Compositions and methods for preventing microbial-mediated disease

Abstract

The disclosure provides RNA compositions and methods for reducing human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) secretome mediated disease and an adjuvant for vaccine efficacy.

Description

INTERNATIONAL PATENT APPLICATIONFORCOMPOSITIONS AND METHODS FOR PREVENTING MICROB IAL-MEDIATED DISEASETECHNICAL FIELD

[0001] The disclosure describes compositions and methods to inhibit microbial disruption of the endosomal pathway by preventing Human cytomegalovirus (HCMV) and Epstein Barr virus (EBV) secretome release and prevent microbial evasion from the immune system in uninfected and infected cells.BACKGROUND

[0002] Human cytomegalovirus (HCMV) infection is almost ubiquitous in humans by the eighth decade of life. HCMV enters the body by infecting the oral keratinocytes, olfactory neurons, nasopharyngeal epithelial cells, ocular conjunctiva, or the urogenital and anal epithelium. Infected cells shed virions that infect other cells or are phagocytosed by circulating phagocytosing cells, such as monocytes and macrophages. HCMV remains latent in circulating monocytes and macrophages but can reactivate during immunosuppression and when exposed to inflammatory cytokines released from cancer, infection, or sites of injury, to disseminate HCMV infection in more myeloid cells or other cell types (Smith et al. 2004). For example, inflammatory cytokines stimulate monocytes to differentiate into monocyte-derived dendritic cells, which if infected with latent HCMV induces HCMV lytic replication, virion production and release, resulting in HCMV infection at distant sites, such as endothelial cells in brain microvessels. The Epstein Barr virus (EBV) infection is also ubiquitous, infecting almost 95% of adults worldwide. EBV remains latent in B cells and reactivates during immunosuppression and when exposed to inflammatory cytokines released from cancer, infection, or sites of injury, to disseminate EBV infection more B cells or other cell types. Both HCMV and EBV infected cells release a viral secretome containing viral proteins in the latent state.SUMMARY OF THE INVENTION

[0003] Cells with latent infection of human cytomegalovirus (HCMV) and the Epstein-Barr virus (EBV) release a secretome released in non-infectious enveloped particles (NIEPs), dense bodies, and viral exosomes that can enter uninfected cells, such as immune cells and endothelial cells. The disclosure identifies HCMV ppi 50 and EBV gp350 known to be secreted from infected cells and can enter uninfected cells to disrupt endocytic recycling and multivesicular body biogenesis by binding to the proteins BICD1 and BICD2. Binding to BICD1 and BIDC2can displace Rab6b binding, disrupting anterograde and retrograde vesicle trafficking and endosomal pathway recycling. HCMC ppi 50 and gp350 will inhibit endocytic recycling to prevent the presentation of viral proteins on the cell surface, including antigen presenting monocyte derived dendritic cells. HCMC ppi 50 and gp350 will also inadvertently decrease the presentation of other microbial peptides with pathogen-associated molecular patterns leading to opportunistic infections.

[0004] Chronic exposure to HCMV and EBV microbial secretomes from latent infections can have adverse effects over time, e.g., accelerated senescence, metabolic dysfunction, dedifferentiation, inflammation, and immunosuppression. Depending upon the HCMV and EBV strain type and host genetics, the viral secretome and inflammation from local infection can cause autoimmune disease, cancer, diabetes, ulcerative colitis, pancreatis, neuropathy, nonalcoholic steatosis (NASH), non-alcoholic fatty liver disease (NALFD), sarcopenia, obesity, mononucleosis syndrome, tinnitus, hearing loss, dementia, and vascular disease, such as cardiovascular and neurovascular disease, and increased susceptibility and severity to opportunistic microbial infection.

[0005] Targeting and eliminating the reservoir of HCMV and EBV infected cells producing the viral secretome is more important. ASOs with at least 80% complementary (antisense) sequence similarity to any 15 to 30 contiguous nucleotides of HCMV mRNA ppl50, including 25 nucleotides 5’ to the ATG start site, and 25 nucleotides 3’ from coding sequence for use in treating HCMV-mediated human disease. In some embodiments, the ppi 50 mRNA ASO has at least 80% sequence identity to the sequences disclosed in SEQ ID NOs: 1-14. In some embodiments, the ppl50 mRNA ASO sequence is derived from SEQ ID NO: 15.

[0006] In some embodiments, compositions comprise one or more ppi 50 mRNA ASOs, a nanocarrier encapsulating or complexing the ASOs, or a vector transduced with a construct encoding one or more disclosed ppl50 ASOs (SEQ ID NO: 1-14).

[0007] In some embodiments, the vector comprises an adeno-associated virus (e.g., AAV2) or lentivirus.

[0008] In some embodiments, the ppi 50 RNAi composition further comprises an effective amount of ganciclovir (GCV) or its prodrug valganciclovir (VGCV) for preventing HCMV replication.

[0009] In some embodiments, the disclosed composition further comprises a pharmaceutically acceptable excipient, binder, or enteric coating.

[0010] In some embodiments, the disclosure provides for a method of treating HCMV-mediated disease, comprising administering the disclosed ASO or vector compositions to a subject in need thereof or prophylactically.[OH] The disclosure contemplates ASOs with at least 80% complementary (antisense) sequence similarity to any 15 to 30 contiguous nucleotides of EBV mRNA gp350, 25 nucleotides 5’ to the ATG start site, or 25 nucleotides 3’ from coding sequence for use in treating EBV-mediated human disease.

[0012] In some embodiments, the gp350 ASOs have at least 80% sequence identity to the sequences disclosed in SEQ ID NOs: 16-28. In some embodiments, the sequence is derived from SEQ ID NO: 29.

[0013] In some embodiments, the ASOs are modified or substituted at the backbone, nucleobase, or ribose sugar. In some embodiments, the backbone modification comprises one or more Rp or Sp phosphorothioate linkages or a phosphorodiamidate morpholino oligonucleotide. In some embodiments, one or more nucleobases are modified with a 5-methylcytidine, 5- methyluridine, abasic RNA, or a combination thereof. In some embodiments, one or more ribose is modified with a locked nucleic acid (LNA), 2'-O-methyl, 2'-O-methoxy ethyl, 2'-fluoro, 2'- fluoroarabinonucleic acid, ethylene-bridged nucleic acid (ENA), or combination thereof. In some embodiments, ASO further comprises an endosomal membrane destabilizing cell penetrating peptide (CPP). In some embodiments, the ASO is covalently or non-covalently conjugated to a cell targeting moiety.

[0014] In some embodiments, the composition of one or more ASO targeting ppi 50 and gp350 includes a vaccine to stimulate the immune system. In some embodiments, the composition of one or more ASO targeting ppi 50 includes a vaccine to stimulate the immune system. In some embodiments, the composition of one or more ASO targeting gp350 includes a vaccine to stimulate the immune system.

[0015] In one embodiment, a nanocarrier encapsulates or complexes the ppi 50 or gp350 ASOs, or a combination thereof. In some embodiments, the nanocarrier is a pH sensitive lipid nanoparticle. In some embodiments, the nanocarrier comprises a cell targeting moiety. In some embodiments, the nanocarrier comprising a moiety that targets monocytes, macrophages or B cells to eliminate the reservoir latent infection. In some embodiments the nanocarrier comprises an mRNA that encodes an immunogenic peptide from HCMV or EBV to activate the immune system for targeting and uncloaking an infected cell. In some embodiments, a HCMV or EBV vaccine is administered to a subject after administering the ASO composition targeting the HCMV or EBV endosomal disrupting protein to ensure that only infected cells are targeted and eliminated by the immune system. In some embodiments the nanocarrier comprising the ASO composition targeting the HCMV or EBV endosomal disrupting protein also comprises an mRNA that encodes an immunogenic protein or peptide expressed in cancer cells to activate the immune system for targeting the cancer.

[0016] In some embodiments, the disclosed gp350 RNAi compositions further comprise an effective dosage of tenofovir (e.g., 25 mg) for preventing EBV replication.

[0017] In some embodiments, the disclosed compositions comprise effective RNA therapy concentration in a unit dosage form e.g., tablet, capsule, or ampule.

[0018] The disclosure provides a method of treating EBV-mediated disease, comprising administering to a subject in need thereof the disclosed gp350 mRNA ASO compositions comprising one or more sequences from SEQ ID NOs 16-29.

[0019] In some embodiments, the composition is delivered by oral administration, intravenous injection, or inhalation.

[0020] In some embodiments, treating EBV infection in a subject with the disclosed composition prevents or ameliorates dementia, comprising administering one or more of the disclosed gp350 mRNA ASO compositions, or a combination thereof.

[0021] In some embodiments, the disclosure provides a method for treating cancer in a subject, comprising administering the disclosed exemplary ASO compositions or ASO with the same effect. In some embodiments, the ASO composition for treating cancer is administered locally at the cancer site, or intravenously, orally, by inhalation, or a combination thereof.

[0022] In some embodiments, the disclosure provides a method of treating microbial infection in a subject, comprising administering the disclosed ASO compositions.

[0023] In some embodiments, the disclosure provides gram-negative bacteria recombinantly engineered with an expression cassette that encodes one or more disclosed ASOs (SEQ ID NOs: 1-29). In a preferred embodiment, the cassette encodes an ASO fused to a tRNAlys scaffold. In yet another preferred embodiment, the engineered bacterial genus is Akkermansia. The recombinant engineered bacteria are freeze-dried (lyophilized) with stabilizers. The disclosure provides a method of treating HCMV and EBV infection in a subject comprising oral administration of recombinant bacteria engineered to express the disclosed ppi 50 or gp350 ASOs in OMVsBRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1A shows a UniProt Clustal alignment of C-terminal ppl50 (P08318) (614-842) aligned with the Rab6b. EMBOSS 001 is ppl50 and EMBOSS 002 is Rab6b. An asterisk indicates an identical amino acid; a colon indicates an amino acid with highly conserved properties, a dot indicates an amino acid with conserved properties.

[0025] FIG. IB shows a UniProt Clustal alignment of C-terminal gp350 (552-697) aligned with the Rab6b. EMBOSS 001 is gp350 and EMBOSS 002 is Rab6b. An asterisk indicates an identical amino acid; a colon indicates an amino acid with highly conserved properties, a dotindicates an amino acid with conserved properties.

[0026] FIG. 2A shows average tissue expression of amyloid precursor protein (APP) in women; graphic obtained from The Human Protein Atlas; (Uhlen et al. 2015).

[0027] FIG. 2B shows the average expression of amyloid precursor protein (APP) in men; graphic obtained from The Human Protein Atlas; (Uhlen et al. 2015).

[0028] FIG. 3 shows a graph depicting the levels of HCMV protein hcmvIL-10 detected in the blood of healthy HCMV seropositive donors; thesis figure presented with permission from author Vivian Young.

[0029] FIG. 4 is a schematic showing miR124, aging, IE1 variants inhibiting STAT3 phosphorylation and nuclear transport or an lElonc variant that increases STAT3 -mediated proliferation.

[0030] FIG. 5 shows a BLAST sequence alignment between IE1 (UniProt ID YP 081562) with 27% identity and 36% positives to phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C) (UniProt ID 060331.2) aligned at the C-terminal end.

[0031] FIG. 6A is a schematic showing IE1, with sequence similarity to PIP5K1C, phosphorylating PIP4 to induce PIP2-mediated inhibition of CK2.

[0032] FIG. 6B is a schematic showing IE2 directly interacting with hnRNPA2 and increases hnRNPA2Bl expression to decrease GLUT1 mRNA stability, translation, or transport.

[0033] FIG. 7A shows the average tissue expression of GLUT1 in women; The Human Protein Atlas (Uhlen et al. 2015).

[0034] FIG. 7B shows the average tissue expression of GLUT1 in men; The Human Protein Atlas (Uhlen et al. 2015).

[0035] FIG. 8 shows the sequence similarity of C-terminal HCMV ppi 50 (Query) and Epstein Barr virus (HHV-4) Gp350 (Subject). The total BLAST score is 35 with an Expect value of 6e- 05. The percent sequence identity is 32% with 49% positives.

[0036] FIG. 9 shows the interactome of BICD 1. Lines are drawn from BICD1 to other proteins shown to interact with BICD1 (diagram obtained from STRING.org; Szklarczyk D. et al., 2023).

[0037] FIG. 10 shows exosomes released from HCMV infected gastrointestinal enterocytes are released from the basolateral membrane with GLUT2, ApoE alleles, and HCMV receptors on the exosome surface. The schematic was produced using Biorender.

[0038] FIG. HA shows the sequence alignment result from a DELTA-BLAST of the entire ppi 50 sequence in UniProtKB / Swiss-Prot database exclusive for Homo sapiens with default parameter setting.

[0039] FIG. 11B shows the sequence alignment result from a DELTA-BLAST of ppi 50 amino acid sequences 838-1003 in UniProtKB / Swiss-Prot database exclusive for Homo sapiens with adefault parameter setting.

[0040] FIG. 11C shows the sequence alignment result from a DELTA-BLAST of the ppi 50 amino acid sequences 811-931 in UniProtKB / Swiss-Prot database exclusive for Homo sapiens with a default parameter setting.

[0041] FIG. HD shows a second sequence alignment result from a DELTA-BLAST of the ppi 50 amino acids sequence 811-931 in UniProtKB / Swiss-Prot database exclusive for Homo sapiens with a default parameter setting.

[0042] FIG. 12A shows the amino acid alignment between human BICD1 (Q96G01) and human BICD2 (Q8TD16-1) with high sequence similarity between BICD1 and BICD2 between amino acids 630 to 820, the location that binds to ppl50.

[0043] FIG. 12B shows BICD2 protein expression level in different immune cell types (data obtained from The Human Protein Atlas).DETAILED DESCRIPTIONDefinitions

[0044] Unless defined otherwise, the technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art. However, if there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0045] The terminology used herein is for the purpose of describing an embodiment only and is not intended to be limiting. Reference to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” in the present disclosure means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessary all embodiments. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are used herein, they mean “comprising”.

[0046] Unless otherwise specified, the words “comprising”, “comprise”, “comprises”, “having”, “have”, “has”, “including”, “includes”, “containing”, “contains”, and “contain” are or open- ended and do not exclude additional unrecited elements. The term “between” means the range of numbers including the first and the last number in a range.

[0047] The term “cloaking” as used herein means to prevent presentation of microbial proteins at the cell surface.

[0048] The term “secretome” encompasses HCMV and EBV proteins and miRNA contained in viral exosomes, non-infectious enveloped particles (NIEPs), and dense bodies. In addition,specialized secretory cells, such as enteroendocrine cells, may secrete viral proteins in secretory granules or through transporters.

[0049] “G,” “C,” “A”, “U” and “T” or “dT” respectively, each generally stand for a nucleotide that contains guanine, cytosine, adenine, uracil and deoxythymidine as a base, respectively. However, the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. Sequences comprising such replacement moieties are embodiments of the invention.

[0050] As used herein “RNA interference” (RNAi) refers to inhibition of mRNA translation with an RNAi molecule. The “RNAi molecule” may comprise an antisense oligonucleotide (ASO) with sequence complementary to the targeted RNA molecule.

[0051] The RNAi molecule may comprise a morpholino. A “morpholino” as used herein is an ASO comprised of single stranded DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups.

[0052] As used herein, the RNAi molecule may comprise chemically modified single-stranded DNA antisense to mRNA.

[0053] RNAi in this disclosure may include RISC-dependent or RISC-independent.

[0054] RISC-dependent RNAi requires Argonaute, which uses double-stranded (dsRNA) or single-stranded short-interfering RNA activates (siRNA) and cleave the complementary mRNA.

[0055] The siRNA may be polyadenylated to increase RNAi efficiency by increased stability.

[0056] RISC-independent RNAi can target the single-stranded DNA (sDNA) or RNA antisense oligomer to the 5’ cap site for steric interference with the binding of RNA polymerase complex and subsequent translation.

[0057] RISC-independent RNAi can target the single-stranded DNA or RNA antisense oligomer 5’ to the 3’ placement of the poly A tail, wherein the antisense oligomer inhibits polyadenylation or deadenylation of the transcript.

[0058] The RNAi molecule may comprise a short hairpin RNA (shRNA). A “shRNA” as used herein is a double-stranded oligonucleotide which comprises a segment antisense to the target the mRNA or miRNA that may be cleaved into siRNA by DICER.

[0059] In some embodiments, dsRNA molecules may also comprise “overhangs”, i.e., unpaired, overhanging nucleotides which are not directly involved in the RNA double helical structure normally formed by the herein defined pair of “sense strand” and “antisense strand”. Often, such an overhanging stretch comprises the deoxythymidine nucleotide, in most embodiments, two deoxythymidines in the 3' end.

[0060] As used herein, the term “consensus sequence” refers to at least 7 contiguous nucleotides, preferably at least 17 contiguous nucleotides, most preferably at least 19 contiguousnucleotides, which is highly conserved among select herpesvirus genomic sequences.

[0061] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of a HCMV ppi 50 or EBV gp350 mRNA molecule that is complementary to the ASO but may also include RNA from an unspliced primary transcription product.

