19, wherein the CDN is cyclic[A(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

30. The method according to any of clauses 14 to 29, wherein the method results in increasing STING-dependent type I interferon production in the cell. 31 . The method according to clause 30, wherein the type I interferon is interferon alpha or interferon beta.

32. The method according to any of clauses 14 to 31 , wherein the contacting comprises administration of the agent that increases PI4KB Golgi recruitment to a subject.

33. The method according to clause 32, wherein the subject has a viral infection.

34. The method according to clause 32, wherein the subject has a bacterial infection.

35. The method according to clause 32, wherein the subject has neoplastic disease.

36. The method according to clause 35, wherein the method further comprises administering immunotherapy to the subject.

37. The method according to clause 35, wherein the method further comprises administering chemotherapy to the subject.

38. The method according to clause 35, wherein the method further comprises administering radiotherapy to the subject.

39. The method according to any of clauses 32 to 38, wherein the subject is mammal.

40. The method according to clause 39, wherein the mammal is a human.

41 . A method of reducing STING pathway activation in a cell, the method comprising: contacting the cell with an agent that decreases PI4KB Golgi recruitment to reduce

STING pathway activation in the cell.

42. The method according to clause 41 , wherein the agent increases OSBP activity in the cell.

43. The method according to clauses 41 or 42, wherein the agent decreases ACBD3 activity in the cell.

44. The method according to any of clauses 41 to 43, wherein the agent is selected from the group consisting of a small molecule, a nucleic acid and a polypeptide.

45. The method according to any of clauses 41 to 44, wherein the contacting comprises administration of the agent that decreases PI4KB Golgi recruitment to a subject.

46. The method according to clause 45, wherein the subject has an autoimmune or inflammatory disease linked to aberrant 2’3’-cGAMP signaling.

47. The method according to clause 46, wherein the autoimmune or inflammatory disease is selected from Aicardi-Goutieres Syndrome, Sjogren's syndrome, Singleton- Merten Syndrome, proteasome-associated autoinflammatory syndrome, SAVI (STING- associated vasculopathy with onset in infancy), CANDLE syndrome, chilblain lupus erythematosus, systemic lupus erythematosus, spondyloenchondrodysplasia), rheumatoid arthritis, juvenile rheumatoid arthritis, idiopathic thrombocytopenic purpura, autoimmune myocarditis, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia, psoriasis, Type 1 diabetes and Type 2 diabetes. 48. The method according to clause 45, wherein the method comprises inhibiting CDN uptake locally or systemically to alleviate a toxic effect of a STING agonist administered for anti-cancer treatment.

49. The method according to any one of clauses 45 to 48, wherein the subject is mammal.

50. The method according to clause 49, wherein the mammal is a human.

51 . A composition comprising: a STING agonist; and an agent that increases PI4KB Golgi recruitment. 52. The composition according to clause 51 , wherein the agent inhibits OSBP activity.

53. The composition according to clause 51 , wherein the agent increases ACBD3 activity.

54. The composition according to any of clauses 51 to 53 wherein the agent is selected from the group consisting of a small molecule, a nucleic acid and a polypeptide. 55. The composition according to any of clauses 51 to 54, wherein the STING agonist comprises a CDN.

56. The composition according to clause 55, wherein the CDN comprises a 2'-5' phosphodiester linkage.

57. The composition according to clause 56, wherein the CDN comprises two 2’-5’ phosphodiester linkages.

58. The composition according to clause 56, wherein the CDN comprises a 2’-5’ phosphodiester linkage and a 3’-5’ phosphodiester linkage.

59. The composition according to clause 58, wherein the CDN has the formula:

wherein X and Y are each independently:

or a salt thereof.

60. The composition according to clause 55, wherein the CDN comprises a 2'-5' thiophosphate linkage.

61 . The composition according to clause 60, wherein the CDN comprises two 2’-5’ thiophosphate linkages.

62. The composition according to clause 55, wherein the CDN is cyclic[G(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

63. The composition according to clause 55, wherein the CDN is cyclic[A(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

64. The composition according to clause 55, wherein the CDN is cyclic[G(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

65. The composition according to clause 55, wherein the CDN is cyclic[A(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

66. A kit comprising: a first composition comprising a STING agonist; and a second composition comprising an agent that increases PI4KB Golgi recruitment.

67. The kit according to clause 66, wherein the agent inhibits OSBP activity.

68. The kit according to clause 66, wherein the agent increases ACBD3 activity.

69. The kit according to any of clauses 66 to 68, wherein the agent is selected from the group consisting of a small molecule, a nucleic acid and a polypeptide.

70. The kit according to any of clauses 66 to 69, wherein the STING agonist comprises a CDN.

71 . The kit according to clause 70, wherein the CDN comprises a 2'-5' phosphodiester linkage. 72. The kit according to clause 71 , wherein the CDN comprises two 2’-5’ phosphodiester linkages.

73. The kit according to clause 71 , wherein the CDN comprises a 2’-5’ phosphodiester linkage and a 3’-5’ phosphodiester linkage. 74. The kit according to clause 73, wherein the CDN has the formula:

wherein X and Y are each independently:

or a salt thereof. 75. The kit according to clause 70, wherein the CDN comprises a 2'-5' thiophosphate linkage.

76. The kit according to clause 75, wherein the CDN comprises two 2’-5’ thiophosphate linkages.

77. The kit according to clause 70, wherein the CDN is cyclic[G(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

78. The kit according to clause 70, wherein the CDN is cyclic[A(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

79. The kit according to clause 70, wherein the CDN is cyclic[G(2’5’)pA(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage. 80. The kit according to clause 67, wherein the CDN is cyclic[A(2’5’)pG(3’5’)p], wherein each prefers to a phosphate, thiophosphate or dithiophosphate internucleotide linkage.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

What is claimed is:

2. The method according to claim 1 , wherein the method comprises increasing PI4KB Golgi recruitment to enhance STING pathway activation in the cell.

3. The method according to claim 2, wherein the method comprises increasing acyl- coenzyme A binding domain containing 3 (ACBD3) activity in the cell.

4. The method according to any of claims 2 to 3, wherein the method further comprises contacting the cell with a STING agonist.

5. The method according to claim 4, wherein the STING agonist comprises a cyclic dinucleotide (CDN).

6. The method according to claim 5, wherein the method results in increasing STING- dependent type I interferon production in the cell.

7. The method according to claim 6, wherein the type I interferon is interferon alpha or interferon beta.

8. The method according to claim 1 , wherein the method comprises decreasing PI4KB Golgi recruitment to reduce STING pathway activation in the cell.

9. The method according to claim 9, wherein the method comprises inhibiting ACBD3 activity in the cell.

10. The method according to any one of claims 1 to 9, wherein the cell is in vitro.

11 The method according to any one of claims 1 to 9, wherein the cell is in vivo.

12. A method of enhancing STING pathway activation in a cell, the method comprising: contacting the cell with an agent that increases PI4KB Golgi recruitment to enhance STING pathway activation in the cell.

13. The method according to claim 12, wherein the agent increases ACBD3 activity in the cell.

14. The method according to any of claims 12 to 13, wherein the method further comprises contacting the cell with a STING agonist.

15. A composition comprising: a STING agonist; and an agent that increases PI4KB Golgi recruitment.