Decoy ICAM-5 receptor proteins and treatment methods

Abstract

Expression vectors, preferably mRNA expression vectors, encoding for soluble ICAM-5 for increasing ICAM-5 levels in a subject in need thereof, as decoy receptor proteins for endogenous transmembrane ICAM-5. Therapeutic or prophylactic formulations of the same. Therapeutic or prophylactic treatment methods using the same, such that the administration of the composition results in expression, translation, and / or activity of the protein encoded by the expression vector in the subject, wherein the protein is soluble ICAM-5. Uses of mRNA expression vectors to deliver soluble ICAM-5 for both the prevention and treatment of RSV to reduce RS V disease severity and prevent death from viral infection.

Description

DECOY ICAM-5 RECEPTOR PROTEINS AND TREATMENT METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 632,129, filed April 10, 2024, entitled DECOY ICAM-5 RECEPTOR PROTEINS AND TREATMENT METHODS, incorporated by reference in its entirety herein.SEQUENCE LISTING

[0002] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "SequenceListing_60212.xml," created on April 10, 2025, as 13,424 bytes in size, the contents of which are incorporated herein.BACKGROUNDTechnical Field

[0003] The present disclosure relates to synthetic expression vectors, preferably mRNAs, encoding ICAM-5 for expression as soluble decoy receptor proteins to block viral infections, such as RSV or EV-D68 infection, as well as inhibit inflammation.Description of Related Art

[0004] Respiratory syncytial virus (RSV) infects nearly all children by the age of two. Each year in the United States, 2.1 million children see a doctor because of RSV and 58,000 kids are hospitalized as a result. Unfortunately, between 100-500 children under the age of five die each year from RSV. Preterm infants and all infants under 6 months old are at especially high risk for severe RSV disease. Elderly individuals are also at risk for severe RSV infections. The RSV F protein is critical for binding host cells and mediating infection and is the target of most candidate vaccines.

[0005] There are currently five preventative treatments for RSV approved by the Food and Drug Administration. Three vaccines currently exist, mRESVIA®, ABRYSVO™, and AREXVY®. Additionally, two monoclonal antibody products, palivizumab and nirsevimab, are approved for use in preventing RSV in infants, though the immunity is short-term. Palivizumab must be administered monthly, and nirsevimab is thought to provide 5 months of effectiveness. While there are now preventative tools for RSV, there are no targeted antivirals for use during RSV infection. Thus, developing both preventative and therapeutic tools to prevent RSV infection and severedisease are of urgent need to save lives and reduce the burden of RSV infection.

[0006] Intercellular Adhesion Molecule-5 (ICAM-5), also known as telencephalin, is a membrane-spanning adhesion molecule. Unlike the other members of the ICAM family, ICAM- 5 is specifically expressed in the central nervous system and was first purified from the telencephalic regions of a rabbit brain. Further it is believed that its expression is restricted to only certain types of neurons. As shown herein, it is discovered for the first time that it is also expressed in respiratory tissues.

[0007] It has been shown that ICAM-5 is a viral receptor for Enterovirus D68 (EV-D68), a member of the Picornaviridae family, which is a neurotropic virus that is the putative cause of Acute Flaccid Myelitis (AFM) in children. The genus Enterovirus of the family Picornaviridae contains many important pathogens for humans and animals. This genus consists of 12 species: four human enterovirus species (EV-A, B, C, and D), five animal enterovirus species, and three human rhinovirus species. EV-D68 contains a single-strand positive RNA genome and shares biological features with both enteroviruses and rhinoviruses. Unlike most enteroviruses, which are acid-resistant and multiply in the human gastrointestinal tract, EV-D68 is an acid-sensitive enterovirus that replicates in the respiratory tract, similar to rhinovirus. EV-D68-associated infections have become more common and like rhinovirus are frequently associated with respiratory illness. However, unlike rhinoviruses, EV-D68 infection has also been linked to clusters of polio-like neurological disorders, such as acute flaccid myelitis and cranial nerve dysfunction in children, implicating EV-D68 as an emerging public health-threatening neurophathogenic agent.

[0008] It has been shown that neuronal ICAM-5 can inhibit microglia adhesion in response to inflammation (LPS stimulation). Further, recent work has suggested that RSV, and indeed many other viruses, can actually infect the nervous system and impair neuronal function (even though these viruses are more frequently associated with respiratory illness).SUMMARY

[0009] The present disclosure provides, for the first time, data demonstrating that ICAM-5 is a host receptor for RSV infection.

[0010] The present disclosure is broadly concerned with an alternative protein-based antiviral to monoclonal antibodies, which counterintuitively seeks to increase soluble ICAM-5 in the treated subject. The secreted ICAM-5 acts as a decoy to compete for receptor-binding sites so that viruses, such as RSV or enterovirus, in an infected subject will bind to the soluble ICAM-5 decoy, insteadof the natural or native / endogenous ICAM-5 transmembrane bound receptor on neural cells or respiratory cells in the subject. In this manner, less virus remains available for binding with hostcell surface receptor proteins meaning less opportunities for virus to use these transmembrane proteins to infect the subject’s cells. Therefore, viral titers will remain low and the infection can be inhibited.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] Figure (FIG.) 1 A is a map of the ICAM-5 encoding synthetic mRNA constructs.