[0062] As used herein, the term “vector” means a viral vector that has been modified to be non- immunogenic and engineered to contain an expression cassette comprising one or more ASOs that targets ppi 50 and / or gp350. Two conceived viral vectors are adeno-associated virus and lentivirus subtypes for infecting enterocytes, and other gut barrier cells, e.g., enteroendocrine cells. Such a vector comprises a regulatory sequence operably linked to nucleotide sequence that encodes at least one antisense strand of a dsRNA of the disclosed exemplary ASOs. Methods for engineering viral vectors with expression cassettes are known in the art (Poletti and Mavilio 2021).

[0063] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

[0064] The term “double-stranded RNA”, “dsRNA molecule”, or “dsRNA”, as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5' end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5' end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The nucleotides in said “overhangs” may comprise between 0 and 5 nucleotides, whereby “0” means no additional nucleotide(s) that form(s) an “overhang” and whereas “5” means five additional nucleotides on the individual strands of the dsRNA duplex. These optional“overhangs” are located at the 3' end of the individual strands. dsRNA molecules which comprise only an “overhang” in one of the two strands may be useful and even advantageous in context of this invention. The “overhang” comprises preferably between 0 and 2 nucleotides. Most preferably two “dT” (deoxythymidine) nucleotides are found at the 3' end of both strands of the dsRNA. Two “U” (uracil) nucleotides can be used as overhangs at the 3' end of both strands of the dsRNA. Accordingly, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3 '-end of one strand of the dsRNA extends beyond the 5 '-end of the other strand, or vice versa. For example, the antisense strand comprises 23 nucleotides and the sense strand comprises 21 nucleotides, forming a two-nucleotide overhang at the 3' end of the antisense strand. Preferably, the two- nucleotide overhang is fully complementary to the mRNA of the target gene. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

[0065] The term “antisense strand” refers to the strand of an RNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated outside nucleotides 2-7 of the 5' terminus of the antisense strand.

[0066] The term “sense strand,” as used herein, refers to the oligonucleotide that includes a region that is substantially complementary to a region of the antisense strand. “Substantially complementary” means preferably at least 85% of the overlapping nucleotides in sense and antisense strand are complementary.

[0067] The phrase “targeting moiety”, targeting moieties”, or “target moiety” refers to a molecule that has affinity for a cell surface receptor on specific cell types with susceptibility to HCMV or EBV infection. The molecules with affinity to the cell surface receptor can be an antibody, e.g., a polyclonal fragment antigen-binding (fab) to CD34+, a peptide, a lectin, a glycolipid, a nucleic acid aptamer, a molecule (e.g., glucose), or glycoprotein.

[0068] The term “immunogenic” as used herein refers to the biologic response of body tissue to injury, irritation, or disease which can be caused by harmful stimuli, for example, pathogens, damaged cells, or irritants.

[0069] The term “cancer” as used herein refers to cell over proliferation disorder. Throughout this application, the term “cancer” refers to any disease / disorder marked by unwanted or aberrant proliferation of tissue.

[0070] The terms “silence”, “inhibit the expression of’ and “knockdown”, as refer to herein refer to the at least partial suppression of the expression of e.g., HCMV-ppl50 and EBV-gp350 viral gene expression, as manifested by a reduction of the amount of mRNA or translated protein.

[0071] Alternatively, the degree of inhibition may be given in terms of an increase in a cell surface receptor, indicating normal endosomal pathway function. The increase in cell surface receptors can be determined by fluorescent activated cell sorting (FACS). In some embodiments, the degree of inhibition is determined by the elimination of infected cells when the endosomal pathway delivers the immunogenic microbial antigen to the cell surface. In other embodiments, the level viral protein targeted by the antisense oligonucleotide is decreased in the blood or infected or non-infected cell type, which can be measured by immunoassays.

[0072] The terms “treat”, “treatment”, and the like, mean in context of this disclosure the prevention, relief from or alleviation of a microbial disorders, e.g., dysbiosis of the microbiome, related to latent or active HCMV or EBV infection. The terms “treat”, “treatment”, and the like, mean in context of this disclosure the prevention, relief from or alleviation of inflammation, vascular degeneration, endosomal dysfunction, immunosuppression, and cancers related to latent or active HCMV or EBV infection.

[0073] The term “nanocarrier” as used herein refers to any composition that complexes with ASOs for non-immunogenic delivery to cells. For example, a nanocarrier can be a pH sensitive nanoparticle (e.g., a solid lipid NP), liposomes, micelles, and natural and non-natural exosomes (Morales-Becerril et al. 2022).

[0074] As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an antisense oligonucleotide effective to produce the intended pharmacological, therapeutic, or preventive result.

[0075] The term “dementia” as referred to herein encompasses neurodegeneration with mixed pathology, e.g., cerebral amyloid angiopathy (CAA), endosomal dysfunction, synapse loss, neuron death, microglia dysfunction, astrogliosis, and myelin degeneration, and may also include disruption of microtubules, as indicative by tau filaments, and alpha-synuclein aggregation. Dementia types include vascular dementia, Alzheimer’s disease (AD), Parkinson’s disease, frontotemporal dementia, or Lewy body disease.

[0076] The term “cancer” as referred to herein can include all types of cancers known to those in the art. The cardinal definition of cancer is cell over proliferation and metastasis (migration and growth at another location).

[0077] The disclosure comprises the following description, embodiments, and figures. Those skilled in the art will understand that the embodiments described do not limit the disclosure, asvariations and modifications can be made. Although various features are described in context of an embodiment, various aspects of an embodiment can be arranged with aspects of another embodiment.PROBLEM TO SOLVEAge-related increase in viral secretome increases susceptibility to infection and disease

[0078] In most young immunocompetent people, HCMV remains in latent state and does not reactivate to cause symptomatic viremia. However, lytic HCMV replication occurs intermittently in dissemination reservoirs, such as in the keratinocytes of oropharyngeal cavity, salivary glands, urogenital epithelium, and gastrointestinal tract. The frequency of HCMV systemic reactivation depends on the extent of the disseminated HMCV viral load, HCMV strain, secondary infections, and host variables such as stress, age, and genetic variants. HCMV reactivation frequency increases dramatically after 70 years of age (Parry et al. 2016).

[0079] Increased HCMV reactivation disseminates HCMV infection throughout the body thereby increasing viral load. Increased HCMV viral load increases the number of cells infected with HCMV in a latent state. Latent HCMV infected cells secrete viral proteins and RNA and alter the secretion of host proteins. The viral secretome can decrease host immunity against new opportunistic microbial infections, produce chronic low-grade inflammation, and alter systemic metabolism. The connection between HCMV-secreted factors and altered host protein expression and the pathogenesis of HCMV-related diseases in immunocompetent individuals is currently under appreciated. No therapeutic drugs are currently in development to prevent HCMV-secretome release or increased HCMV viral load with age in immunocompetent individuals. Indeed, lifetime HCMV infection produces an immunocompromised state in the elderly due to limited naive T cells and exhaustion.

[0080] One reason that HCMV evades innate and adaptive immunity is because HCMV virions and viral particles are enveloped in the host cell membrane, such as that from the endoplasmic reticulum, and display non-immunogenic host proteins. These host proteins can even increase tropism, allowing them to enter cell types that do not express the viral cognate receptor. The host proteins on the virion surface may even contain immune checkpoint molecules that block immune activation allowing HCMV to infect new cells unimpeded by antibody-dependent cell- mediated cytotoxicity, and detection by NK and T-cells. The combination of unimpeded invasion, variant HCMC strains, and opportunistic timing explains why the vaccine approach is unlikely to be a successful in restricting HCMV infection. Currently, no treatments are in development to prevent HCMV-mediated secretion of viral factors or altered host protein secretion that can cause physiological and developmental defects.

[0081] HCMV strain variants have specific cell tropism allowing infection of a range of celltypes. HCMV strain variants may interact with the host allelic variants to change gene expression resulting in disease heterogeneity. HCMV seropositivity is significantly associated with stroke, type-2 diabetes, autoimmune type-1 diabetes, schizophrenia, cardiovascular disease, cancer, fever of unknown origin, hypertension, and neurodegenerative diseases, such as Alzheimer’s disease and vascular dementia (Cheng et al. 2009; Huang et al. 2012; Schmidt et al. 2018; Barnes et al. 2015; Michaelis et al. 2009; Jurgen Geisler et al.; Torrey et al. 2006; Wang et al. 2022; Li et al. 2011; Pak et al. 1988). The present disclosure provides evidence connecting specific HCMV- and EBV secreted proteins with disease and compositions and methods of treatment and prevention of HCMV- and EBV-secretome mediated diseases.Evidence of HCMV-secretome mediated non-cell autonomous disease mechanism

[0082] HCMV infected cells secreted at least 13 viral proteins in a virus-free culture supernatant, indicating that HCMV infected cells secrete HCMV proteins during latency (Dumortier et al. 2008; Luganini et al. 2016; Mocarski et al. 1988). HCMV proteins, such as IE1 (UL123; P13202 ), IE2 (UL122; P19893), ppl50 (UL32; P08318) and hcmvIL-10 (ULI 11 A; P17150 or US27; P09703) are released from cells with latent HCMV infection (Turner et al. 2020; Dumortier et al. 2008). Turner et al., 2020 showed viral proteins are released in exosome and virion fractions from HCMV infected MRC5 primary fetal lung fibroblasts (ATCC CCL- 17). The complete HCMV secretome includes factors within non-infectious enveloped particles (NIEPs), dense bodies, and viral exosomes. For example, ppl50 is secreted within NIEPs and viral exosomes and the secreted form is found cleaved from 150 kDa to 65 kDa. HCMV viral strains can differ in the amount of secretome released. For example, the AD 169 strain produces 10-fold more NIEPs than other strains (Irmiere and Gibson 1983).ROLE OF HCMV PROTEIN PPI 50 IN DISRUPTING ENDOCYTIC TRANSPORT

[0083] Rab6a (UniProt: P20340) and Rab6b (UniProt: Q9NRW1) are small RAS-like GTPases that regulate recruitment of the dynactin complex to golgi, endoplasma reticulum, and vesicle membranes to transport endosomal vesicles containing APP-SORLA from the trans-golgi- network to cell surface focal adhesions or neurites (Fourriere et al. 2019). The Rab6b protein sequence has 91% sequence identity to Rab6a and a cell-type specific expression pattern predominantly in the brain, e.g., in microglia, pericytes, and Purkinje cells (Opdam et al. 2000). Rab6b binds a spliced isoform of APP -binding family A member 1, previously called Mintl 826, which colocalizes with APP during vesicle transport (Thyrock et al. 2013). Rab6b also binds to the C-terminus of the protein bicaudal D homolog 1 cargo adaptor (BICD1; UniProt:Q96G01), a dynein motor adaptor required for the recruitment of cytoplasmic dynein- dynactin complex, and dynactin (Short et al. 2002), a multisubunit protein complex that binds and activates dynein, the microtubule traversing motor. During lytic replication, the HCMVprotein ppi 50 (P08318) and small capsid protein are required for the final envelopment of the nucleocapsid, wherein the lytic expressed small capsid binds to ppl50-BICDl complex to transfer Rab6a vesicles to the viral assembly compartment (Indran et al. 2010; Indran and Britt 2011; Tandon and Mocarski 2012; Dai et al. 2013). Binding of ppl50 to BICD1 was demonstrated by yeast two-hybrid screening (see Table 1 in Indran et al. 2010), wherein cells transfected BICDl-gfp (+ anti-BIDCl) and ppl50-myc constructs and stained for anti-myc showed ppi 50 and BICD1 colocalized as visualized by fluorescence confocal microscopy, (see Fig. 3 in Indran et al., 2010). The yeast-two hybrid screens identified ppl50 amino acids 614- 842 interacting with BICD1 amino acids 630 to 820 (Figs. 2 and 5, Indran et al. 2010). The micrographs of Indran et al. Fig. 3A demonstrated that ppl50 colocalized with BICD1 and Rab6a in uninfected cells. Lasar scanning confocal microscopy demonstrated colocalization of ppl50-BICDl-Rab6a, and coimmunoprecipitation with the addition Rab6a, resulting in a ternary complex (see Figs. 1, 2 and 3 in Indran and Britt 2011). Therefore, the authors asserted that ppi 50 did not displace Rab6b binding to BICD1.

[0084] BICD1 amino acid sequence (663-803) in the third coiled coil region (CC3) are highly conserved and corresponds to the Rab6a binding site region (Terawaki et al. 2015). BICD1 molecules form homodimers that bind two Rab6 molecules. Therefore, one BICD1 CC3 homodimer could bind ppi 50, while the other CC3 homodimer binds Rab6. Thus, ppi 50 could still displace Rab6a binding and exist as a ternary complex (ppi 50, Rab6a, and BICD1). The ppl50 amino acids, which bind to BICD1 (614-842) have 22.48% sequence similarity to Rab6b (see Clustal alignment FIG. 1 A). The Epstein Bar virus protein gp350 has a similar sequence to the ppi 50 sequence that binds to BICD 1 (see FIG. IB). BICD1 is predominantly expressed in the neuronal tissue cluster, which interacts with STAT3, DISCI, and GSK3p. Ppl50 and gp350 have sequence similarity to Rab6b, indicating that ppi 50 and gp350 bind to dynactin and may displace Rab6b.

[0085] Human BICD2, a paralog of BICD 1, also functions as a dynein adaptor protein. Figure 12A shows the amino acid alignment between human BICD1 (Q96G01) and human BICD2 (Q8TD16-1) with high sequence similarity between BICD1 and BICD2 at BICD1 amino acids 630 to 820, which is the BICD1 location that binds to ppl50. The BICD2 protein has high expression in squamous epithelium and immune cells (Fig. 12B). A heterozygous mutation in this region of BICD2 (p.Glu774Gly) causes autosomal-dominant proximal spinal muscular dystrophy (SMA) (Peeters et al. 2013). The SMA phenotype is a slow progression of muscle wasting predominantly in the lower extremities, resembling the muscle wasting in vascular dementia and frontotemporal dementia patients, but with little cognitive impairment. Both BICD1 and BICD2 bind to dynactin I (DCTNl)(Fig. 9). BICD1 loss-of -function variants areassociated with hearing loss and neuropathy (Hirsch et al. 2023). Hearing loss and neuropathy are associated with dementia. Mutations in dynactin I cause neurodegenerations that include Perry syndrome, distal hereditary motor neuronopathy type 7B (dHMN7B), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy. Perry syndrome is characterized by parkinsonism, neuropsychiatric symptoms. BICD2 facilitates cytoplasmic trafficking with Rab6, and BICD2 depletion induces an increased innate immune response to viral infection from disrupted trafficking of viral pathogens, e.g., HIV (Dharan et al., 2017). The disruption of BICD 1 and BICD2 in uninfected cells by HCMV secreted ppi 50 and EBV secreted gp350 could disrupt Rab6 cytoplasmic trafficking of immunogenic peptides to the cell surface to increase susceptibility to microbial infection (Huber et al. 2020). Figure 9 shows Rab6A interaction with BICD1 and BICD2.

[0086] Rab6b and Rab6a are encoded by different genes with 91% sequence identity, however Rab6b is predominantly expressed in neuronal cells, e.g., microvessel pericytes, microglia, and Purkinje cells, which are affected in AD. Rab6b is also expressed in immune cells and is required to upregulate inflammatory TNF signaling (Dominguez Cadena et al. 2021). BICD1 and Rab6b colocalize in neurites and are involved in bidirectional cargo transport (Wanschers et al. 2007).

[0087] Drosophila Rab6 attaches to BICD at amino acid K12FK; the analogous amino acid in human Rab6a and Rab6b is K13FK. As shown in table 1 below, other microbes have sequence similarity to C-terminal ppi 50 and may also displace Rab6a and Rab6b binding to enable immunity cloaking. BICD1 binds to neuronal protein tyrosine phosphatase, non-receptor type 23 (PTPN23) (Budzinska et al., 2020). PTPN23 is part of the endosomal sorting complex required for transport (ESCRT) machinery that functions in the biogenesis of multivesicular bodies, such as exosomes. Thus, ppl50 and gp350 could inhibit the interaction of BICD1-PTPN23 to disrupt MVB formation, sequestration, and release of exosomes in uninfected cells. Moreover, decreasing ppi 50 and gp350 expression in infected cells could prevent viral secretome release in infected cells since Rab6b engages in targeting and docketing of secretory vesicles to the cell surface with BICD1. The BICD1 knockdown phenotype of swollen vacuole-like compartments resembles the swollen autophagic vacuoles seen in neurons from AD patients (Bolen et al., 2008).

[0088] The HCMV secreted protein ppi 50 can enter uninfected cell to mediate BICD1-Rab6b disruption of endosome trafficking from trans-golgi-network (TGN) to the cell surface, resulting in functional deficiency of APP and SORLA, and consequently reduced aAPP-NGF -mediated synaptic plasticity, neuron survival, acetylcholine stores, proper tau phosphorylation, and iron dyshomeostasis (Barthelson et al. 2020). The ppl50 mediated dysfunction of BICD1-Rab6bendosomal trafficking prevents SORLA-APP transport and subsequent a-secretase cleavage, resulting in APP remaining in cytoplasmic compartments for P- and y-secretase processing into Ap (McConlogue et al. 1996).

[0089] Upregulated Rab6 expression in AD brain may be driven by functional deficiency from ppl50 binding the BICD1-Rab6 complex (Scheper et al. 2007). The dysfunction of BICD1-Rab6 vesicles transport can affect neurite maintenance and presynaptic vesicle transport, since the restriction of Rab6a vesicles to the centrosome prevents anterograde transport (Schlager et al. 2010). In the absence of Rab6b, the presynaptic bouton has approximately 25% less vesicles in a presynaptic bouton and a 3-fold increase of vesicles in the axon (Nyitrai et al. 2020). Thus, HCMV ppi 50 displacement of Rab6b from BICD1 can disturb the delivery of synaptic cargo. Accordingly, vesicle density is significantly decreased in the synaptic boutons from AD brain compared to boutons from age-matched cognitive control brain (Wang et al. 2023), and axons from AD brain contain swellings with an accumulation of vesicles and transport proteins (Stokin et al. 2005). The loss of cargo transport to presynaptic boutons can contribute to decreased synapses — an early hallmark of AD. The non-cell autonomous effects of the HCMV secretome proteins, e.g., ppi 50 and IE1, can explain the differential activation of the unfolded protein response (UPR) in non-tangle bearing neurons with increased Rab6 expression, wherein neurons bearing tangles have no increased Rab6(?) expression (Scheper et al. 2007). The loss of normal APP / SORLA vesicle transport and function by ppl50-mediated disruption of BICD1-Rab6b vesicle trafficking is consistent with AD pathology, and likely causal to all CAA related dementias. Accordingly, a deep learning approach recently identified that BICD1 variants confer a high genetic risk factor for AD (Jo et al. 2022). (The question mark after Rab6 in this paragraph indicates that the disclosure did not specify Rab6a or Rab6b).

[0090] The secretion of ppi 50 and other microbial proteins that bind BICD1 may have evolved prevent cell surface display of viral immunogenic proteins, essentially cloaking the infected cell from the immune system and preparing cells for infection. HCMV ppi 50 binding to BICD1 could disrupt the BICDl-interactome mediated transport of late endosomes to the lysosome, which is disrupted in AD and other neurodegenerative disease (Small et al. 2017). For example, BICD1 depletion results in the intercellular sequestration of the BDNF receptor (TrkB) into enlarged endosomal compartments (Terenzio et al. 2014; Budzinska et al. 2020). This finding of enlarged endosomes with BICD1 replicates neuronal pathology found in early AD (Cataldo et al. 2004).

[0091] HCMV infection increases the release of viral exosomes containing ppi 50 and other viral proteins. The binding of ppi 50 to BICD1 may also upregulate the sequestration and release of viral exosomes, NIEPs, and dense bodies since Rab6b is involved in targeting and docketingof secretory vesicles to the cell surface (Patwardhan et al. 2017). Moreover, BICD1 is a component of the endosomal sorting complex (ESCRT) machinery, which is involved in the formation of multivesicular bodies from which exosomes are derived. In some embodiments, the disclosed RNAi compositions inhibit HCMV ppi 50 protein expression reducing ppi 50- mediated endosomal dysfunction. In some embodiments, reducing ppl50-mediated endosomal dysfunction increases viral protein immunogenic display on the cell surface eliciting immune cell mediated elimination (immunotherapy). In some embodiments, the disclosed RNAi compositions inhibit HCMV ppi 50 protein expression reducing release of viral exosomes, NIEPS, or dense body release from latently HCMV infected cells. In some embodiments, the disclosed RNAi compositions inhibit HCMV ppl50 protein expression reducing HCMV virion dissemination (Meyer et al. 1997). In some embodiments, the disclosed ppi 50 RNAi compositions reduce HCMV viral load, and thus the HCMV secretome.

[0092] HCMV ppl50 also interacts with cyclin A2 (UniProt ID Q13561) in the cell to block the progression of the cell cycle, and can essentially produce uninfected quiescent cells. (Bogdanow et al. 2013). Accordingly, astrocyte and cerebrovascular senescence is associated with AD (Bryant et al. 2020; Bhat et al. 2012). Biomarkers of abnormal cell cycle progression are found in neurons and lymphocytes of AD patients (Song et al. 2012; Moh et al. 2011). The extent of quiescent / senescent cells would be determined by the secretome amount of HCMV or other herpesvirus types, which is determined by the viral load, and is already present in HCMV or herpesvirus type seropositive healthy young adults (Redeker et al. 2017; Turner et al. 2014). In some embodiments, the disclosed ppi 50 RNAi compositions derepress HCMV-mediated systemic quiescence or senescence.

[0093] The ppi 50 BICD1 binding can also disrupt the interaction of BICD 1 with CELF4 (UniProt ID Q9BZC1), which is involved in pre-mRNA splicing, and DDX11 (UniProt ID Q96FC9). The ppl50 BICD1 binding can also disrupt the interaction of BICD1 with DDX11 (UniProt ID Q96FC9). DDX11 functions in translation initiation, nuclear and mitochondrial RNA splicing, and ribosome and spliceosome assembly (see FIG. 9; diagram obtained from STRING.org; Szklarczyk D. et al., 2023).

[0094] HCMV ppi 50 or other herpesvirus protein mediated misprocessing of APP in the brain and periphery can explain systemic HCMV-mediated disease. In the decades leading up to cognitive impairment, Ap peptides accumulate to form aggregates in the brain. Ap peptides are also detected in the intestinal cells and blood of healthy adults. Normally microvascular perfusion and microglia phagocytosis eliminate Ap, however microvascular degeneration and impaired microglia phagocytosis from increased cmv-IL-10 and IL-10 levels can reduce Ap disposal. For example, IL-10 impaired microglia phagocytosis in an APP mouse model,increasing the amyloid plaque burden and worsening cognitive behavior (Chakrabarty et al. 2015). In some embodiments, the disclosed ppi 50 RNAi compositions reduced Ap plaque formation.

[0095] Herpesviruses are well known for evolving multiple mechanisms to ensure successful transmission. Herpesviruses have sequence conservation in their glycoprotein B, which has sequence similarity to APP. APP is found associated with the HIV Gag polyprotein in lipid rafts to prevent HIV particles from leaving lipid rafts. However, HIV Gag induces y-secretase cleavage of APP for release in HIV-infected macrophages and microglia (Chai et al., 2017; Hategan et al., 2019). Herpes simplex virus appears to have evolved a similar viral restriction method to HIV. aAPP is found associated with HSV-1 glycoprotein B and glycoprotein B interacts with Rab5 and early endosomal antigen 1 (EEA1) to disrupt the MHC-II processing pathway (Bourgade et al. 2022; Niazy et al. 2017). HSV-1 glycoprotein B may induce the cleavage of the C-terminal fragment C99 for release. The evidence disclosed indicates that HCMV disrupts the endosomal pathway by binding to BICD1, thereby diverting APP endosomal targeting to the cell surface to prevent viral restriction, in addition to cloaking viral immunogenic proteins at the cell surface.Evolutionary Conserved Sequences for Disrupting Endosome Vesicle Retrograde Trafficking and Recycling

[0096] Microbes have evolved a common cloaking mechanism to hide from the immune system, and in turn, host cells evolved mechanisms to detect endosomal dysfunction for microbial cloaking and disrupt the localization of the viral restriction protein APP. There is evidence that the amyloidogenic products trigger cell suicide, e.g., caspase-2 / 3 mediated suicide (Troy et al. 2000; Rissman et al. 2004; Zhao et al. 2016; Gervais et al. 1999; Nishimura et al. 2002; Andreas Weidemann et al.; Ayala-Grosso et al. 2002; Marin et al. 2000; Balusu et al. 2023).

[0097] Performing a BLAST on UniPort.org using the blastp program, matrix BLOSUM62, to search the uniprotkb swissprot database identified proteins with signification alignment to the C-terminal end of HCMV ppl50 (P08318) as shown in Table 1 below. The HCMV sequences used in the sequence alignments are from the AD169 strain (Genbank reference no. FJ527563). The C-terminal ppi 50 sequence shown to bind BICD1 overlaps with high sequence similarity to protein sequences in diverse microbes, indicating that diverse microbes have evolved a common cloaking mechanism by targeting BICD1 to evade immune detection by inhibiting delivery of ubiquitously expressed MHC-I bound immunogenic molecules to the cell surface (Raffatellu 2018)Table 1. Sequence identity to C-terminal ppl50 in diverse microbes

[0098] The BLAST results indicate that diverse microbes have evolved a common cloaking mechanism by targeting BICD1 to evade immune detection by inhibiting delivery ubiquitously expressed MHC-I bound antigenic molecules to the cell surface (Raffatellu 2018). The C-terminal end of HCMV ppl50 (YP 081491.1 or P08318), from amino acids 729 to 881, is within ppl50-BICDl binding domain and has sequence identity to the Epstein Barr virus (EBV) protein gp350 (P03200.1) with 49% positives and an E-value of 6x 10-5 (FIG. 8). The roseoloviruses (HHV6 / HHV7) protein ppi 00 also has sequence similarity to HCMV ppi 50 but the alignment occurs at the N-terminal end. The conservation between HCMV and EBV indicates that EBV gp350 may also bind to BICD1 with the ability to divert BICD1-Rab6b endosomes containing APP / SORLA to the viral assembly complex or to the endosomal sorting complex required for transport (ESCRT) for production of multivesicular bodies for viral exosomes, which benefits viral transmission. The gp350 protein is present in the blood of healthy adults; derived from the secretion of gp350 in exosomes from EBV latent infected cells (Carcaba 2020). Thus, HCMV ppi 50 and EBV gp350 proteins are secreted from latent infected cells and can enter uninfected cells to disrupt the endosomal transport disrupting for example, APP processing. Examples 2 and 3 describes experiments to demonstrate that ppi 50 and gp350 disrupt APP processing and blocking their expression using antisense oligonucleotides disclosed in Table 2 and 3 prevent Ap formation.EFFECTS OF ENDOCYTIC RECYCLING DYSFUNCTION ON APP PROCESSING

[0099] Mutations in the amyloid precursor protein (APP; P05067) and the sortilin-related receptor (SORLA; Q92673) mutations are significantly associated with risk of developing multiple neurodegenerative diseases such as early- and late-onset AD, Parkinson’s disease, Lewy body dementia, and frontotemporal dementia (Pottier et al. 2012; Reitz et al. 2011; Rogaeva et al. 2007). (Ciani et al. 2019; Dai et al. 2020).

[0100] The APP-SORLA complex is transported from the TGN to the cell surface, dendrites, and axons where SORLA assists in APP cleavage to C-terminal sAPPa by a-secretase and y- secretase in the non-amyloidogenic pathway (Bauereiss et al. 2015; Choy et al. 2012; DelBove et al. 2018; Mehmedbasic et al. 2015; Fjorback et al. 2012). APP and SORLA mutations likely disrupt the trafficking of APP and SORLA to the cell surface, which regulates the transport of nerve growth factor receptor (TrkA; P04629) to the cell surface (Zhang et al. 2013). Reduced nerve growth factor signaling causes neurite outgrowth collapse and cholinergic neuron degeneration. SORLA also regulates the trafficking of the brain-derived neurotrophic factor receptor (TrkB; QI 6620 ) and BDNF (P23560) -TrkB signaling regulates tau phosphorylation and localization in neurites (Elliott et al. 2005; Chen et al. 2014; Rohe et al. 2013). SORLA- mediated BDNF-TrkB signaling was shown to phosphorylate APP at Y687 in the TGN to prevent APP amyloidogenic cleavage (Xia et al. 2021). Cleavage of APP at the cell surface by a- secretase releases the neuroprotective extracellular fragment sAPPa. In contrast, amyloidogenic proteolytic fragments appear to prevent NGF retrograde endosomal trafficking required forsynaptic signal transduction.

[0101] In addition, APP binds and regulates the activity of the high-affinity choline transporter. The high-affinity choline transporter recycles choline back into the presynaptic terminal in the rate-limiting step for cholinergic neurotransmission (Wang et al. 2007). APP also stabilizes the iron efflux pore, ferroportin, at the cell surface (Wong et al. 2014). Reduced cell-surface ferroportin increases intracellular iron stores and the susceptibility to cell death by ferroptosis (Ashraf and So 2020). Cell-surface ferroportin deficiency in macrophages could explain why higher ferritin levels but low serum iron are associated with anemia in AD patients (Faux et al. 2014).

[0102] The Notch receptor signaling functions in the adult brain in neurogenesis, cell differentiation, synaptic plasticity, migration, and survival (Ables et al. 2011; Lampada and Taylor 2023). Notch is delivered to the cell surface by the endosomal pathway where it interacts with its cognate ligands and is cleaved by y-secretase to release the Notch intracellular domain. After y-secretase cleavage, the Notch intracellular domain travels to the cell nucleus to initiate the expression of genes, such as SOX2 through the upregulation Hesl and STAT3 phosphorylation (Yamamoto et al. 2010). The y-secretase null mutation R352 prevents APP and Notch cleavage and individuals with this mutation develop familial frontotemporal dementia (Amtul et al. 2002).

[0103] Misprocessed APP results in amyloid-P (AP) plaques — a hallmark of AD and other dementias. HCMV infected human foreskin fibroblast produce amyloid-P (Lurain et al. 2013a). The C99 cleavage product of APP, which contains Ap, interacts with SORLA to block APP association, further increasing Ap formation and preventing exocytosis and virion egress (Uemura and Greenlee 2001; Spoelgen et al. 2006). The C99 fragment was also shown to induce impaired lysosomal-autophagic function (Lauritzen et al. 2016). Amyloid-P accumulates in vessel walls to cause cerebral amyloid angiopathy (CAA), resulting in microbleeds, lobar intracerebral hemorrhage, and microinfarction leading to microvessel degeneration.Upregulation of APP expression occurs with the detection of pathogen-associated molecular patterns (PAMPS) and damage-associated molecular pattern (DMAPS), which may function to induce cell suicide, providing viral restriction to the host (Lingel et al. 2020). The high prevalence of HCMV infection and increased lytic activity in the aged explains why CAA- mediated microbleeds and other vascular pathology is almost ubiquitous in brains from elderly individuals (Vemooij et al. 2008; Fan et al. 2021).

[0104] HCMV IE1 promotes Hesl degradation thereby decreasing SOX2 transcription, and HCMV pp71 dysregulates Notch signal transduction by disrupting the cell surface location of the Notch ligand Jagl and the Notch intracellular domain (Huang et al. 2021). SOX2 hasimportant functions in specific differentiated neurons and in Bergmann glia of the cerebellar cortex. For example, SOX2 regulates circadian rhythm in neurons in the superchiasmatic nucleus and is required for removal of extracellular glutamate by Bergmann glia (Mercurio et al. 2019). lEl-mediated and pp71-mediated dysfunction of Notch signaling replicates some pathological disturbances in dementias, e.g., circadian rhythm disruption (Cronin et al. 2017), tau in Bergmann glia (Stopschinski et al. 2021), and decreased neurogenesis (Moreno- Jimenez et al. 2019).HCMV AND EPSTEIN BARR VIRUS INFECTION CAN CAUSE IMMUNOSUPPRESSION

[0105] Rab6b functions in TNF secretion and delivering MHC II molecules to the cell surface in B cells and macrophages (Briken et al. 1997; Micaroni et al. 2013), so to reiterate, HCMV ppl50 sequences likely evolved to displace Rab6b on BICD1 for immune evasion. IL-10 is a cytokine that reduces immune activity during infection by inhibiting Thl cells, NK cells, and macrophages (Couper et al. 2008; Brooks et al. 2008). HCMV encodes an HCMV protein with sequence similarity to human IL-10, cmvIL-10 (Kotenko et al. 2000). HCMV infection also induces human IL-10 expression (Avdic et al. 2016). In addition to hcmv-IL-10 and human IL- 10 induction, HCMV also prevents antigen presentation by class I MHC proteins (Abdelaziz et al. 2019). Furthermore, HCMV infected immune cells, and their infection status is associated with oxidative stress, shortened telomeres of lymphocytes, reduced B cell lineages, and T-cell senescence, and reduced interferon response (Pham et al. 2021). The subsequent effects of HCMV infection on immunity may increase host susceptibility to other opportunistic infections, which are increasingly found to be associated with age-related neurodegeneration and cancer (Chang and Barry 2010).

[0106] The HCMV protein hcmvIL-10 can be detected in the blood of healthy HCMV seropositive donors, see FIG. 3 (data presented herein with permission of Vivian Young, “Detection of HCMV viral IL- 10 (vIL-10) in healthy blood donors, Master’s Theses, USFCA (https: / / repository.usfca.edu / thes / 155 / ). The globular region of IE1 was shown to interact with the PML nuclear bodies that are critical components of host viral resistance, including regulating IFN-y induced gene expression (Raghavan et al. 2014; Scherer et al. 2016). The delivery of IE1 in viral exosomes would decrease immunity to other pathogenic microbes.

[0107] The HCMV protein pp71 (UL82) is found in exosome fractions (Turner et al. 2020). Transfection of mRNA encoding pp71 decreased MHC-I expression at the cell surface (Trgovcich et al. 2006). Thus, pp71 delivered in viral exosomes would lead to immunosuppression in uninfected cells by failure of microbial antigen presentation at the cell surface. HCMV proteins or miRNAs secreted from latent infected cells can influence theinfection susceptibility at targeted cell types.