[0013] FIG. IB shows a sequence of a construct (SEQ ID NO: 1), showing the N-terminal signal peptide sequence (which is ultimately cleaved) circled and underlined, the extracellular ICAM-5 sequence, the underlined linker sequence, and the C-terminal IgGl Fc portion with the mutated serine residue underlined.

[0014] FIG. 2 is a histogram showing ICAM-5 gene expression in cells 24 hours after exposure to RSV or control (mock virus).

[0015] FIG. 3 is a histogram showing the neutralization of RSV in A549 lung cells treated with Opg / ml (RSV only), lOOpg / ml, and 200pgg / ml of ICAM-5 peptide.

[0016] FIG. 4 is a histogram showing the adherence of calcein-AM labelled neutrophils to A549 lung cells when incubated with or without sICAM-5 (200 pg / ml).

[0017] FIG. 5 is a computational model of ICAM-5 and RSV F protein interactions.

[0018] FIG. 6 is a histogram showing Human ICAM-5 gene expression by tissue type.

[0019] FIG. 7 is a histogram comparing gene expression of ICAM-5 and ICAM-1 in central nervous system tissues.

[0020] FIG. 8A shows brightfield and fluorescence imaging of SH-SY5Y cells infected with RSV with a green fluorescent protein (GFP) tag at an MOI of 1 for 24 hours.

[0021] FIG. 8B is a histogram of GFP luminescence in RSV infected and control (mock infected) SH-SY5Y cells.

[0022] FIG. 9A shows fluorescence imaging of primary human excitatory neurons infected with RSV at an MOI of 1 at 24, 48, and 72 hours following infection.

[0023] FIG. 9B shows the frequency, above background, of primary human excitatory neuron cells with detectable RSV at 24, 48, and 72 hours following infection with RSV or control.

[0024] FIG. 10 is a heatmap showing the concentration (pg / ml) of proinflammatory cytokines CCL2, CCL4, CCL3, IL-2R, TNFa, IFNa, IL-6, 1-8, CCL5, CXCL10, and CXCL9 in primary human excitatory neuron cells (neurons) infected with control, RSV at MOI 1 , and / or RSV at MOI 0.1, and in SH-SY5Y and A549 cells infected with control or RSV.

[0025] FIG. 11 is a histogram showing ICAM-5 concentration in neuronal cell line SH-SY5Y infected with RSV and in primary human excitatory neurons (neuron) infected RSV at an MOI of 1 or 0.1 where indicated compared to control (mock infection) for 72 hours.

[0026] FIG. 12 is a histogram showing the neutralization of RSV in SH-SY5Y cells treated with Opg / ml (RSV only), lOOpg / ml, and 200pg / ml of ICAM-5 peptide.DETAILED DESCRIPTION

[0027] In one or more embodiments, the disclosure concerns a therapeutic agent that inhibits RSV or EV-D68 infection in a subject in need thereof by acting as a decoy receptor protein for RSV orEV-D68 in the subject to inhibit viral activity in the subject. Preferably, wherein the subject has or is at risk of a viral infection, or at risk of severe morbidity due to viral infection, preferably RSV or EV-D68 infection. The therapeutic agent comprises ICAM-5 and specifically binds to the virus in the subject and thereby blocking ICAM-5 receptor-binding sites on the virus in the subject, thus inhibiting the ability of the virus to bind to the natural or native / endogenous ICAM-5 transmembrane bound receptor on neural cells or respiratory cells in the subject (which are normally used by the virus to gain entry into and infect these cells). Thus, the therapeutic agent seeks to increase the amount of soluble ICAM-5 circulating in the subject to inhibit viral activity through this binding / blocking mechanism.

[0028] In one or more embodiments, the disclosure concerns synthetic messenger ribonucleic acid (mRNA) encoding an ICAM-5 polypeptide configured for translation in a cell of a subject to yield a functional ICAM-5 polypeptide that acts as a decoy receptor protein for RSV. In one or more embodiments, the mRNA encodes for an ICAM-5 polypeptide comprising a disclosed sequence (e.g., SEQ ID NOB), or a sequence having at least 95%, preferably at least 98% sequence identity with a disclosed sequence and retaining binding ability with the virus. For example, another reported ICAM-5 amino acid sequence is GenBank: AAH26338.1, which has 99% sequence identity to SEQ ID NOB (NCBI Accession No.: NP_003250).

[0029] When the mRNA is delivered to a cell, the mRNA will be processed into a polypeptide by the endogenous intracellular machinery of the subject. Preferably, the mRNA encodes soluble ICAM-5 (sICAM-5), meaning that the translated ICAM-5 polypeptide is secreted and nottransmembrane bound. In particular embodiments, the mRNA is derived from cDNA (e.g., SEQ ID NO: 7). In one or more embodiments, the mRNA may include one or more modified nucleosides, such as such as where some or all of the uridines in the mRNA sequence are replaced during mRNA synthesis by a uridine analog, e.g., pseudouridine / N(l)-methylpseudouridine or 5- methoxyuridine. In particular embodiments, the mRNA may be codon-optimized using standard protocols.