[0108] Non-infectious dense bodies are secreted from HCMV infected cells. The HCMV protein pp65 (pUL83) makes up 95% of dense bodies. The HCMV pp65 inhibits the activation of interferon response factor 3, which is required for the type I interferon response. Thus, dissemination of pp65 blocks the interferon response alarm that initiates innate viral immunity in uninfected cells.

[0109] The age-related increased frequency of HCMV reactivation increases HCMV viral load and hence the level of HCMV secretome. Accordingly, individuals with HCMV IgG levels in the upper tertile had 3.4 greater odds of active tuberculosis (Stockdale et al. 2020). HCMV IgG seropositivity was associated severe COVID-19 in young patients (Weber et al. 2022); a reason for the association not being detectable in elderly is likely because the elderly population have high HCMV seropositivity, reaching 100% in some populations. Taken together the HCMV secretome derived from replicating reservoirs could influence both the integrity of distant microcapillary beds, inflammation, and infection susceptibility that may explain the phenotypic diversity of neurodegenerations and increased mortality (Savva et al. 2013). In summary, the HCMV secretome explains the immune system dysfunction seen in dementia patients (Busse et al. 2017).

[0110] Cells with latent EBV infection also secrete a protein with sequence similar to human IL- 10, ebvILlO (Yoon et al. 2012)DECREASED GLUT1 PROTEIN IN AD BRAIN EXPLAINED[OHl] Glucose enters endothelial cells of microvessels in the brain through the glucose transporter GLUT1 (solute Carrier Family 2; UniProt ID Pl 1166) (Morgello et al. 1995). HCMV-infected cells secrete exosomes containing the immediate-early protein (IE1), which can suppress endothelial GLUT1 protein expression (Dumortier et al., 2008; Yu et al., 2011). GLUT1 is significantly reduced in cerebral microvessels from AD brains compared with age- matched controls (Kalaria and Harik, 1989; Horwood and Davies, 1994; Simpson et al., 1994; Mooradian et al., 1997). Decreased glucose transport into microvessels decreases glycolysis in endothelial cells and reduces glucose transport into the brain’s interstitial neuropil, resulting in decreased glycolysis in regions with high GLUT1 expression. Accordingly, glucose metabolism imaged by positron emission tomography with radionuclide called fluorodeoxyglucose ([18F]FDG) is decreased in select brain regions of AD patients compared to brains from cognitively healthy people (Marcus et al. 2014). Without glucose to fuel synaptic activities, decreased neurotransmission leads to synapse loss (Baloyannis 2009; Mergenthaler et al. 2013). The loss of GLUT1 compromises the blood-brain barrier and induces capillary degeneration (Winkler et al., 2015).

[0112] GLUT1 is also the glucose transporter expressed in astrocytes, while GLUT3 is the predominant glucose transporter in neurons (Maher et al. 1991.). Interestingly, astrocytes from subjects who are cognitively normal but have AD-like brain pathology maintain GLUT1 expression (Kobayashi et al., 2018). Thus, HCMV-IEl-mediated GLUT1 downregulation may be a prerequisite for developing AD and other AD-like dementias. The GLUT1 mRNA level is unaffected in brain regions from AD patients compared to same brain regions of cognitively healthy people, indicating that repressed translation results from post-transcriptional modification (Mooradian et al. 1997).

[0113] The loss of GLUT1 expression in mice overexpressing the APP results in the blood-brain barrier breakdown through microvessel degeneration, reduced neuron activity, and neurodegeneration that replicated AD pathology (Winkler et al. 2015). Deficient GLUT1 expression in microvessels may explain why AD brain tissue shows profound disturbance of the microvessels when examined using electron microscopy (Miyakawa 1997). Individuals bom with mutations in the gene encoding GLUT1, SLC2A1 (GLUT1-DS) have phenotypes observed in dementia patients, including cognitive deficits, motor dysfunction, fatigue, epilepsy, hypersomnia and / or insomnia (Olivotto Sara et al.; Klepper et al. 2020; Vossel et al. 2013).

[0114] The cause of decreased GLUT1 and GLUT3 protein expression in AD like dementias is unknown. From the perspective of a productive HCMV infection, reduced GLUT1 / GLUT3 expression would reduce a cell-mediated immune response (Fu et al. 2004). However, except for erythrocytes, no studies have reported on the expression of GLUT1 / GLUT3 in immune cells from AD patients compared to cognitive controls. Erythrocytes have innate immune function and erythrocyte GLUT1 protein expression was found similar between AD patients and cognitive controls (Harik 1992); however, GLUT1 translation does not occur in mature circulating erythrocytes, rather it occurs in the bone marrow during erythropoiesis.

[0115] Herpesviruses are well known to evolve multiple mechanisms to accomplish important tasks. As shown in FIG. 4, IE1 secretion could cause the decreased GLUT1 expression found in microvessels by interacting with STAT3 (Reitsma et al. 2013; Wu et al. 2018). The association of IE1 with STAT3 transfers tyrosine 705 unphosphorylated STAT3 to the nucleus, which affects its ability to transactivate gene expression. STAT3 inhibition is associated with reduced GLUT1 expression (Shang et al. 2020; Li et al. 2022; Xia Zheng et al.). Unfortunately, blocking IE1 expression is not a viable target for treating HCMV-mediated GLUT1 downregulation because IE1 also functions to block apoptosis mediated by pathogen induced unfolded protein response (UPR), i.e., downregulating IE1 expression could cause apoptosis of the uninfected cells receiving IE1 ASO, leading endothelial cell death.

[0116] The disturbance of BICDl-rab6b cellular localization by ppi 50 may also affect STAT3function because BICD1 also interacts with STAT3.

[0117] MicroRNAs can prevent translation without affecting mRNA stability (Thermann and Hentze 2007). HCMV infection significantly increases the expression of human miR124 during latency in human embryonic lung fibroblast cells (Fu et al. 2014). MicroRNA 124 downregulates STAT3 (Wei et al. 2013) and GLUT1 expression levels by RNAi (Zhao et al. 2017), see miR124 depicted in the Fig. 4 schematic. HCMV infected cells express and secrete increased levels of miR124. HCMV infection can also upregulate STAT3 activity depending on the cell type and HCMV strain, which may be deterministic for the HCMV -mediating disease type, i.e., cancer or vascular degeneration (Roca Suarez et al. 2018; Reitsma et al. 2013).

[0118] Clinical strains show sequence variants in the HCMV secreted protein IE1 at amino acids 199-206 (Prod'homme et al. 2003b). This region is close to the ATP binding region required for IE1 to phosphorylate substrates (Pajovic et al. 1997). A BLAST of the IE1 sequence from accession number YP 081562 identified phosphatidylinositol 4-phosphate 5-kinase type-1 gamma isoform (Accession: 060331.2) aligned at the C-terminal end with 27% sequence identity and 36% positives (see FIG. 5 for sequence alignment). The sequence identity of IE1 may function by mimicry to alter the phosphoinositide signaling pathway. Phosphatidylinositol phosphate kinase type ly is a major type of brain PI(4)P5-kinase, and is concentrated in the synapse and involved in actin assembly of signaling scaffolds, in the nucleation of actin, synaptic endocytosis, and exocytosis (Wenk et al. 2001). Type I phosphatidylinositol 4- phosphate 5-kinase catalyzes phosphorylation of phosphatidylinositol 4-phosphate, producing phosphatidylinositol 4, 5 -bisphosphate (also known as PIP2). Phosphatidylinositol signaling can inhibit protein casein kinase 2 (CK2 activity) (Korol chuk et al. 2005). Accordingly, CK2 immunoreactivity pattern is altered and reduced in AD brain (limoto et al. 1990). Two ribonucleoproteins, hnRNPA2 and hnRNPL, bind the 3' end of GLUT1 to regulate mRNA transport and protein expression (Hamilton et al. 1999; White et al. 2008). The binding of hnRNPA2 to AU response element (AURE) region at nucleic acids 1885-1906 increases the degradation of GLUT1 mRNA. CK2 is a constitutively active serine / threonine kinase that associates and phosphorylates hnRNPA2 independently of RNA binding. CK2 is also known to phosphorylate the carboxyl -terminal domain of RNA polymerase II to regulate elongation and modify synaptic plasticity by directly or indirectly activating NMDA receptors in the hippocampal synapse in step with synaptic activity (Kimura and Matsuki 2008). CK2 phosphorylates hnRNPA2 to regulate mRNA stability, transport, and translation, see FIG. 6A (Pancetti et al.). PIP2 signaling also activates the tyrosine kinase Fyn, which is highly expressed in cells with hyperphosphorylated tau (Shirazi and Wood 1993). Tyrosine phosphorylation of hnRNPA2 by Fyn releases hnRNPA2 from GLUT1 mRNA allowing translation (White et al.2008). This process may explain the up-regulation of Fyn as a bio-feedback mechanism to increase GLUT1 translation.

[0119] IE2, and likely IE1 as well (IE1 and IE2 contain the same first three exons and differ only at exon 4 or 5), directly interacts with hnRNPA2 and increases hnRNPA2Bl expression, which decreases GLUT1 mRNA stability and prevents translation, see FIG. 6B (Liang et al.2019). GLUT1 is expressed in multiple tissues, including kidney, liver, skin, rectum, cerebral cortex, esophagus, bone marrow, urinary bladder, retina, choroid plexus, adipose tissue, skeletal muscle, etc. (see FIGS. 7A and 7B). The systemic delivery of IE1 / IE2 in viral exosomes or NIEPs to these tissues could disrupt GLUT1 expression, resulting in systemic reduced glycolysis-mediated ATP production. This scenario could explain the frailty associated with dementia and HCMV seropositivity (Schmaltz et al. 2005; Borda et al. 2019).

[0120] HCMV infection is associated with diabetes and cardiovascular disease risk

[0121] The risk of type 2 diabetes is 47% higher in individuals with HCMV seropositivity and this association is dependent on age and sex. Thus, age-related increase in HCMV activity can increase susceptibility type 2 diabetes, as well as genetic risk alleles, diet, and a sedentary lifestyle (Sijia Chen et al.; Fleck-Derderian et al. 2017; Huda Mohammed Hasan 2021; Yoo et al. 2019). Individuals with type 2 diabetes and the ApoE4 allele have significantly increased risk of AD (RR of 5.5 (CI 2.2-13.7)) (Peila et al. 2002). Metformin is a gold standard medication for lowering blood sugar in diabetics. Metformin’s mechanism of action could result from inhibition of complex IV of the mitochondria electron transport (LaMoia et al. 2022). Metformin also appears to reduce HCMV in vitro titers by interfering with electron transport complex I (Combs et al. 2021), which was the initial mechanism described, but the clinical concentration cannot inhibit complex I, suggesting another mechanism (LaMoia and Shulman 2021). However, since HCMV significantly upregulates mitochondria respiration, infected cells may be more sensitive to complex I inhibition (Karniely et al. 2016).

[0122] The benzylisoquinoline alkaloid berberine reduces hyperglycemia in type 2 diabetes (Yin et al. 2008). Interesting, berberine can reduce HCMV replication by interfering with IE2 transactivation (Luganini et al. 2019). Berberine has low intestinal absorption (Habtemariam2020), and thus could inhibit HCMV replication in intestinal barrier cells. Administering berberine to human subjects in a 16-week randomized double-blind study showed berberine treated subjects with altered gut microbiome and decreased hypoglycemia in newly diagnosed type 2 diabetes patients (Ming et al. 2021). In RhCMV infected rhesus macaques, genera of bacteria that produce short chain fatty acids (SCFA) levels were reduced compared to uninfected macaques (Chin et al. 2022). SCFA, such as butyrate, are produced from select bacteria and may be beneficial for brain homeostasis, whereas HCMV infection in rhesus monkeys and humaninfants decreases the abundance of SCFA generating bacteria (Chin et al. 2022).

[0123] Diabetes is thought to result from upregulated gluconeogenesis through the dysregulation of insulin repression on Pckl and G6pc gene expression (Hatting et al. 2018). While the cause of this insulin resistance to transcriptional regulation is unknown, the HCMV secretome can alter glucose regulation to mediate hyperglycemia to fuel HCMV replication (Shao et al. 2005). Hyperglycemia may result from reduced GLUT1 protein translation in muscle cells by secreted HCMV IE1. Accordingly, GLUT1 protein expression and glucose uptake is reduced in leg muscle from individuals with type 2 diabetes but total body glucose uptake was higher compared to controls (Ciaraldi et al. 2005). Higher HCMV viral load and / or a higher metabolically active HCMV strain type could increase total body glucose consumption since HCMV upregulates expression of the high glucose transporter GLUT4 and glucose metabolism (Yu et al. 2011, 2014). GLUT1 is utilized in resting muscle, whereas GLUT4 is active during exercise, thus exercise is an important source of glucose disposal and is known to normalize blood glucose levels (Zorzano et al. 2005; Richter and Hargreaves 2013).

[0124] The HCMV protein UL28 is secreted by HCMV infected cells and induces IL-6 and VEGF expression (Slinger et al. 2010). Increased IL-6 levels can induce insulin resistance in type 2 diabetes (Rehman et al. 2017). VEGF is upregulated in diabetic retinopathy and the myocardium of diabetic patients (Tahergorabi and Khazaei 2012). Biomarkers of increased angiogenesis is associated with an increase in white matter hyperintensities, which is found in both AD and vascular dementia, and thought to be an ischemic origin (WMH) (ladecola 2013; Callahan et al. 2020). Type 2 diabetes increases the risk of developing vascular dementia (Thomassen et al. 2020). Glucose transport in mouse brain and insulin secretion is inhibited by Ap in cell culture and in vivo, which can contribute to the connection between the HCMV and diabetes, and why AD resembles type 3 diabetes in the brain (Mark et al. 1997; Shigemori et al. 2022; Kandimalla et al. 2016). Cardiovascular disease (CVD), coronary heart disease, or atherosclerosis is highly associated with dementia (Stampfer 2006; Brain et al. 2023). CMV seropositive individuals have higher CRP levels compared to seronegative individuals and are at a substantially higher risk for cardiovascular-related mortality and stroke (Simanek et al. 2011; Wang et al. 2017a). HCMV is infects the endothelial cells and smooth muscle cells in the heart and the inflammatory response induces migration of foam cells into the intima to form plaques (Du et al. 2018). In another connection with CMV activity, being underweight is more of a risk for CVD than being overweight (Park et al. 2017).GLUT TRANSFER VIA EXOSOMES

[0125] The human gastrointestinal tract is a major reservoir of HCMV infected epithelial cells, fibroblasts, smooth muscle cells, enteroendocrine cells, and endothelial cells. Thegastrointestinal tract can be a major source of viral secretome, including non-infectious and infectious HCMV and EBV particles (Fig. 10).

[0126] HCMV infected cells increase their constitutive production of viral exosomes in fibroblasts, and exosomes markers are found at the basolateral membrane of human enterocytes (Lin et al. 2005; Streck et al. 2020). The glucose transporter GLUT2 is a low affinity glucose transporter expressed in the gastrointestinal tract basolateral membrane and in the endosomal membrane (Merigo et al. 2018). Thus, HCMV-mediated enhanced exosome release could carry GLUT2 from gut barrier cells (e.g., enterocyte or endothelial cell) to the brain, see FIG. 10. This process would redistribute GLUT2 to distant sites, such as the brain or liver without increasing GLUT2 mRNA. Accordingly, GLUT2 protein is increased by 2-fold in AD brain compared to brains from healthy controls (Liu et al. 2008), while GLUT4 is not increased. HCMV infection reduces GLUT1 expression but upregulates GLUT4 expression in fibroblast (Yu et al. 2011). Although GLUT4 is expressed in the hippocampus, GLUT4 expression does not significantly differ between AD patient brains and cognitive control brains. The lung is a major reservoir for HCMV (Chen and Hudnall 2006). GLUT2 is also expressed in human lung epithelial cells and therefore viral exosomes released from lung epithelial cells could contribute to increased GLUT2 in the AD brain. Many AD patients die of pneumonia (Manabe et al. 2019), which in combination with dysphagia, may be related to localized HCMV-mediated immunosuppression aiding opportunistic bacterial infection in the lungs, or directly through HCMV viremia. The loss of GLUT2 from the basolateral membrane of gut epithelial cells could release less glucose into circulation, contributing to the loss of weight and muscle mass in AD patients.

[0127] GLUT12 protein is dramatically upregulated in AD disease brain compared to healthy control brain (Pujol -Gimenez et al. 2014). GLUT12 mRNA is highly expressed in the human heart, skeletal muscle, and prostrate, with lower levels in the brain, placenta, and kidney.GLUT 12 protein was detected in human skeletal muscle, fat, heart myocardium, and small intestines but not in the brains from cognitively normal adults (Waller et al. 2013).Cardiomyocytes release GLUT in exosomes, which is likely upregulated in HCMV infected GLUT12 expressing cardiomyocytes, adipocyte stem cells, enterocyte of small intestines, or myoblasts (Yadav et al. 2023; Zwezdaryk et al. 2016; Couillard et al. 1991; Yeh et al. 2022), leading to exosomal transfer of GLUT12 to the brain (Garcia et al. 2016).HCMV-mediated pathological diseasesAPOE ALLELE CONFERS NASH and NAFLD RISK

[0128] ApoE4 is the highest genetic risk variant for AD, other dementias, and viral infection, with the ApoE4 allele increasing AD risk by as much as 50% (Riedel et al. 2016). ApoE4 is less lipidated and has lower expression than other ApoE alleles. Thus, cells expressing ApoE4 wouldhave increased intracellular lipids for vesicle and virion production. Another contribution of differential risk with ApoE allele types could result from the presence of ApoE on the surface of virions, non-infectious viral particles, and exosomes. For example, hepatitis C virus infected cells release lipoviral particles and HCMV infected cells could do this as well (Felmlee et al. 2013). ApoE3 homozygous individuals would have HDL-lipoviral particles that preferentially bind to receptors in the liver, ovaries, and adrenal glands (Brunham et al. 2006; Nguyen et al. 2009). Accordingly, homozygous ApoE3 is significantly overrepresented in nonalcoholic steatohepatitis (NASH) patients (Sazci et al. 2008). NASH is a more severe form of nonalcoholic fatty live disease (NAFLD) of unknown cause characterized by excessive fat deposits in the liver. Since glucose is the major fuel for HCMV and EBV replication, HCMV and EBV may have evolved mechanisms to modulate gluconeogenesis like the hepatitis C virus (Deng et al. 2011). Accordingly, gluconeogenesis is significantly upregulated in NASH patients (Sunny et al. 2011). HCMV and EBV virion, non-infectious particles, and viral exosomes targeting the liver could explain liver inflammation in NASH and NAFLD patients (Boeckmans et al. 2021; Da Cunha and Wu 2021). Accordingly, infectious mononucleosis is associated with an increased incidence of NAFLD (Loosen et al., 2022). In some embodiments, compositions comprising ASOs to ppl50 mRNA or gp350 are for use in the treatment of NASH or NAFLD. In some embodiments, methods of NASH and NAFLD treatment comprise administering to a patient an effective dosage of compositions comprising antisense oligonucleotides to ppi 50 or gp350.HCMV and EBV mediated cancer

[0129] Viral infection is estimated to play a role in 20% of human cancers. While, the explanation for an association between HCMV infection and dementias remains unknown, evidence for the association HCMV and cancer pathogenesis has been known for some time (Sinclair et al. 2000; Prod'homme et al. 2003; Poma et al. 1996; Zhang et al. 2003). For example, the HCMV protein IE1 binds the Rb-related protein pl 07 resulting in the derepression of E2F- responsive promoters and induction of the cell cycle progression in quiescent cells, which can lead to cancer (Poma et al. 1996). Accordingly, HCMV proteins, RNA, and DNA are found associated with many types of human tumors (Soderberg-Naucler 2022).

[0130] There is evidence that genetic variants in HCMV proteins can explain divergent disease pathogenesis. For example, 48% of various tumors surveyed contained a HCMV strain variant expressing an MIE gene lacking intron 2 (Mohammad 2017); this variant was detected less frequently in healthy individuals, or in individuals with HCMV viremia or myocardial infarction. In the same study, three clinical isolates contained both wildtype and variant HCMV strains, but infection of endothelial cells with the variant HCMV strain produced plaques withonly dense particles expressing IE proteins of different sizes. HCMV strain mutants have mutations in the IE1 globular region (Region A), and these mutations may define the virulence of the HCMV strain and determine whether IE1 upregulates (cancer) or downregulates STAT3 (vascular degeneration; CAA) (Retiere et al. 1998). See lElonc depicted in Fig. 4

[0131] HCMV strains differ in IE1 and hcmvIL-10 protein sequence variants. Indeed, cmvIL-10 (ULI 11 A) is one of the most frequently mutated genes identified among HCMV clinical strains (Suarez et al. 2019), and has been shown to increase MCF-7 breast cancer cell proliferation and chemotaxis in culture (Bishop et al. 2015). Thus, the delivery of hcmvIL-10 and IE1 sequence variants in exosomes, NIEPs, or dense bodies can increase cancer risk. For example, HMCV strains types have different cmv-IL-10 transcript isoforms or differ in their ability to induce of hIL-10 expression (Poole et al. 2020). hIL-10 blocks the PBMC proliferation and differentiation of dendritic cells from monocytes (Allavena et al. 1998). With monocyte differentiation inhibited, HCMV does not reactivate from latency at the tumor site to infect the tumor related cells. Similarly, HCMV is not distributed to distant sites, e.g., HCMV does not infect endothelial cells in the brain. In this scenario, an individual has increased cancer risk but decreased risk of vascular-related dementia. Alternatively, if the HCMV strain expresses a cmvIL-10 variant or has reduced hIL-10 induction (perhaps through a glycoprotein ULI 1 sequence variant), HCMV reactivates during monocyte-derived dendritic cell differentiation to infect the tumor and tumor related cells, leading to cancer eradication, but also endothelial cells, pericytes, smooth muscle cells, and astrocytes in the brain (Zischke et al. 2017; Poole et al. 2020). Reactivation of monocyte-derived dendritic cells would kill the cells; accordingly myeloid-derived dendritic cells are reduced in AD patients compared to cognitive controls (Ciaramella et al. 2016). Thus, lower levels of pULl 1 or defective CD45 phosphatase signaling would reduce hIL-10 upregulation, effectively increasing cancer immunity but also targeting HCMV infected microvessel cells in the brain (Magalhaes et al. 2017). In addition, to reducing pUL-11 efficacy, the ability of HCMV to spread through homing monocytes-derived dendritic cells could result in an HCMV strain that prevents cancer but induces HCMV-mediated systemic disease, e.g., AD, NASH, and diabetes. Measuring the HCMV pULl 1 expression level and / or variant genotyping, see two ULI 1 genotypes in Fig. S5A in Kiileki et al. 2022 (Kiilekci et al. 2022) is likely to be predict whether an HCMV strain can eradicate cancer but cause vascular disease. Another important factor is whether the host expresses the IL-10 receptor variant a and P (or IL-10R1 and 2). IL-10R1 signaling capacity is decreased compared to IL-10R2 (Gruber et al. 2008).

[0132] Predicting the type of HCMV-mediated disease in an individual, e.g., vascular disease / cancer eradication or immunosuppression with increased probability of cancer, will include determining the predominant cmvIL-10 and UL-11 genotype of the individuals HCMVstrains, and the genotype of host alleles, e.g., IL-10R, IL-10, BICD1, HLA (Jo et al. 2022; Pandey et al. 2021; Jones et al. 2002; Ma et al. 2005). These host and viral variants combine to determine immune escape. Genotype combinations producing low immune escape will eradicate cancer but cause insidious vascular disease, which is the cornerstone for many HCMV-mediated diseases.

[0133] The secretion of cmvIL-10 and / or HCMV-mediated induction of host IL- 10 and TGFP expression signals a shift in microglia phenotype from inflammatory to anti-inflammatory resulting in reduced microglia migration and phagocytic activity (Paglinawan et al. 2003; Michelson et al. 1994; Avdic et al. 2016; Chang and Barry 2010; Deere et al. 2019). Norepinephrine regulates microglia activity, and cell death in the locus coeruleus, which is ubiquitous in AD, would gradually reduce norepinephrine levels throughout the brain, leading to decreased clearance of Ap. (Heneka et al. 2010). Lastly, microglia express high levels of GLUT1 and loss of GLUT1 expression in microglia could inhibit microglia activity (Wang et al. 2019). Accordingly, AD like pathology is commonly found in brain tissue from glioblastoma multiforme (GBM) patients, for example increased GLUT1 mRNA but decreased GLUT1 protein in GBM tumors (Nelson 2002; Tsukamoto et al. 1996); and no inverse association is found between AD and GBM. The foregoing explains the robust inverse relationship between dementia and cancer risk within individuals.

[0134] The ability of ppi 50 and gp350 to disrupt membrane receptor cell surface trafficking may explain why immune checkpoint inhibitors are ineffective in treating cancer in some patients. The combination of ppi 50 mRNA and gp350 mRNA ASOs with immune checkpoint inhibitors may significantly increase the efficacy of immune checkpoint inhibitors.

[0135] EBV infection is associated with a growing number of epithelial and lymphoid cancer types, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, gastric carcinoma, a lymphoepithelioma-like carcinomas, T cell lymphomas, post-transplant lymphoproliferative disease, hepatic, gastric, smooth muscle, and pulmonary carcinomas (Shannon-Lowe and Rickinson 2019; Han et al. 2021).Ppi 50 sequence similarity with host proteins implicates HCMV-secreted ppi 50 in AD and obesity

[0136] Viral proteins have multiple splice variants, post-transcriptional modifications, and domains that interact with distinct proteins to enhance infection, evasion, and transmission. For example, viral proteins can produce an external phenotype in the host to increase nutrients for replication by increasing hunger drive. Another example of an external phenotype is the tropism for sensory neurons in the brainstem that coordinate swallowing. Dysfunction and death of these sensory neurons disrupts coordination of swallowing leading to dysphagia. Dysphagia increasesviral transmission by coughing, which explains why spouses and caretakers of dementia patients have a higher rate of dementia.

[0137] A DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) of the entire ppi 50 sequence in the UniProtKb-Swiss-Prot database exclusive for Homo sapiens identified the protein microtubule-actin cross-linking factor 1, isoforms 6 / 7, MACF1 (094854.5), with an Expect value of 2e-27 and 24% positive amino acid identity to ppl50 amino acids 535-1031 (see FIG. 11 A for alignment). MACF1 regulates microtubule stability by inducing GSK3B phosphorylation of microtubule stability proteins, e.g., tau. MACF1 regulates dendritic arborization and axon outgrowth (Ka and Kim 2016). Thus, the presence of ppi 50 could disrupt GSK3 phosphorylation in uninfected cells to conserve resources for HCMV infected cells. A position specific iterated BLAST of ppl50 amino acid sequences 838-1003 in the UniProtKb- Swiss-Prot database exclusive for Homo sapiens identified with the protein triple functional domain protein, TRIO (075962.2) with an Expect value of le-04 (see FIG. 1 IB for sequence alignment). TRIO plays a key role in neurite outgrowth and lamellipodia formation (Barbosa et al. 2018). Thus, the presence of ppl50 in uninfected cells could disturb neurite outgrowth, which is required for memory formation, the depletion of which is a hallmark of AD. In summary, the dissemination of ppi 50 iteralia secreted proteins can produce pathology resembling age-related neurodegenerations, e.g., AD.

[0138] The enteroendocrine cell is a specialized secretory cell of the gut barrier infected by HCMV. Accordingly, phosphorylated tau is found in enteroendocrine cells (Chapelet et al. 2023). A DELTA-BLAST of ppl50 amino acids 811-931 in Swiss-Prot database exclusive to Homo sapiens identified sequence similarity with the protein dedicator of cytokinesis protein 4, DOCK4 (Q8N1I0.3) with an Expect value of 7e-05 and 41% positivity (see FIG. 11C for sequence alignment). The protein DOCK4 functions as a guanine nucleotide exchange factor (GEF) in neuron polarization and axon regeneration. DOCK4 is also expressed in enteroendocrine cells where it can induce secretion of the hunger hormone gherlin, which is increased in morbidly obese individuals (Huang et al. 2022; Wblnerhanssen et al. 2017). Thus, ppi 50 could function in enteroendocrine cells to produce an external phenotype that drives hunger, which would increase resources for HCMV replication, e.g., glucose. An increased hunger drive could increase obesity risk. Accordingly, several research groups have concluded that obesity risk appears to be a geographical phenomenon, which could be explained by a contagion (Datar and Nicosia 2018; Tun et al. 2018), e.g., a particular HCMV or EBV strain, wherein ppi 50 (or gp350) cleavage is increased or has amino acid variants simulating DOCK4.

[0139] A DELTA-BLAST of ppi 50 amino acids 811-931 in Swiss-Prot database exclusive to Homo sapiens also identified a sequence similarity with the protein Dynactin subunit 1,(Q14203.3) with an Expect value of le-05 and 49% sequence positivity (see FIG. 1 ID). Dynactin subunit 1 is part of the dynactin complex that is essential for targeting dynein to microtubule plus ends. The ppi 50 sequence could interact with dynein and prevent dynein- mediated cargo trafficking along microtubules in axons.

[0140] The sequence alignments demonstrate that ppl50 is a pleiotropic protein capable of interacting with many host proteins to disrupt cell processes, the extent of which are determined by host protein sequence variants, HCMV sequence variants, and viral load, which increases with age. The analyses indicate the futility in approaching one protein disruption as a therapeutic approach.EMBODIMENTS

[0141] In some embodiments, a pharmaceutical composition comprises ASOs complementary to ppi 50 mRNA to block ppi 50 expression and therefore prevent ppi 50 from interacting with BICD1 and disrupting of the endosomal pathway, and subsequently the viral restriction function of APP and SorLa receptor neurotropic function as it regulates the location of the BDNF receptor, TrkB.

[0142] In some embodiments, administering compositions comprising ppi 50 mRNA ASO to subjects decreases HCMV-ppl50 mediated ppl50-MACFl mimicry and subsequent reduction of GSK3B phosphorylation. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject reduces HCMV-ppl50 mediated ppl50-TRIO mimicry and subsequent disruption of neurite outgrowth and lamellipodia formation. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject in need of treatment reduces ppl50-DOCK4 mimicry and subsequent induction of the hunger hormone gherlin. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject reduces ppl50-Dynactin subunit 1 mimicry and subsequent disruption of vesicle movement towards the microtubule plus ends.

[0143] In some embodiments, the disclosure provides a composition for use in treating HCMV- cmv-IL-10 mediated immunosuppression comprising oligonucleotides antisense to ppl50 and for inhibiting expression by RNAi. In some embodiments, a subject is administered a composition for use in treating HCMV-mediated immunosuppression comprising oligonucleotides antisense to ppl50 for inhibiting expression by RNAi. In some embodiments, inhibiting ppi 50 expression by RNAi reduces risk and severity of infection with diverse microbes, i.e., fungi, yeast, pathogenic bacteria, and endogenous and zoonotic viruses.

[0144] . In some embodiments, the disclosure provides compositions for use in treating ebvIL- 10 mediated immunosuppression comprising ASOs to gp350 to inhibit gp350 expression byRNAi. Inhibiting gp350 expression could increases EBV protein presentation at the cell surface eliciting immune cell mediated elimination of EBV infected cells and therefore reducing ebvILlO levels. In some embodiments, the disclosed EBV gp350 ASOs reduce gp350-mediated endosomal dysfunction. In some embodiments, reducing gp350-mediate endosomal dysfunction increases viral protein immunogenic display on the cell surface eliciting immune cell mediated elimination (immunotherapy). In some embodiments, the disclosed EBV gp350 ASOs inhibit reducing release of viral exosomes, NIEPS, or dense body release from latently EBV infected cells. In some embodiments, the disclosed EBV gp350 ASOs reduce EBV virion dissemination by increasing death of infected cells (Meyer et al. 1997). In some embodiments, the disclosed EBV gp350 ASOs reduce EBV viral load, and thus the EBV secretome.

[0145] In some embodiments, a subject is administered a composition for use in treating EBV- mediated immunosuppression comprising OSAs to gp350 for inhibiting expression by RNAi. In some embodiments, use of OSAs to gp350 are used in the manufacture of a medicament for the treatment of immunosuppression, or conversely immune stimulation. In some embodiments, inhibiting gp350 expression by RNAi reduces risk and severity of infection with diverse microbes, i.e., fungi, yeast, pathogenic bacteria, and endogenous and zoonotic viruses.

[0146] In some embodiments, the disclosed exemplary ppi 50 ASOs decrease HCMV viral exosome secretion (HCMV secretome) as explained above; therefore, inhibiting secretion of IE1 and IE2 in the HCMV viral secretome will increase GLUT1 expression. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASO to a subject will increase brain GLUT1 expression, as measured by positron emission tomography using 2- [18F] -fluorodeoxy glucose before and after treatment. In some embodiments, the disclosed ppl50 mRNA ASO (SEQ ID NOs 1-15) are used in the manufacture of a medicament for increasing GLUT1 protein expression in the brain.

[0147] In some embodiments, the disclosed exemplary ppi 50 mRNA ASOs are used in the manufacture of a medicament for treating HCMV-mediate type 2 diabetes. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject decreases HCMV-mediated type 2 diabetes severity, e.g., reduce refractory HbAlc level. In some embodiments, administering compositions comprising the disclosed exemplary ppl50 mRNA ASOs to a subject reduces HCMV-mediated increased IL-6 levels as compared to before treatment. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject reduces HCMV-mediated VEGF upregulation. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject increases GLUT1 expression, as measured by positron emission tomography using 2-[18F]-fluorodeoxy glucose.

[0148] In some embodiments, the disclosed exemplary ppi 50 mRNA ASOs are used in the manufacture of a medicament for treating cardiovascular disease (heart disease). In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject decreases risk of myocardial infarction. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject reduces HCMV-mediated increased CRP levels as compared to before treatment. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject reduces cardiovascular-related mortality. In some embodiments, administering compositions comprising the disclosed exemplary ppi 50 mRNA ASOs to a subject reduces secondary stroke risk. In some embodiments, the ppi 50 mRNA ASOs are used in the manufacture of a medicament for treating secondary stroke.