[0030] Any suitable mRNA expression vector may be used, including those more recently developed to deliver the COVID-19 vaccines, as well as any NextGen mRNA expression vectors capable of delivering the coding sequences to the subject and inducing in vivo translation of the recombinant ICAM-5 polypeptide (preferably sICAM-5).

[0031] In one or more embodiments, the mRNA expression vectors may be complexed or encapsulated for delivery. In one or more embodiments, lipid nanoparticles (LNPs) can be used as the delivery vehicle. In one or more embodiments, the mRNA composition is formulated in an LNP. In one or more embodiments, a lipid nanoparticle comprises the mRNA, as well as lipids including an ionizable lipid (such as an ionizable cationic lipid), a structural lipid, and / or a phospholipid. LNP formulations are commercially available, and are prepared by self-assembly mixing of the mRNA in aqueous solution rapidly with the lipids and ethanol, which contain an ionizable cationic lipid / phosphatidylcholine / cholesterol / PEG-lipid.

[0032] In one or more embodiments, the mRNA comprises a translatable region encoding the recombinant ICAM-5 under conditions such that the nucleic acid is localized into a cell of the subject after administration, and the recombinant polypeptide is capable of being translated in the cell from the nucleic acid, and more preferably secreted from the cell as functional, soluble ICAM- 5. In one or more embodiments, the mRNA construct comprises a signal peptide coding sequence that encodes for a signal peptide (e.g., SEQ ID NO: 4) that directs the ICAM-5 polypeptide to be secreted by the cell. The signal peptide sequences are ultimately cleaved from the resultant expressed and secreted functional polypeptide. Signal peptides can be optimized to improve the secretion efficiency of and synthesized as artificial constructs, or can be derived from common existing eukaryotic signal sequences, such as Secrecon, Human IgKVII, CD33, human serum albumin, and the like. In one or more embodiments, the mRNA construct comprises a coding sequence for a signal peptide as disclosed herein. In one or more embodiments, the mRNA construct comprises a coding sequence for a 23-bp signal peptide from tissue plasminogen activator (tPA) (SEQ ID NO:4).

[0033] In one or more embodiments, the ICAM-5 coding sequence used in the mRNA constructis modified to remove the native transmembrane domain from the ICAM-5 coding sequence. The protein is preferably translated and then secreted as a soluble form, which means that it does not adopt its normal endogenous transmembrane localization, but instead is secreted and released from the transfected cells as soluble ICAM-5. In one or more embodiments, the mRNA is translated in a host cell, processed, and released, wherein a precursor form of the polypeptide contains a signal peptide that is first cleaved for the functional protein to be secreted and released from the transfected cell.

[0034] In one or more embodiments, the mRNA expression vector further comprises a coding region for an Fc (fragment crystallization) domain, preferably an IgG Fc domain, and preferably conjugated to the ICAM-5 coding sequence via a short peptide linker (e.g., 3-8 aa residues). In one or more embodiments, the Fc domain can be a native sequence, or it can be a modified Fc sequence. Fc fragments are commonly used and investigated for use with monoclonal antibody therapies. In one or more embodiments, the mRNA expression vector comprises an Fc fragment of the full native sequence, such as residues P100 to K330 of the native Fc sequence. A variety of strategies are known for optimizing physicochemical properties and functions mediated by the Fc fragment through targeted mutations or chemical modifications of the Fc fragment used in the fusion. Any of these optimizations can be applied to Fc domains used in the described mRNA constructs. In one or more embodiments, the mRNA expression vector comprises a coding sequences for an Fc domain (SEQ ID NO:6) comprising a sequence disclosed herein or a sequence having at least 95% sequence identity, preferably at least 98% sequence identity, and retaining Fc functions thereof. In one or more embodiments, the mRNA expression vector comprises a coding region for the linker (SEQ ID NO:5). In one or more embodiments, the peptide linker comprises a sequence (SEQ ID NO: 5) disclosed herein. Thus, in such embodiments, the ICAM-5 polypeptide is translated as an ICAM-5-Fc fusion polypeptide (SEQ ID NO:1, SEQ ID NO:2). Such fusion polypeptides can be characterized as having improved fusion protein stability and half-life could be enhanced through the stability of the Fc region (SEQ ID NO: 6) and the Fc receptor interactions in the immune system. Moreover, the extended half-life may reduce drug dosage and administration frequency, also minimizing potential side effects or toxicity.

[0035] In one or more embodiments, a particularly preferred ICAM-5 fusion protein comprises a sequence disclosed herein (SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3), or a sequence having at least 95% sequence identity, preferably at least 98% sequence identity, and retaining the ability to act as a decoy for binding with the virus.