[0149] In some embodiments, the disclosed ppi 50 mRNA ASOs compositions are used in the manufacture of a medicament for treating cancers such as GBM, colon cancer, pancreatic cancer, hepatocellular carcinoma (HCC), hepatoblastoma, a mixed liver cancer, a cancer derived from mesenchymal tissue, liver sarcoma or cholangiocarcinoma. The ppl50 mRNA ASO would increase plasma membrane recycling to the cell surface, which should increase immunogenicity of GBM, which are notoriously non-immunogenic; thus, ppi 50 ASOs compositions function as immunotherapy. In some embodiments, ppi 50 mRNA ASOs compositions comprise immune checkpoint inhibitors.

[0150] In some embodiments, administering compositions comprising ppi 50 mRNA ASOs to a subject reduces HCMV-mediated cancer risk. In some embodiments, administering compositions comprising ppl50 mRNA ASOs to a subject reduces HCMV-mediated cancer growth. In some embodiments, administering compositions comprising ppi 50 mRNA ASOs to a subject reduces HCMV-mediated cancer metastasis. In some embodiments, compositions comprising ppi 50 mRNA ASOs are administered in combination with immune checkpoint inhibitors.

[0151] In some embodiments, the disclosed gp350 mRNA ASOs compositions are used in the manufacture of a medicament for treating cancers, such as such Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. The gp350 mRNA ASOs would increase plasma membrane recycling to the cell surface, which would increase cancer cell immunogenicity; thus, gp350 ASOs compositions function as immunotherapy. In some embodiments, gp350 mRNA ASOs compositions comprise immune checkpoint inhibitors.

[0152] In some embodiments, administering compositions comprising gp350 mRNA ASOs to asubject reduces EBV-mediated cancer risk. In some embodiments, administering compositions comprising gp35O mRNA ASOs to a subject reduces EBV-mediated cancer growth. In some embodiments, administering compositions comprising gp350 mRNA ASOs to a subject reduces EBV-mediated cancer metastasis. In some embodiments, administering compositions comprising ppi 50 mRNA ASOs to a subject reduces HCMV-mediated VEGF upregulation. In some embodiments, administering compositions comprising ppi 50 mRNA ASOs to a subject increases GLUT1 expression in immune cells to increase immune cell activity and subsequently tumor immunity.

[0153] In some embodiments, compositions comprising ppi 50 mRNA and / or gp350 mRNA ASOs are administered to cancer patients simultaneously or sequentially with immune checkpoint inhibitors.

[0154] In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject decrease HCMV-mediated gut dysbiosis as measured by increased microbiome diversity and beneficial bacteria. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject reduces HCMV-mediated gut dysbiosis as measured by increased microbiome diversity and beneficial bacteria as measured by 16S ribosomal RNA. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject reduces gastrointestinal inflammation, as measured by reduced cytokine levels. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject reduces HCMV viral secretome release from the gut barrier cells e.g., enteroendocrine cells. In some embodiments, administering compositions comprising ppi 50 mRNA ASO reduces HCMV virions dissemination from gastrointestinal barrier cells, e.g., enteroendocrine cells. In some embodiments, administering compositions comprising ppi 50 mRNA ASO to a subject normalizes bowel movement frequency and stool consistency. In some embodiments, the ppi 50 mRNA ASOs are used in the manufacture of a medicament for treating gut dysbiosis. Exemplary ASO sequences that inhibit ppl50 protein expression are provided in SEQ ID NOs: 1-15 (Table 2).

[0155] In some embodiments, administering compositions comprising gp350 mRNA ASOs decrease EBV-mediated gut dysbiosis as measured by increased microbiome diversity and beneficial bacteria by 16S ribosomal RNA compared to before treatment. In some embodiments, administering compositions comprising gp350 mRNA ASOs to a subject reduces gastrointestinal inflammation, as measured by reduced cytokine levels compared to before treatment. In some embodiments, administering compositions comprising gp350 mRNA ASOs to a subject reduces HCMV viral secretome release from the gut barrier cells e.g., enteroendocrine cells compared to before treatment. In some embodiments, administering compositions comprising gp350 mRNAASOs to a subject reduces EBV virions dissemination from gastrointestinal barrier cells, e.g., enteroendocrine cells. In some embodiments, administering compositions comprising gp350 mRNA ASOs to a subject normalizes bowel movement frequency and stool consistency as compared to before treatment. In some embodiments, the gp350 mRNA ASOs are used in the manufacture of a medicament for treating gut dysbiosis. Exemplary ASO sequences that inhibit gp350 protein expression are provided in SEQ ID NOs: 16-29 (Table 3).

[0156] In some embodiments, administering compositions comprising ASO to ppi 50 mRNA to a subject decreases HCMV-mediated NASH as measured by liver biopsy before and after treatment. In some embodiments, the ppi 50 mRNA ASOs are used in the manufacture of a medicament for treating NASH. Exemplary ASO sequences that inhibit ppi 50 protein expression are provided in SEQ ID NOs: 1-15 (Table 2). In some embodiments, administering compositions comprising ASO to gp350 mRNA to a subject decreases EBV-mediated NASH as measured by liver biopsy before and after treatment. In some embodiments, the ASO gp350 mRNA are used in the manufacture of a medicament for treating NASH. Exemplary ASO sequences that inhibit gp350 protein expression are provided in SEQ ID NOs: 16-29 (Table 3).

[0157] The disclosure provides connections between major diseases with known risk factors but unknown etiology. In addition, the disclosure describes how HCMV ppl50 and EBV gp350 can mediate the endosomal pathway disruption that is seen in many diseases, such as early and late- onset dementias, (e.g., vascular related dementias, AD, Parkinson’s disease, and frontotemporal dementia, LBD). The disclosure describes the mechanism for HCMV-mediated type-2 diabetes, autoimmune type-1 diabetes, NASH, NAFLD, cancer, obesity, and neuropathy. The embodiments disclosed can be used to treat HCMV viremia, HCMV congenital syndrome (hearing loss, developmental delay, and cognitive impairment) (Arav-Boger 2015), atherosclerosis-mediated cardiovascular disease (e.g., heart disease and stroke), advanced senescence, immunosuppression, obesity, gut dysbiosis, accelerated senescence, and HCMV viral syndrome in transplant patients leading to organ failure (Azevedo et al. 2015). HCMV seropositivity is also significantly associated with schizophrenia (Calkova et al. 2022), major depressive disorder (Ford et al. 2019), autoimmune disease (Gugliesi et al. 2021), and frailty (sarcopenia) in older women (Thomasini et al. 2017). In view of HCMV causing congenital hearing loss, the HCMV secretome could be ototoxic and cause tinnitus of unknown etiology in adults. It is also possible that HCMV ppi 50 could cause early hair graying, through its disruption of the BICD1-Rab6b melanosome secretory pathway.

[0158] The disclosure thus provides pharmaceutically acceptable compositions and methods of treatment for all HCMV-mediated diseases, including early and late on-set dementias, type-2 diabetes, autoimmune type-1 diabetes, NASH, NAFLD, cancer, tinnitus, HCMV viremia,HCMV congenital syndrome, hair graying, schizophrenia, atherosclerosis-mediated cardiovascular disease (e.g., heart disease and stroke), advanced senescence, immunosuppression, obesity, gut dysbiosis, and accelerated senescence, adult on-set hearing loss, and transplanted-related HCMV viremia, major depressive disorder, autoimmune disease and frailty.

[0159] In one embodiment, the disclosure provides compositions and methods for prophylactic prevention of all the foregoing HCMV-mediated diseases. In one preferred embodiment, the disclosed compositions and methods of treatment eradicate all strains of HCMV in a subject by immunotherapy, wherein the endosomal pathway resumes function, MHC-1 presents a viral antigen on the cell surface, and CD8+ T cells and NK cells kill the infected cell. HCMV proteins (US6, US2, ULI 8, US3, US11) disrupt MHC-1 function with redundancy during early and late phase of viral gene expression (Halenius et al. 2015; Gabor et al. 2020). Thus, HCMV infected cells may not express the MHC-1 disrupting proteins during latency. In some embodiments, the ASO compositions are delivered with antiviral nucleosides to prevent HCMV replication. In a preferred embodiment, ppi 50 ASOs are administered with the HCMV antiviral agent ganciclovir (GCV) or its prodrug valganciclovir (VGCV). In another preferred embodiment, the ASOs dosage is determined by the HCMV load, which can be determined from the quantity of viral protein released in the secretome, which can be measured in a subject’s blood, semen, or saliva. For example, subjects with higher cmv-ILlO levels in Fig. 3 are likely to have higher HCMV viral load. The novel ppi 50 ASO compositions provided herein have the capacity to eradicate multiple major diseases plaguing humankind.

[0160] EBV is associated with diverse autoimmune syndromes, such as multiple sclerosis (MS), systemic lupus erythematosus, Sjogren’s syndrome, systemic vasculitis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, systemic sclerosis, and celiac disease (Harley et al. 2018; Parks et al. 2005; Croia et al. 2014; Serafini et al. 2007; Mechelli et al. 2015; Machhua et al. 2022). Regarding MS, for which myelin degeneration is the cardinal pathology, myelin degeneration also occurs in AD, and MS and AD pathology coexist in some patients (Luczynski et al. 2019). Moreover, the diagnosis with any one of these EBV-related diseases significantly increases the risk of dementia (Hou et al. 2019; Luczynski et al. 2019; Chen et al. 2019; Zhao et al. 2018). The diversity of EBV-mediated disease phenotypes is likely due to a combination of EBV’s high capacity for genetic variation (Correia et al. 2018) and genetic variants in the subject. The disclosure thus provides pharmaceutically acceptable compositions and methods of treatment for all EBV related diseases, including diverse autoimmune syndromes, such as multiple sclerosis (MS), systemic lupus erythematosus, Sjogren’s syndrome, systemic vasculitis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, systemic sclerosis, and celiacdisease (Harley et al. 2018; Parks et al. 2005; Croia et al. 2014; Serafini et al. 2007; Mechelli et al. 2015; Machhua et al. 2022). The disclosure provides for compositions and methods for prophylactic prevention of the foregoing EBV-mediated diseases.

[0161] . In one preferred embodiment, the disclosed compositions and methods of treatment eradicate all EBV strains in a subject by immunotherapy, wherein the endosomal pathway becomes functional, MHC-1 presents a viral antigen on the cell surface, and NK cells or CD8+ T cells kill the infected cell. Like HCMV, EBV encodes with proteins redundancy to disrupt MHC-1 function during early and late phase of viral gene expression. Thus, MHC-1 disruption in latent EBV infected cells may not express other MHC-1 disruption proteins. In some embodiments, the ASO compositions are delivered with an EBV antiviral agent for preventing EBV reactivation. In a preferred embodiment, the ASO compositions are delivered in combination with EBV replication inhibitor tenofovir (Drosu et al. 2020).

[0162] In preferred embodiments, the ASO dosage is determined by the EBV load, which can be determined from the quantity of viral protein released in the viral secretome. The viral protein can be measured in samples in the subject’s blood, semen, or saliva using ELISA. For example, subjects with higher ebvILlO levels are likely to have higher EBV viral load. The novel gp350 ASO compositions provided herein have the capacity to eradicate multiple major diseases plaguing humans.

[0163] The ppi 50 and gp350 mediated disruption of the endosomal pathway may reduce the levels of immune checkpoint inhibitors at the cell surface increasing autoimmune disease risk, whereas the loss of checkpoint inhibitors would enhance immune targeting of cancer cells (another mechanism to explain the robust inverse association between cancer and neurodegeneration) and make checkpoint inhibitors less effective in cancer. Indeed, Alzheimer’s disease is now characterized as an autoimmune disease.HCMV-MEDIATED DISEASE TREATMENT

[0164] Provided herein are compositions and methods for reducing ppi 50 HCMV mRNA / protein expression or function by RNA interference (RNAi) for use in reducing HCMV- mediated disease pathology. In some embodiments, ppi 50 RNAi is achieved by delivering ASOs, short hairpin RNA molecules encoding the antisense target sequence, morpholinos, or a vector comprising a construct encoding the antisense target sequence, wherein the antisense oligomer sequence is fully complementary or substantially complementary to any contiguous ppi 50 mRNA sequence or sequences 5’ upstream or 3 ’downstream from ppi 50 CDS from 15- 25 nucleotides in length that block ppi 50 protein translation. In some embodiments, the RNAi molecule is a morpholino targeting the 5’ UTR and AUG start codon. Inhibiting ppi 50 expression reduces endosomal dysfunction of infected and uninfected cells, cell death,immunosuppression, GLUT1 repression, cancer, diabetes, gastrointestinal dysbiosis, HCMV disease in transplant patients, congenital HCMV disease, vascular pathology, dementia, systemic inflammation. The reduction of these HCMV-mediated pathologies results from inhibiting ppi 50 in viral exosomes, inhibiting viral exosome release, and / or virion egress. In some embodiments, the viral secretome is reduced by eliminating the number of HCMV infected cells through ppi 50 ASO mediated immunotherapy, wherein reducing ppi 50 protein expression restores endosomal trafficking to the cell surface to deliver of MHC-1 baring viral epitopes, wherein the infected cells are removed by recognition by the innate or adaptive immune system. Compositions

[0165] Composition comprising RNA therapy can be achieved by delivering antisense oligonucleotides (ASO), which are single stranded oligonucleotides that are antisense to the target RNA and can reduce mRNA expression by (1) binding to target RNA to cause steric interference for ribosome complex binding, (2) splicing disruption, (3) inhibits polyadenylation to accelerate decay, (4) RNase H cleavage by use of gapmers to form DNA / RNA duplex, (5) Ago2 mediated slicing (Castanotto et al., 2015), or a combination thereof.

[0166] An antisense oligonucleotide (ASO) with the sequence complementary to the targeted RNA molecule may comprise a morpholino. A “morpholino” as used herein is an ASO comprised of single stranded DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups.

[0167] The ASO may comprise a gapmer. A “gapmer” as used herein is an ASO comprising e.g., five nucleotides of RNA-like residues flanking a central 10-nucleotide DNA region. In some embodiments, the number of RNA and DNA nucleotides of the gapmer varies, e.g., 4-6 RNA nucleotides flanking 8-12 DNA nucleotides.

[0168] In some embodiments, the ASO may comprise single-stranded DNA antisense to mRNA. In some embodiments, the ASO may comprise chemically modified single-stranded DNA antisense to the target RNA.

[0169] RNA interference using siRNA to knockdown IE1 / IE2 inhibited HCMV infection (Xiaofei et al. 2012), however, targeting IE1 and IE2, which block apoptosis could lead initiate apoptotic cell death (Zhu et al. 1995). In contrast, inhibiting ppl50 expression using ppl50 mRNA antisense expressing cells did not induce apoptosis after HCMV infection (Meyer et al. 1997). HCMV is known to regulate Dicer expression, so providing short hairpin RNAs or pre- miRNA for processing into siRNA or miRNA may not be as effective as ASOs in HCMV or EBV infected cells (Sleman et al. 2022). Furthermore, Dicer diurnal expression pattern and level decreases with age (Yan et al. 2013).

[0170] The RNA molecule or RNA-DNA hybrid molecule (gapmer) can be naked orcomplexed or contained within a nanocarrier. Solutions for facilitating uptake or absorption into the cell are known by those skilled in the art. Absorption or uptake of antisense oligonucleotide can occur through unaided diffusive or active cellular processes, or by auxiliary agents or a targeting moiety. The antisense oligonucleotide may also be introduced into a cell in vivo by local or systemic delivery. For example, for in vivo delivery, antisense oligonucleotide, whether double stranded RNA (dsRNA), single-stranded DNA (ssDNA), or a morpholino, gapmer, or cRNA can be injected into a tissue site or administered orally for enteric or intravenously for systemic dissemination. The ASO molecules of this disclosure can be administered to a subject prophylactically or a subject in need of medical intervention. Such an administration may comprise the injection of one or more ASOs, or a viral vector of this disclosure, into a diseased site in a subject, for example in the case of cancerous tissues / cells, the injection is preferably in proximity to the cancerous tissue.

[0171] A phosphorodiamidate morpholino oligomer or morpholino with neutral phosphorodiamidate linkages that replace the anionic phosphodiester linkage. Morpholinos ASOs for research are available through GeneTool LLC and a morpholino drug is approved as an FDA treatment for Duchenne muscular dystrophy. In some embodiments, the morpholinos can be modified conjugated / coupled to 7-14 guanidinium head groups (Morcos et al. 2008), penetrating peptides (e.g., TAT, penetratin, arginine-rich peptide NH2-R9F2C-CONH2), or oligonucleotide enhancing compounds (OEC, e.g., UNC7938) to improve cell entry / biological activity. In some embodiments, one or more disclosed ASOs are morpholino oligomers are combined in a composition for in vivo delivery (with <4 complementary sequences at 5’ and 3’ ends). In some embodiments, the disclosed morpholinos include variants, for example, antisense oligomers with phosphodiesterase linkages, modified sequences, and sequence identities within 80% identity, with 5’ or 3’ extended or shortened sequences, or different conjugated molecules.

[0172] In some embodiments, the ASO has locked-nucleic acid modified, e.g., nucleotides are modified with a 2'-O-methyl, 2'-O-Methoxy ethyl, 2'-fluorine at 2'-ribose (OH), and 2'- fluoroarabinonucleic acid increase annealing affinity to complementary RNA. A locked nucleotide contains a methylene bridge between the 2' and 4' position of the ribose further increasing binding affinity. A phosphorothioate or amide nucleotide linkage increases RNase resistance.

[0173] It is conceived by those skilled in the art that an ASO sequence targeting ppl50 or gp350 can inhibit the expression by at least about 60%, by at least 70%, by at least 80%, preferably by at least 90%. Expression silencing measured as described in Example 1 using the exemplary ASOs targeting pp!50 and gp350 disclosed in Tables 2 and 3, respectively. The compositioncomprises a pharmacologically effective amount of antisense RNA molecule, for which the antisense RNA may target one or more locations on the mRNA, 5’ upstream or 3’ downstream from the CDS, and a pharmaceutically acceptable carrier. Such a pharmaceutical composition may also comprise antisense oligonucleotides (ASO), e.g., dsRNA, siRNA, shRNA, sDNA, morpholinos, or shRNAs, or the herein described viral vector(s) comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one sense strand encoding the disclosed exemplary ASOs. The composition comprises a pharmaceutically acceptable excipient for in vivo administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, delayed-release coatings, disintegrating agents, binding agents, stabilizers, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives, e.g., antioxidants, as known to persons skilled in the art.

[0174] It is particularly envisaged that the pharmaceutically acceptable carrier allows for the systemic or enteric administration of the RNA molecules, vectors or engineered human or bacteria cells by oral administration to all aspects of the gastrointestinal system, e.g., keratinocytes of the oral cavity. The composition can be delivered to the salivary glands by cannulation of the salivary gland ducts. In addition to oral administration parenteral administration includes transdermal, transmucosal, inhalation, insufflation, eye drops, nasal spray, buccal, vaginal, and anal administration as feasible ways of administering to a patient in need of therapeutic intervention the disclosed compositions. When parenteral administration is employed, this can comprise the direct injection of the compounds of this invention into the diseased tissue or at least in proximity. However, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intradermal, intrathecal, and other administrations of the disclosed compositions are within the skill of the artisan.

[0175] For intramuscular, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of RNAi molecule in the cells infected with HCMV or EBV. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. The pharmaceutical compositions usefulaccording to the invention also include encapsulated formulations to protect the ASOs against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Oral formulations may include a mucoadhesive, such as a polydopamine (pD) coating. In vivo dosage of ASO can range from 1 mg / kg to 200 mg / kg and may be administered biweekly or monthly.

[0176] The preparations of such formulations are apparent to those skilled in the art. Nanocarriers can encapsulate or complex the ASOs and can be prepared according to methods known to those skilled in the art, for example, as described in PCT publication W091 / 06309 and WO201 1 / 003780 which is incorporated by reference herein. In some embodiments, oligonucleotides are delivered by nanoparticles disclosed in patent no. US 11622972.

[0177] It can be expected that a shorter ASO comprising one of the sequences in Table 2 and 3 minus only a few nucleotides on one or both ends may be similarly effective as compared to the disclosed ASO sequences. Likewise, it can be reasonably expected that a longer ASO comprising one of the sequences in Table 2 and 3 plus only a few nucleotides on one or both ends may be similarly effective compared to the disclosed ASO sequences.

[0178] Exemplary ASOs that target HCMV ppi 50 mRNA to prevent endosomal pathway disruption are disclosed in SEQ ID NOs: 1-15 (Table 2).

[0179] Exemplary ASOs that target EBV gp350 mRNA to prevent endosomal pathway disruption are disclosed in SEQ ID NOs: 16-29 (Table 3).

[0180] In some embodiments, the dsRNA molecules provided herein comprise a duplex length (i.e., without “overhangs”) of about 16 to about 30 nucleotides. In some embodiments, the dsRNA duplex lengths range from about 19 to about 25 nucleotides. In a preferred embodiment, the duplex structure is 19 nucleotides in length. In some embodiments, the antisense strand of the dsRNA molecule is at least partially complementary to the sense strand.

[0181] The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 13 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located within nucleotides 2-7 of the 5' terminus of the antisense strand. In another embodiment, it is preferable that the area of mismatch is not located within nucleotides 2-9 of the 5' terminus of the antisense strand. As mentioned above, at least one end / strand of the dsRNA may have a single-stranded nucleotide overhang of 1 to 5, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. dsRNA having only one overhang hasproven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. In one embodiment, the siRNA has one nucleotide overhang. In preferred embodiments, the single-stranded overhang is located 3' of the antisense strand, or 3' of the sense strand. The dsRNA may also have a blunt end, preferably located at the 5 '-end of the antisense strand. In another embodiment, the antisense strand of the dsRNA has a nucleotide 3' overhang, and the 5 '-end is blunt. In yet another embodiment, one or more of the nucleotides in the overhang contains a nucleoside thiophosphate. The foregoing siRNA modifications are well known in the art to increase expression knockdown.

[0182] The dsRNA of the present invention may also be chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and / or modified by methods well established in the art. Chemical modifications may include, but are not limited to 2' modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages, inverted deoxythymidines. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Preferably, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, preferably bis-(2-chloroethyl)amine; N-acetyl-N'-(p-glyoxylbenzoyl) cystamine; 4-thiouracil; and psoralen. In one preferred embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA is produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams D J and Hall K B, Biochem. (1996) 35: 14665-14670). In a particular embodiment, the 5'-end of the antisense strand and the 3'-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate group. The chemical bond at the ends of the dsRNA is preferably formed by triple-helix bonds.VECTORS

[0183] In some embodiments, viral vectors deliver expression constructs encoding the ASOs or a ASO sequence within the shRNA sequence. In some embodiments, the ASO can be encoded viral vector can be a self-inactivating lentivirus vector (Zufferey et al. 1998; Shin et al. 2006). In some embodiments, the viral vector is an AAV vector. In some embodiments, the vector contains multiple expression cassettes to express multiple ASOs or shRNAs that target different locations in the ppl50 or gp350 mRNA, i.e., multiple sites on one transcript. In some aspects,the expression construct comprises multiple ASOs, such as a shRNA, which are co-expressed using intervening internal ribosome entry sites with an integrated ATG start codon. In some aspects, the nucleotide sequences are modified for codon optimization.DELIVERY AND TARGETING

[0184] In some embodiments, the vector comprises a targeting moiety, such as a peptide, fab, or aptamer, which targets the vector to an HCMV or EBV infected cell or susceptible cell type (Korbelin et al. 2016).

[0185] The oligonucleotides may be modified to enhance affinity and nuclease resistance. The oligonucleotides may be delivered complexed in a lipid nanoparticle or encapsulated within a liposome. The oligonucleotide, lipid nanoparticle, or liposome may be conjugated with a cell targeting moiety. In some embodiments, the oligonucleotides are inserted in an expression construct within a viral vector and delivered by transduction in vivo.

[0186] In some embodiments, the ASOs are delivered in nanocarriers, e.g., exosomes, nanoparticles, liposomes or micelles (Mitchell et al. 2021). Many liposomes comprising lipids with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art (Moncalvo et al. 2020). In a preferred embodiment, siRNA is complexed with MPEG-PCL- CH2R4H2C for delivery to colonic enterocytes (Ibaraki et al. 2022).

[0187] U.S. Pat. No. 5,540,935 and U.S. Pat. No. 5,556,948 describes PEG-containing liposomes derivatized with functional moieties on their surfaces. In some embodiments, nanocarriers are conjugated with any of the functional targeting ligands described below. In some instances, HCMV infected endothelial cells are targeted using glucose as the targeting moiety, which can be conjugated to PEG and incorporated into a nanocarrier (Min et al. 2020). Techniques for conjugating ligands to the liposome surface, both covalently and non-covalently, known in the art. In some instances, oligonucleotides are labeled with azide using 3- azidoproprionic acid and reacted with an antibody functionalized with a dibenzocyclooctyne (DBCO) click group (Wiener et al. 2020

[0188] In some embodiments, a cell targeting moiety facilitates introducing into a cell the exemplary disclosed ASOs. A cell targeting moiety can be a peptide, lectin, antibody, antibody fragment, glycolipid, nucleic acid aptamer, or glycoprotein. For example, B cells can be targeted with the AchR-binding peptide rabies viral glycoprotein (RVG), and epithelial cells and HCMV infected endothelial cells can be targeted with an antibody, antibody fragments, or peptide targeting the transferrin receptor (TfRl) or integrin avP6 (Tabata et al. 2008; Di Fan et al. 2023); incorporated by reference from Provisional application no. 63 / 519,840 filed August 16, 2023. TfRl targeted antibody ASO or nanocarrier conjugate can cross the gut barrier by transcytosis. Monocytes are a reservoir for HCMV and were identified as the source of immunosuppressivefactors in cell culture (Rice et al. 1984). HCMV infected monocytes present in the gut associated lymphoid tissue (GALT) can secrete exosomes containing viral factors into lymph circulation and GI tract. In some embodiments, HCMV infected monocytes, endothelial cells, and hemopoietic stem or progenitor cells can be targeted by their cell surface expression of CD 14, CD36, and CD34+ cell surface receptors (Liang et al. 2009). For example, ppl50 ASOs can be covalently or non-covalently conjugated to a fragment antibody binding region (Fab antibody), peptides, or aptamer binding CD14, CD36, or CD34+. In one embodiment, ppl50 ASOs are covalently or non-covalently conjugated to a targeting moiety for reactive endothelial cells, e.g., cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)](Tian et al. 2018). In some embodiments, a nanocarrier encapsulated or complexed with ppl50 ASOs has a targeting moiety, e.g., cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)] or Fab targeting CD14, CD36, or CD34+.

[0189] EBV utilizes the entry receptors CD21 (CR2), CD35 (CR1), EphA2, integrin receptors (avP5, avP6, avP8), NRP1, NMHC-IIA, and HLA-DRB1 (Cruz et al. 2022). In some embodiments, the EBV gp350 ASO is conjugated to a targeting moiety, such as a Fab-antibody, peptide, or aptamer with high affinity to any of the recited EBV entry receptors for delivering gp350 ASO to EBV infected cells. In one embodiment, chemically modified gp350 ASO is conjugated to cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)] for delivery to activated endothelial cells. In some embodiments, a nanocarrier encapsulated or complexed with gp350 ASOs has a targeting moiety, e.g., cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)] or Fab- antibody targeting cell surface receptors selected from CD21 (CR2), CD35 (CR1), EphA2, integrin receptors (avP5, avP6, avP8), NRP1, NMHC-IIA, and HLA-DRB1. HCMV and EBV can infect hematopoietic cells to provide a latent reservoir that can modify immune cell differentiation, function, and survival of differentiated immune cells.

[0190] In some instances, the targeting moiety is conjugated to the ASO molecule or nanocarrier using an amide bond formation, thiol bond formation, hydrazone bond formation, ester bond formation, or an avidin-biotin bond (Eroglu and Ibrahim 2020).

[0191] In some embodiments, the compositions described herein are designed to enter HCMV or EBV infected cells, e.g., endothelial cells, epithelial cells, pericytes, astrocytes, neurons, oligodendrocytes, microglia, monocytes, macrophages, dendritic cells, lung epithelial cells, retinal epithelial cells, adipocytes, enterocytes, smooth muscle cells, erythrocytes, and stem cells. In some embodiments, the compositions can be delivered to epithelial cells, fibroblasts, and endothelial cells of the lung and nasopharyngeal region with an aerosol inhaler or intranasal mist. In other embodiments, the compositions can be administrated by intravenous injection, muscular injection, eye drops, genital or rectal suppository, or oral administration. RNAi molecule formulations can be delivered orally in phosphate-buffered saline (Arora et al. 2002) orin a delayed release formulation to target the lower gastrointestinal tract.

[0192] Screening HIV patient’s gastrointestinal tract for HCMV identified the colon as the site with the highest concentration of giant cell inclusions in epithelial cells and mesenchymal cells, including histiocytes, smooth muscle cells and endothelial cells. Therefore, in some embodiments, a delayed release formulation comprising enteric coated capsules can target RNAi molecules to release in the lower GI tract, such as the colon and duodenum. Delayed release formulations for colonic drug delivery are well known in the art (Gazzaniga et al. 2022).

[0193] Delivering ppi 50 or gp350 ASOs to cells of the gastrointestinal tract (GI), such as the enteroendocrine cells, enterocytes, fibroblasts, endothelial cells, stromal cells, GALT monocytes and smooth muscle cells can prevent endosomal dysfunction allowing MHC presentation of microbial immunogenic peptides. Delivering ppi 50 or gp350 ASOs to HCMV or EBV lytic replicative cells of the gastrointestinal tract (GI), such as the enterocytes, fibroblasts, endothelial cells, stromal cells, and smooth muscle cells can prevent virion release and therefore infection spread. Delayed release compositions, consisting of enteric coated tablets or capsules, are formulated to target oligomer release in the lower GI tract, such as the colon (Lee et al. 2020). Enteric coatings include pH responsive polymers, such as methacrylic resins, polyvinyl acetate phthalate, hydroxypropyl methylcellulose derivatives and polysaccharides. Conventional formulations for targeting the gastrointestinal tract are well known (Hua 2020). Conceived oral formulations for local and systemic delivery of nucleic acid are described by (Kumari et al. 2023).

[0194] In some embodiments, ppi 50 or gp350 ASOs are conjugated with endosome membrane disrupting cell penetrating peptides (CPP), such as octa-arginines or nona-arginines. In some embodiments, the cell penetrating peptides fused to ASOs are conjugated to cell targeting moieties.ENGINEERED BACTERIA

[0195] Gram-negative bacteria can be engineered to overexpress ppi 50 nad / or gp350 ASOs pre- shRNA and secrete the shRNA in bacterial outer membrane vesicles, enter gut barrier cells, or systemic circulation function to reduce ppi 50 or gp350 expression in HCMV and / or EBV infected cells of the gastrointestinal tract by RNAi (Carvalho et al. 2019). In one embodiment, bacterial strains from the genus Akkermansia are engineered to over express ASOs that target ppi 50 or gp350 mRNA. Gram-negative bacteria can be engineered to over-express shRNA sequence by fusing shRNA with a tRNAlys scaffold increasing miRNA concentration in outer membrane vesicles (0MV)(Cui et al. 2022).

[0196] In one embodiment, a bacterial vector construct contains one or more shRNAs encoding the antisense oligonucleotides of SEQ ID NOs 1-29 fused to a tRNAlys scaffold. In a preferredembodiment, an Akkermansia species is transformed with a vector comprising a shRNA encoding an ASO with SEQ ID NOs: 1-29 fused to a tRNAlys scaffold, selected for transformation, expanded to high density, and lyophilized. In one embodiment, the freeze-dried lyophilized transformed bacteria are encapsulated in an enteric coated delayed release capsule and administered orally. Stabilizers are used during the freeze-drying process to increase bacteria survival, media containing 6% skim milk, 8% trehalose and 4% sodium ascorbate. In some embodiments, the gram-negative engineered bacteria are tested for survival by periodically testing stools for the engineered bacteria. In some embodiments, it is necessary to administer the engineered bacteria composition to subjects periodically to sustain constitutive release of ppi 50 or gp350 ASO in OMVs.PROOF OF PRINCIPLE

[0197] Example 1 discloses exemplary ASOs that target HCMV ppi 50 (Table 2) and gp350 (Table 3) and describes methods to measure the inhibition level of ppi 50 and gp350 protein expression. Example 2 describes methods for showing that the proteins ppi 50 and gp350 can induce amyloidogenesis, while Example 3 demonstrates that ASO targeting ppi 50 and gp350 mRNA can prevent amyloidogenesis. Example 4 will demonstrate the effects of ppi 50 ASO in HCMV infected cell culture, wherein transfected ASOs targeting ppi 50 are expected to reduce Ap in uninfected cells. Example 5 describes experiments designed to demonstrate that inhibiting ppi 50 expression by ASO targeting ppi 50 prevents the formation, sequestration, and release of multivesicular bodies, such as exosomes, in uninfected cells. Blocking uninfected cells from releasing exosomes would evolve increase infection success by preventing cells from communicating the invasion. Example 6 describes methods for demonstrating efficacy of the exemplary ASOs targeting ppi 50 in a humanized mouse model infected with HCMV.