[0036] In the embodiments, after administration, the approach increases the level of circulatingICAM-5 in the treated subject, and this soluble, circulating ICAM-5 will be more readily available to interact with and bind to the virus upon infection and block its ability to bind to the endogenous, transmembrane bound ICAM-5 in the subject. In this manner, the soluble ICAM-5 interferes with the ability of the virus to infect the neural cell, reducing viral spread as well as associated symptoms of infection, including disruptions in neuronal function and other neurologic sequelae.

[0037] In one or more embodiments, the mRNA comprises an ICAM-5 polypeptide coding sequence that is modified to increase affinity binding and interaction with RSV F protein. In one or more embodiments, the modifications yield a translated ICAM-5 polypeptide having one or more mutated amino acid residues for increased affinity binding. In one or more embodiments, the mRNA expression vector is modified to increase translation levels of the ICAM-5 polypeptide, including via increased solubility and secretion of the translated ICAM-5 polypeptide rather than bound to the cellular membrane. For example, the coding sequence for ICAM-5 can be modified to remove the transmembrane domain and / or the coding sequence can include a signal peptide for increased secretion. For example, the mRNA expression vector (SEQ ID NO:7) is modified to include a coding region for an IgG-FC domain (SEQ ID NO:6), wherein said ICAM-5 polypeptide (SEQ ID NO:3) is translated as an ICAM-5-Fc fusion polypeptide (SEQ ID NO:1 or SEQ ID NO:2).

[0038] In one or more embodiments, provided herein are therapeutic or prophylactic formulations comprising the therapeutic agent comprising soluble ICAM-5 or encoding ICAM-5, preferably, soluble ICAM-5 in an amount effective to bind and block viral infection normally mediated by endogenous membrane bound ICAM-5 on host neural or respiratory cells. In one or more embodiments, provided herein are therapeutic or prophylactic formulations comprising synthetic mRNA encoding ICAM-5, preferably, soluble ICAM-5 in an amount effective to bind and block viral infection normally mediated by endogenous membrane bound ICAM-5 on host neural or respiratory cells. Moreover, it will be appreciated that since ICAM-5 can act as a decoy protein, once bound, the virus will likewise be unable to interact with and infect any other endogenous transmembrane proteins, such as ICAM-1 expressed on epithelial cells. The formulations comprise the mRNA formulated in a pharmaceutically-acceptable carrier, which includes any suitable diluents, excipients, vehicles, and the like, in which the mRNA may be dispersed for administration. Suitable carriers will be pharmaceutically acceptable. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any ofthe other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would naturally be selected to minimize any degradation of the mRNA or other agents and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration. For example, carriers suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer. Exemplary carriers include aqueous solutions such as normal (n.) saline (-0.9% NaCl), phosphate buffered saline (PBS), sterile water / distilled autoclaved water (DAW), various oil-in- water or water-in-oil emulsions, as well as dimethyl sulfoxide (DMSO) or other acceptable vehicles, and the like.

[0039] In one or more embodiments, the therapeutic or prophylactic formulations are configured for delivery to nasal and respiratory mucosa (i.e., the point of entry for RSV and other viruses). In one or more embodiments, various strategies for enhancing delivery of the mRNA to the central nervous system can be employed. In fact, neural pathway between the nasal mucosa and the brain provide a unique pathway for noninvasive delivery of therapeutic agents to the CNS. Developing formulations with a lower molecular weight and higher lipophilicity are favorable characteristics for rapid intranasal uptake of small molecules, such as nucleic acids, into the CNS. In one or more embodiments, the mRNA can be directly delivered to the CNS by lumbar puncture or intraventricular injection in a suitable formulation. This mode of delivery is particularly suited for getting sICAM-5 into the cerebral spinal fluid, especially when systemic immunization does not optimally target sICAM-5 to the CNS. Potentially, the composition of the lipid nanoparticle delivery vehicle could also be altered to target the CNS (e.g., coating with ionic liquid).

[0040] In some embodiments, the therapeutic or prophylactic formulations can be provided in unit dosage form in a suitable container. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of the mRNA (and / or other active agents) in the carrier calculated to produce the desired effect. In other embodiments, mRNA can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject. A kit comprising the therapeutic or prophylactic formulations is also disclosed herein. The kit further comprises instructions for administering the therapeutic or prophylactic formulations to a subject. The mRNA can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable carrier, or it can be provided separately from the carrier. The kit can further comprise instructions for preparing the mRNA or the therapeutic orprophylactic formulations for administration to a subject. Other ingredients may be included in the formulations, such as adjuvants, other active agents, preservatives, buffering agents, salts, other pharmaceutically-acceptable ingredients.