[0198] Antisense oligonucleotides as defined herein encompass all types of RNA antisense therapy molecules for inhibiting ppl50 and gp350 protein expression. The ASOs in Table 2 and 3 are exemplary and other ASO sequences targeting ppi 50 and gp350 may also decrease expression to produce a similar result. Therefore, the scope of the disclosure encompasses using antisense RNA therapy with any complementary sequence 5’ to the CDS, the CDS, or 3’ of CDS of ppi 50 or gp350 to decrease expression of HCMV ppi 50 or EBV gp350 to inhibit ppi 50 or gp350 in the viral secretome. The ASO-mediated decrease of ppi 50 and gp350 expression in infected cells will decrease their secretion and delivery to uninfected cells and prevent ppl50 and gp350-mediated endocytic transport dysfunction. Decreasing endocytic transport dysfunction will increase the presentation of microbial pathogen-associated molecular patterns (PAMPs) at the cell surface to prevent or treat microbial infections or enhance vaccine efficacy.EXAMPLESExample 1. Testing ASO knock-down of pp!5O and gp35O mRNA expression

[0199] Table 2 below lists ASOs 5' to 3' with sequence complementary to the ppl50 mRNA nucleotide sequence, including the 5’UTR and translation start site. The ppi 50 coding sequence is nucleotides 39874-43020 derived from HCMV strain AD169 (FJ527563); Genbank protein accession no. ACL51112. The coding sequence begins with ATG at nucleotide no. 43020 on the sense strand from the 3’ to 5’ direction. Herpesviruses transcribe mRNA from the antisense and sense strand. The ASOs (SEQ ID NOs: 1-15) were designed to silence UL32 mRNA translation without binding off-target sequences in the host transcriptome. An ASO can be selected from any contiguous 20-25 nucleotide sequence in SEQ ID NO: 1-14. An ASO can be selected from any contiguous 20-25 nucleotide sequence in SEQ ID NO: 15, which contains the 5’ UTR and 25 nucleotides 3’ to the start codon. The ASO sequence is at least 80% identical to the 20-25 contiguous nucleotide sequence disclosed in SEQ ID NOs: 1-15. able 2. Antisense oligonucleotides designed to knockdown ppi 50 mRNA translation

[0200] . Table 3 below shows exemplary ASOs 5' to 3' with sequence complementary to the EBV gp350 mRNA nucleotide sequence, including the 5’UTR and translation start site. The gp350 coding sequence nucleotides range from 77142-79865 and are derived from wildtype EBV strain AJ507799.2; Uniprot accession no. Q777F0. The coding sequence begins with ATG at nucleotide no. 79865 on the sense strand in the 3’ to 5’ direction. The ASOs (SEQ ID NOs: 16-30) were designed to silence gp350 / BLLFl translation without binding off-target sequences in the host transcriptome. An ASO can be selected from any contiguous 20-25 nucleotide sequence in SEQ ID NO: 16-28. An ASO can be selected from any contiguous 20-25 nucleotide sequence in SEQ ID NO: 29, which contains the 5’ UTR and 25 nucleotides 3’ to the start codon. The ASO sequence is at least 80% identical to the 20-25 contiguous nucleotide sequence disclosed in SEQ ID NOs: 16-29.Table 3. Antisense oligonucleotides designed to knockdown gp350 mRNA translation

[0201] Cultured endothelial cells (HUVEC), astrocytes, or THP-1 monocytic cells (freshly isolated of immortalized cell lines) are simultaneously transfected with ppl50 mRNA, gp350 mRNA or a control RNA and the test ASO(s). To test the efficacy of select ASOs, the ppi 50 and gp350 mRNA quantity is measured 24 hours after transfection by reverse transcribing the mRNA to cDNA and then performing a (real-time) quantitative PCR assay with complementary primers. Efficacy of a select ASO is also determined by measuring ppi 50 and gp350 protein levels using Western blot or ELISA in the test ASO and control (scrambled ASO) transfected cells. Alternatively, ppi 50 mRNA and gp350 mRNA are fused with T2A-GFP. T2A is a protease susceptible linker. GFP expression is then used as a surrogate measure of ppi 50 andgp350 translation inhibition. Expression knockdown can also be tested by measure ppi 50 and gp350 mRNA using quantitative reverse transcriptase PCR. The ASO and combination thereof with the highest expression knockdown are selected for use in the following studies (Examples 2-6). Combinations of different ASOs targeting different regions of ppl50 and gp350 will also be tested for maximum expression knockdown. In addition, since many people are infected with both HCMV and EBV, cells with be cotransfected and ppi 50 and gp350 and ASOs selected that when combined work effectively to knockdown both ppi 50 and gp350 expression.

[0202] Example 2, Testing pp!50- and gp350-mediated endosomal pathway disruption by levels of APP and APP proteolytic peptide productionCultured endothelial cells (HUVEC), astrocytes, or THP-1 monocytic cells (freshly isolated of immortalized cell lines) are simultaneously transfected with ppi 50 mRNA, gp350 mRNA or a control RNA. After 48 hours, differences in the amount of cellular APP, soluble aAPP isolated from the culture medium, and amyloid-P are detected by Western blotting or ELISA. The transfection of ppi 50 and gp350 mRNA and subsequent translation is expected to change the levels of cellular APP, soluble aAPP isolated in culture medium, and amyloid-P by disrupting the endosomal pathway and thereby induce amyloidogenesis.Example 3, Testing the inhibition of endosomal pathway disruption by transfecting ASO in cell culture

[0203] Optimal ASOs or combinations selected from the Example 1 knockdown studies or a scrambled control ASO are simultaneously transfected with ppl50 or gp350 mRNA as performed in Example 1. After 48 hours, differences in the amount of cellular APP, soluble aAPP isolated from the culture medium, and amyloid-P are detected by Western blott or ELISA. The transfection of the optimal ppi 50 and gp350 ASOs, and combinations thereof, is expected to inhibit the ppi 50- and gp350-mediated changes in cellular APP, soluble aAPP, and Ap isolated from culture medium and cell lysis by preventing endosomal pathway disruption and thereby APP amyloidogenesis.Example 4, Confirmation of ppi 50 ASO knockdown in endothelial cells infected with TB40- UL32-HCMV / E.

[0204] Cultured human umbilical vein endothelial cells (HUVECS) are grown to confluence on fibronectin coated coverslips and infected with TB40-UL32-HCMV / E strain (ATCC - VR- 3348) at a multiplicity of infection (MOI) of 5 or 20, or mock infected, as performed in Bentz et al. The HCMV strain TB40-UL32-HCMV / E is engineered with enhanced GFP fused to C-terminal UL32 marking expression of the gene product ppl50 (Bentz et al. 2006; Sampaio et al. 2005) and measuring Ap production at 24-, 48-, and 72-hours post infection as described (Lurain et al. 2013a; Barbian et al. 2020), or by ELISA (ThermoFisher Scientific Catalog # KHB3491).

[0205] Alternatively, cultured human fetal astrocytes (HF A) or astroglioma (U251) cells are infected with HCMV clinical isolates from early- or late Alzheimer’s disease patients, or the HCMV AD 169, strain or mock infected, and amyloidogenesis is compared as described (Lurain et al. 2013a; Barbian et al. 2020).

[0206] The most effective ppi 50 ASOs selected from Example 1 will be transfected into HCMV infected cells and the change in GFP expression and Ap measured at 3-7 days post-ASO transfection by anti-Ap antibody staining on Western blots, anti-Ap immunohistochemistry, or ELISA (ThermoFisher Scientific Catalog # KHB3491).

[0207] The ability of ppi 50 ASOs to block infection spread in cell culture will be tested. Cultured confluent endothelial cells are infected with TB40-UL32-HCMV / E (MOI, 20) or EBV strain AJ507799.2, and simultaneously transfected with ppi 50 ASOs, or control ASOs by electroporation or an RNAi transfection agent. HCMV infectivity (particle formation and translocation) will be measured by live imaging of GFP fluorescence 3-7 days after transfection (Sampaio et al. 2005). Transfection of ppl50 ASOs are expected to reduce Ap staining in uninfected cells.Example 5, Inhibition of viral exosome release

[0208] The binding of ppi 50 to BICD-rab6b-PTPN23 is expected disrupt the biogenesis of multivesicular bodies preventing sequestration and release of viral exosomes since Rab6b is involved in targeting and docketing of secretory vesicles to the cell surface and BICD1 interacts with PTPN23 which is involved in the endosomal sorting complex (ESCRT) machinery required for exporting exosomes derived from multivesicular bodies (MVB). To demonstrate that ppl50 disrupts MVB biogenesis, the human intestinal epithelial cell line Caco-2 will be grown in culture and infected at MOI 20 with AD 169 HCMV strain (scrambled sequence) and incubated for 48 hrs (Esclatine et al. 2000). The culture supernatants from HCMV infected cells comprising viral exosomes and (mock) non-infected cells will be isolated from the culture medium as described by Cao et al., 2020 (Cao et al. 2020). The level of HCMV proteins found in viral exosomes IE1, glycoprotein B, or pp71 proteins from the HCMV infection supernatant will be measured using the methods described by Streck et al. (Streck et al. 2020). Uninfected cultured Caco-2 cells will then be exposed separately to supernatant from the HCMV infected or uninfected Caco-2 cells. The supernatant incubated cultured Caco-2 cells rinsed from the culture dish are washed by centrifugation and resuspended 24 hours after exposure to the infected andmock infected supernatant. The supernatant of the Caco-2 cells will be isolated 48 hrs after exposure to the infected and mock infected supernatants. Caco-2 cells exposed to supernatants from the HCMV infected Caco-2 cells should exhibit disrupted MVB biogenesis (exosomes), which will be evident a reduced level of host exosome proteins CD63, CD81 or TsglOl in the supernatant of the test cultures. Blocking ppl50 delivery to uninfected cells by transfecting HCMV infected cultures with ppi 50 ASOs compared to control (scrambled ASO) will establish that the secretome comprising ppi 50 from infected cell supernatant disrupts MVB biogenesis, sequestration, and release. The ppi 50 protein has a size variant and this modification could prevent MBV biogenesis disruption in infected cells.

[0209] Glucose utilization will be compared in Caco-2 cells exposed to supernatant from HCMV infected and uninfected Caco-2 cells. The HCMV secretome comprises the proteins IE1 and IE2, which was shown to reduce GLUT1 expression. The Caco-2 cells exposed to the supernatant from HCMV infected cells are expected to show reduced GLUT1 expression compared to the Caco-2 cells exposed to supernatant from mock infected cells. GLUT1 expression level will be measured by western blotExample 6, Inhibition of HCMV viral exosome release by in vivo delivery of ASOs in humanized mice

[0210] Optimally modified ASO dosages and combinations for in vivo delivery will be tested for efficacy and maximum tolerated dose in a HCMV (AD 169) infected humanized mouse model (Jackon Laboratory: CD34+ hu-NSG-FLT3) (Stein et al. 2008; Theobald et al. 2018; Koenig et al. 2020) or NOD-scid ZL2Rycnull (Smith et al. 2010). The humanized mice will be infected by intraperitoneal injection of HCMV infected fibroblasts and administered granulocyte-colony stimulating factor for 7 days by osmotic pump (see Smith et al., 2010 for methods). A composition comprising chemically modified ASO conjugated with a CD34+ Fab formulated with pharmaceutically acceptable excipients will be delivered to mice by intravenous tail injection or intraperitoneal injection at varying concentrations. Mice will be administered intravenous modified ASO composition formulated in a nanocarrier, such as a lipid nanoparticle, for either immediate release, delayed release, or a combined immediate and delayed release formulation on the day of HCMV and mock infection. Various oral formulations for local and systemic delivery of nucleic acid are described by (Kumari et al. 2023). On days 7, 14, and 28 after infection and administering the ppi 50 and control ASO compositions, the HCMV viral secretome and viral load with be assessed with known methods in the art (see Smith et al., 2010 for methods).

[0211] Examples described here are directed to confirm the approach of using ASOs targetingHCMV protein ppi 50 and reduced systemic disease from the HCMV secretome. However, examples 3-6 can also apply to confirming the efficacy of ASOs targeting gp350 and the reduction of systemic disease from the EBV secretome.

[0212] The methods described in the Examples are known by a person with ordinary skill in the art and do not require undue experimentation.

Claims

CLAIMSWhat is claimed:

1. An antisense oligonucleotide with at least 80% complementary sequence similarity to any 12 to 30 contiguous nucleotides of HCMV mRNA ppl50 for use in treating HCMV-secretome mediated microbial disease.

2. The oligonucleotide of claim 1, wherein the oligonucleotide sequence has at least 80% sequence identity to the sequences disclosed in SEQ ID NOs: 1-14.

3. The oligonucleotide of claim 1, wherein the sequence is derived from SEQ ID NO: 15.

4. The oligonucleotide of any of claims 1-3, wherein the oligonucleotide is modified or substituted at the backbone, nucleobase, or ribose sugar.

5. The oligonucleotide of claim 4, wherein the backbone modification comprises one or more Rpor Spphosphorothioate linkages or a phosphorodiamidate morpholino oligonucleotide.

6. The oligonucleotide of claim 4, wherein one or more nucleobase is modified with a 5- methylcytidine, 5-methyluridine, abasic RNA, or a combination thereof.

7. The oligonucleotide of claim 4, wherein one or more ribose is modified with a locked nucleic acid (LNA), 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-fluoroarabinonucleic acid, ethylene-bridged nucleic acid (ENA), or combination thereof.

8. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide further comprises an endosomal membrane destabilizing cell penetrating peptide (CPP).

9. The oligonucleotide of any one of claims 1-8, wherein the oligonucleotide is covalently or non-covalently conjugated to a cell targeting moiety.

10. An antisense oligonucleotide with at least 80% complementary sequence similarity to any12 to 30 contiguous nucleotides of EBV mRNA gp350, 25 nucleotides 5’ to the ATG start site, or 25 nucleotides 3’ from coding sequence for use in treating EBV-secretome mediated disease.

11. The oligonucleotide of claim 10, wherein the oligonucleotide sequence has at least 80% sequence identity to the sequences disclosed in SEQ ID NOs: 16-28.

12. The oligonucleotide of claim 11, wherein the sequence is derived from SEQ ID NO: 29.

13. The oligonucleotide of any one of claims 10-12, wherein oligonucleotide is modified or substituted at the backbone, nucleobase, or ribose sugar.

14. The oligonucleotide of claim 13, wherein the backbone modification comprises one or more Rpor Spphosphorothioate linkages or a phosphorodiamidate morpholino oligonucleotide.

15. The oligonucleotide of claim 13, wherein the nucleobase modification comprises one of more 5-methylcytidine, 5-methyluridine, abasic RNA, or a combination thereof.

16. The oligonucleotide of claim 13, wherein the ribose modification comprises locked nucleicacids (LNA), 2'-0-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-fluoroarabinonucleic acid, ethylene- bridged nucleic acid (ENA), or combination thereof.

17. The oligonucleotide of any one of claims 10-16, wherein the oligonucleotide further comprises an endosomal membrane destabilizing cell penetrating peptide (CPP).

18. The oligonucleotide of any one of claims 10-17, wherein the oligonucleotide is covalently or noncovalently conjugated to a cell targeting moiety.

19. A nanocarrier comprising one or more oligonucleotide of any one of claims 1-7 or claims 10-16.

20. The nanocarrier of claim 19, wherein the nanocarrier is a pH sensitive lipid nanoparticle.

21. The nanocarrier of claims 19 or 20, wherein the nanocarrier comprises a cell targeting moiety.

22. A vector comprising an expression construct encoding one or more oligonucleotide sequence of any one of claims 2, 3, 11 or 12.

23. The vector of claim 22, wherein the vector comprises an adeno-associated virus or lentivirus.

24. A composition comprising one or more oligonucleotides of any one of claims 1-9, 10-18, the nanocarrier of any one of claims 19-21, or the vector of claims 22 or 23.

25. The composition of claim 24, wherein the composition further comprises a pharmaceutically acceptable excipient, binder, or enteric coating.

26. A method of treating HCMV-secretome mediated disease, comprising administering to a subject in need thereof, or prophylactically, the composition of claim 25.

27. A method of treating EBV-secretome mediated disease, comprising administering to a subject in need thereof, or prophylactically, the composition of claim 25.

28. The method of any one of claims 26-27, wherein the disease is caused by a microbial infection.

29. The method of any one of claims 26-28, wherein the disease is a neurodegeneration.

30. The method of any one of claims 26-29, wherein the composition is delivered by oral administration, intravenous injection, eye drops, nasal spray, or inhalation.

31. A method of treating or preventing dementia in a subject, comprising administering the composition of claim 25.

32. The method of claim 31, wherein the composition is administered orally, eye drops, intravenously, nasal spray or by inhalation, or a combination thereof.

33. A method of treating cancer in a subject, comprising administering the composition of claim 25.

34. The method of claim 33, wherein the composition is administered locally at the cancer siteor intravenously, orally, nasal spray, inhalation, or a combination thereof.

35. A method of treating microbial infection in a subject, comprising administering the composition of 25.

36. The method of claim 35, wherein the composition is administered locally at the site of infection, intravenously, orally, eye drops, nasal spray, inhalation, or a combination thereof.

37. A gram-negative bacteria recombinantly engineered with an expression vector construct that encodes one or more the complementary sequence of the antisense oligonucleotide of claims 1-3 or 10-12 fused with a tRNAlys scaffold.

38. The gram-negative bacteria of claims 37, wherein the engineered bacterial genus is Akkermansia.

39. A composition comprising the lyophilized recombinant bacteria of claims 37 or 38.

40. A method of treating HCMV secretome mediated disorder in a subject comprising the oral administration of the composition of claim 39.

41. A method of treating EBV secretome mediated disorder in a subject comprising the oral administration of the recombinant bacteria of claim 39.

42. Use of the antisense oligonucleotides of any one of claims 1-9 or 10-12 for in the manufacture of a medicament for treating microbial mediated disease.

43. The composition of claims 25 or 35, wherein the composition further comprises an HCMV or EBV protein or peptide to stimulate an immune response to said viruses.

44. The composition of claim 25 or 25, wherein the composition further comprises an mRNA encoding an HCMV or EBV.

45. A method of vaccinating a subject against microbial infection, wherein the composition of claims 25 or 39 is administered before or concurrently with the vaccine.

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