[0041] In one or more embodiments, the therapeutic or prophylactic formulations are configured for intranasal delivery / administration. In one or more embodiments, the therapeutic or prophylactic formulations are configured as a liquid, powder, or gel, a unit dosage form of which can be delivered to the subject via swab, spray, atomizer, nebulizer, aerosol, rinse, lavage, and the like. In addition to being administered at the site of infection (respiratory mucosa and / or CNS), these delivery modalities eliminate the use of needles for drug delivery. However, it is contemplated that other routes of administration may be used, including oral delivery or even intramuscular or intraperitoneal. In one or more embodiments, the therapeutic or prophylactic formulations are configured for intramuscular or subcutaneous administration. In one or more embodiments, the therapeutic or prophylactic formulations are configured as a liquid, powder (for reconstitution), or other suitable form for injection. In one or more embodiments, the therapeutic or prophylactic formulations are formulated in lipid nanoparticles, such as for intravenous, intramuscular, intraperitoneal, or subcutaneous administration. A variety of lipid nanoparticle formulations for formulating and encapsulating polynucleotides, including mRNA, are available and being developed. Such lipid nanoparticles generally comprise, for example, the therapeutic or prophylactic mRNA, which is encapsulated in a capsule / membrane or encased in a solid matrix composed of a variety of one or more types of lipids, such as cationic lipids, ionizable lipids, helper lipids, phospholipids, cholesterols, polymers (PEG), peptides, glycerol, and the like. Nonlimiting examples of recent developments in mRNA lipid nanoparticular formulations include those described in U.S. Patent No. 11,524,023, incorporated by reference herein. Other suitable delivery vehicles include polymeric nanoparticles, micelles, liposomes, and the like.

[0042] Embodiments described herein contemplate methods of treating or inhibiting viral infection in a subject in need thereof, such as from a respiratory virus, enterovirus, or other viral infection mediated by ICAM-5 transmembrane protein. Embodiments described herein contemplate methods of increasing levels of ICAM-5 (preferably soluble, circulating ICAM-5) in a subject in need thereof. Embodiments described herein contemplate methods of blocking interaction of RSV in an infected host with the host’s (endogenous) transmembrane proteins, e.g., such as ICAM-5 in neural cells and / or ICAM-1 in epithelial cells.

[0043] The methods comprise administering a therapeutically effective amount of mRNA encoding for ICAM-5 to a subject in need thereof. As used herein, a “therapeutically effective”amount refers to the amount of mRNA that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect as against the viral infection by preventing and / or inhibiting and / or blocking interaction and binding of virus to endogenous ICAM-5 of the subject. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the condition is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject. The methods can be used for treatment or prophylaxis of viral infection. The terms “therapeutic” or “treatment,” as used herein, refer to processes that are intended to produce a beneficial change in an existing condition (e.g., RSV infection) of a subject, such as by reducing the severity of the clinical symptoms and / or effects of the infection, and / or reducing the duration of the infection / symptoms / effects and / or reducing the viral titer in the subject. Thus, in some embodiments, the subject is afflicted with or suffering from a respiratory viral infection (e.g., RSV) before the mRNA is administered, wherein methods described herein are useful for treating the condition and / or ameliorating the effects of the condition. Prophylaxis instead refers to methods that are intended to inhibit or ameliorate the effects of a future viral infection or condition to which a subject may be exposed (but is not currently infected with). Thus, in some embodiments, the subject is free of a given condition (or observable signs of morbidity) before administering the mRNA, wherein the methods described herein are useful for inhibiting the occurrence or incidence of the condition and / or inhibiting the effects of the condition, as described here. In some cases, prophylactic administration of the mRNA may inhibit the development of observable morbidity from viral infection (i.e., near 100% prevention) if / when the subject is ultimately exposed to the virus. In other cases, prophylactic administration of the mRNA may only partially lessen the extent of morbidity due to the viral infection (i.e., reduce the severity of the symptoms and / or effects of the infection, and / or reduce the duration of the infection / symptoms / effects). In either case, method is still considered a prophylaxis to target infection or disease mediated by ICAM-5.

[0044] Advantageously, the therapeutic or prophylactic formulations can be administered via a suitable route of administration as a prophylactic before infection (such as during RSV season or as part of a well-visit, or other panel of vaccinations), or as an antiviral treatment against severe disease after the initial infection and even at the time of diagnosis (such as in an emergency room or urgent case). In either case, administration of the therapeutic or prophylactic formulation inhibits viral infection in the subject, such as by reducing viral shedding, reducing viral titers or load in epithelial tissue, reducing damage to lung tissue, reducing symptoms of viral infection,reducing inflammation, and / or by reducing the severity or duration of the symptoms. Again, by artificially increasing the level of soluble, circulating ICAM-5 in the treated subject, virus in the subject’s body will more readily interact with and bind to the circulating sICAM-5 reducing the virus that remains free to interact with the subject’s endogenous transmembrane proteins, such as ICAM-1 or ICAM-5 to infect neural cells or respiratory epithelial cells. Since ICAM-5 is also a native protein found in the body, it can be repeatedly dosed with minimal risk of immune response to the formulation itself, which is often seen with monoclonal antibody therapy and RSV protein vaccines because they are recognized as foreign by the immune system.

[0045] Further, the use of mRNA to deliver soluble protein, particularly via the intranasal route, is complimentary to other vaccine approaches under development. While traditional vaccines need to be delivered months prior to RSV exposure (to ensure development of the immune response), ICAM-5 antiviral can be used to treat an active infection by inhibiting infection and keeping viral titers low, thus giving the subject’s own immune system time to mount an immune response and preventing the subject’s immune system from becoming overwhelmed by high viral load. Since the ICAM-5 antiviral acts directly on the virus itself, the mechanism of action would not interfere with any vaccine elicited immune response. This makes the mRNA formulations particularly attractive in populations that have poor vaccine immune responses or those who are unable to get vaccinated (i.e., immunocompromised, very young children).

[0046] Advantageously, ICAM-5 also is a receptor for certain enteroviruses (EV-D68), so this strategy can also be used to prevent or treat viral infection beyond RSV (essentially for any virus whose infection is mediated by transmembrane ICAM-5). Thus, while the present description is provided above with particular focus on RSV, it will be appreciated that it can be applied to develop therapeutics or prophylactics against any number of viruses where infection is mediated by ICAM-5.

[0047] The data herein also unexpectedly demonstrates that the soluble ICAM-5 can also block recruitment of proinflammatory immune cells. That is, data shows that endogenous upregulation of transmembrane ICAM-5 can recruit immune cells that are the actual drivers of neurologic sequelae. Thus, the mRNA delivered ICAM-5 may also be effective to inhibit inflammation in the CNS, as well as to treat tissue damage from a broad spectrum of infections or other inflammatory conditions, aside from its antiviral properties. Accordingly, in one or more embodiments, methods described herein concern treating, inhibiting, or ameliorating inflammatory conditions of the CNS. Embodiments described herein concern methods for reducing inflammation of the CNS of a subject in need thereof. Embodiments described herein concern method of treating, inhibiting, orameliorating damage to neural tissue due to inflammation of the CNS. Methods comprise administering a therapeutically effective amount of mRNA encoding for ICAM-5 to a subject in need thereof. The mRNA can be administered before, during, or after a subject experiences symptoms of neural inflammation or distress (e.g., cognitive, sensory, or motor deficits that may also manifest as emotional instability and seizures in the most severe cases). The methods can be carried out using similar modes of administration as described above for viral infection.

[0048] Finally, the high-throughput screening assay developed as part of this work can be used to identify other candidate inhibitors for blocking RSV infection in future iterations of the mRNA therapeutic.

[0049] Although described and exemplified herein using mRNA expression vectors, it will be appreciated that aspects of the invention can be carried out using other suitable protein expression vectors for delivering the recombinant protein. For example, the techniques can be adapted for expression of soluble ICAM-5 using a viral (e.g., Adenoviral) expression vector, a DNA expression vector, a bacterial expression vector (e.g., E. coli), baculovirus, insect cells, yeast, and mammalian cells (e.g., CHO or HEK293). Preferred expression vectors are suitable for administration to the subject for in vivo expression of the soluble ICAM-5. However, other expression vectors can be used to express soluble ICAM-5 for subsequent recovery and isolation of a purified protein that can be used or administered therapeutically.

[0050] Additional advantages of the various embodiments described herein will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present disclosure encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0051] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0052] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the disclosure. It should be understood that when numerical ranges areprovided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).EXAMPLES

[0053] The following examples set forth methods in accordance with the disclosure. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.EXAMPLE 1Treatment and Prophylaxis of RSV and Inflammation by Soluble ICAM-5

[0054] In this work, we show that ICAM-5 is a host cell receptor for RSV and investigate the use of soluble ICAM-5 as a decoy receptor protein as a potential therapeutic candidate. As shown below, this work demonstrates that soluble ICAM-5 can bind the RSV F protein, and further that soluble ICAM-5 can block RSV infection. Surprisingly, we also identified a role for ICAM-5 in inflammation and have shown that soluble ICAM-5 can reduce neutrophil recruitment.

[0055] In this work, we have also developed engineered forms of ICAM-5 for mRNA construction. mRNA constructs were synthesized by GenScript Biotech (New Jersey) using provided sequence information (NCBI Accession No.: NP_003250). Each mRNA construct (SEQ ID NO:7) is synthesized with a Cap added at the 5’ end using a standard Capl process and each has a poly A tail of lOOnt (not shown). The uridines are also modified with nucleobase Nl- methylpseudouridine during mRNA synthesis. The signal peptide sequences (SEQ ID NO:4) are cleaved off of the resulting functional protein, but may be important to the expression vector for secretion efficiency and are shown below. The coding sequences and resultant expressed polypeptide sequences are shown in SEQ ID NO: 1 and SEQ ID NO:7 (again noting that the signal peptide is likely not part of the final functional protein expressed by each construct).

[0056] We have also used structure-function studies to generate constructs having targeted mutations to increase affinity of ICAM-5 for RSV F protein. Unlike the direct use of soluble protein, the mRNA constructs expressing soluble ICAM-5 will have a more durable effect. We are evaluating this therapeutic approach in preclinical models as a prophylactic or treatment after infection.

[0057] In this study, we demonstrated that the gene that encodes ICAM-5 is upregulated after RSV infection.

[0058] RNA-seq performed 24 hours after RSV infection or mock infection control. Number of reads aligned to the ICAM-5 gene reported, see FIG. 2.

[0059] We have also confirmed that ICAM-5 can successfully neutralize RSV.

[0060] RSV-GFP was mixed with sICAM-5 or PBS (RSV only control) for 1 hour then added to A549 lung epithelial cells. 24 hours after infection GFP was quantified to determine percent neutralization relative to control. *** P<0.001, * P<0.05, Unpaired t test. Data shown in FIG. 3

[0061] We have also conducted studies to demonstrate that sICAM-5 blocks neutrophil adhesion to lung epithelial A549 cells.

[0062] Differentiated neutrophil-like cells (HL-60) were labeled with a dye (calcein AM) and incubated with or without sICAM-5 (200 pg / ml) for 30 minutes. Then neutrophil-like cells are added to A549 cells for 30 minutes to adhere. Nonadherent neutrophils are washed off and calcein fluorescence is used to determine the level of neutrophil adherence. *** P<0.001, Unpaired t test. Data shown in FIG. 4.

[0063] ICAM-5 mRNA-LNP may provide a novel defensive strategy to reduce RSV infection or disease severity and augment future RSV vaccines that become approved. Engineered ICAM-5 to be delivered to cells using suitable expression vectors, such as using mRNA-lipid nanoparticles, so that ICAM-5 will be produced in the airway and CNS as a soluble decoy receptor that can bind the RSV virus and thereby block its binding to ICAM-5 transmembrane protein on cells.

[0064] Computational modeling of ICAM-5 and RSV F protein interactions to predict interaction sites, see FIG. 5. H-DOCK protein -protein docking based on a hybrid algorithm of template-based modeling and ab-initio free docking. Blue is ICAM-5 (PDB:4OI9); red is RSV F protein (PDB:5C6B). The docking score was -264.26 and the confidence score was 0.9076. When the confidence score is higher than 0.7, the two molecules are most likely to binding. Pink shaded area are the predicted interacting amino acids.

[0065] Discovery Studio software was used to predict the effect of positional mutations within ICAM-5 protein on the entropy of the interaction with RSV F protein. Mutations that decrease any entropy measures are predicted to stabilize the interaction and thus increase affinity of the interaction between ICAM-5 and RSV-F protein. The major column used for classification is the mutational energy column. Any values lower than -0.5 are considered stabilizing (the lower the better). The other columns are measures of Van Der Walls, Electrostatic interactions and Entropy calculation interactions. The lower the more stabilizing for them as well, but these were not usedfor classification as stabilizing. In the tables below, the results from the structure-function analysis are provided:Table 1 : Details of hydrogen bondsTable 2: Details of hydrophobic interaction

[0066] Studies were also carried out to characterize Human ICAM-5 gene expression by tissue type, from which it can be see that ICAM-5 is highly expressed in brain and lung tissue, see FIG. 6.

[0067] Adult RNA-seq tissue data generated by the Genotype-Tissue Expression project (GTEx) reported as normalized protein-coding transcripts per million (nTPM) corresponding to median values of the different individual samples from each tissue. ICAM-5 is highly expressed in both lung and brain tissues. Thus, sICAM-5 could be used to treat a wide range of conditions mediated by endogenous ICAM-5 in these tissues.

[0068] As noted, ICAM-5 is reported as being concentrated in specific subcategories of CNS cells. Studies were carried out to characterize gene expression of ICAM-5 and ICAM-1 in central nervous system tissues, see FIG. 7.

[0069] Adult RNA-seq CNS tissue data generated by the Genotype-Tissue Expression project (GTEx) reported as normalized protein-coding transcripts per million (nTPM) corresponding to median values of the different individual samples from each tissue. ICAM-5 is highly expressedin specific CNS tissues, whereas ICAM-1 is detected at only low levels in all CNS tissues.EXAMPLE 2In vivo studies

[0070] In the first part of the study, ICAM-5 mRNA-LNP are delivered via various routes (intranasal, intramuscular, intravenous, subcutaneous) along with controls to mice as various dosages (e.g., Img / kg bodyweight or 0.04 mg / kg bodyweight) to determine the optimal route and dosage / deli very schedule.

[0071] In the second part of the study, the prophylactic effect of ICAM-5 mRNA-LNP on RSV infection is tested. The mice are grouped as follows:

[0072] On day 0, the mice are administered the ICAM-5 mRNA-LNP treatment or control (PBS). On day 1, the mice are inoculated with RSV or mock RSV via intranasal swab. On day 3, the nasal wash to done on the mice. The mice are weighed daily. At the end of the study, lung, brain, and nasal tissues are analyzed for RSV viral loads, pathology, and immune cell characterization.

[0073] Mice receiving the ICAM-5 mRNA-LNP exhibit improved body weight, reduced RSV viral loads, and reduced lung and brain pathology scores.

[0074] In the third part of the study, the therapeutic effect of ICAM-5 mRNA-LNP on RSV infection is tested. The mice are grouped as follows:

[0075] On day 0, the mice are inoculated with RSV or mock RSV via intranasal swab. On day1, the mice are administered the ICAM-5 mRNA-LNP treatment or control (PBS). On day 3, the nasal wash to done on the mice. The mice are weighed daily. At the end of the study, lung, brain, and nasal tissues are analyzed for RSV viral loads, pathology, and immune cell characterization.

[0076] Mice receiving the ICAM-5 mRNA-LNP exhibit improved body weight, reduced RSV viral loads, and reduced brain and lung pathology scores.EXAMPLE 3RSV infection of neuronal cellsSH-SY5Y

[0077] An RSV Al strain was GFP labelled by adding GFP to the viral genome (RSV Al GFP). Then the RSV Al GFP was added at a multiplicity of infection (MOI) of 1 to a tissue culture plate containing human neuronal cells (SH-SY5Y; ATCC). As a control, PBS was added as a mock infection to the control well. After 24 hours of infection, luminescence was measured by a platereading luminometer to measure GFP levels, see FIG. 8B. Fluorescent microscopy was also used to visualize GFP expression in the cells (20X magnification), see FIG 8A.Primary human excitatory neurons

[0078] Primary human excitatory neurons developed from induced pluripotent stem cells from a healthy donor were purchased. These cells were cultured for 3 days to form mature neuron projections.

[0079] The cells were then infected with RSV Al GFP at MOI=1 and were cultured for 24, 48, and 72 hours. At each timepoint the cells were fixed with Paraformaldehyde solution and immunofluorescence was performed. The cells were stained with DAPI to label cell nuclei (Thermo, DI 306), Anti -RSV antibody (Thermo, PA 1-7240) and Anti-beta-3 -tubulin (Thermo (MAl-118), see FIG 9A. Anti-goat or Anti -mouse secondary antibodies with AF594 (Thermo, A- 11058) and AF647 (Thermo, A-31571) fluorphores, respectively, were used to for immunofluorescence. The cells were imaged with W1 Spinning Disk Confocal Microscope. The frequency of cells with detectable RSV (above background) at each timepoint was quantified, data shown in FIG. 9B. 48 hours post infection showed the greatest frequency of cells with detectable RSV.EXAMPLE 4RSV infection induces innate immune inflammatory cytokine production in neurons and lung epithelia cells

[0080] Neuronal cell line (SH-SY5Y), and respiratory epithelial cell line (A549, ATCC) were infected with RSV at an MOI of 1. Primary human excitatory neurons were infected with RSV at an MOI of 1 and 0.1. Cell culture supernatant from the infected cells was collected after 24 hours of infection. 50pl of supernatant was added to 50pl of Assay Diluent from the Cytokine 25-Plex Human Panel (Thermo, LHC0009M) and the assay protocol was followed. The assay was measured on a Luminex 200 instrument and standard curves were used to determine analyte concentrations. The concentration (pg / ml) of proinflammatory cytokines CCL2, CCL4, CCL3, IL- 2R, TNFa, IFNa, IL-6, 1-8, CCL5, CXCL10, and CXCL9 was determined, results are shown in FIG. 10.EXAMPLE 5RSV infection increases levels of soluble ICAM-5 (sICAM-5) in neurons

[0081] Neuronal cell line (SH-SY5Y) was infected with RSV at an MOI of 1. Primary human excitatory neurons were infected with RSV at an MOI of 1 and 0.1. An ELISA was used to determine soluble levels of ICAM-5 in the cell culture supernatant (Abeam) 72 hours after infection. Optical density determined and assay standards were utilized to interpolate concentration. The results are shown in FIG. 11.

[0082] Both SH-SY5Y and primary human excitatory neurons show increased levels of sICAM- 5 after being infected with RSV at an MOI of 1 .EXAMPLE 6Soluble ICAM-5 blocks RSV infection of neuronal cells

[0083] RSV A2 GFP virus was used at an MOI of 1 to infect SH-SY5Y cells. In the control (RSV only) the virus was incubated with PBS. In the ICAM-5 conditions, the virus was incubated with 100 and 200 pg / ml of ICAM-5 for 1 hour, then mixture added to SH-SY5Y cells and incubated for 24 hours. GFP levels were measured using a fluorescent plate reader.

[0084] The results, see FIG. 12, show that incubation with ICAM-5 at both concentrations significantly ( / K0.0001 ) neutralized RSV infection.

Claims

CLAIMS:

1. An expression vector, preferably an mRNA expression vector, encoding for soluble ICAM- 5 for increasing ICAM-5 levels in a subject in need thereof, wherein said soluble ICAM-5 is translated in vivo from said mRNA and is a decoy receptor protein for transmembrane ICAM-5.

2. The expression vector of claim 1, wherein said soluble ICAM-5 does not inhibit endogenous expression of ICAM-5.

3. The expression vector of claim 1, wherein said soluble ICAM-5 does not block the RSV- F binding site on endogenous transmembrane ICAM-5.

4. The expression vector of claim 1, wherein said soluble ICAM-5 does not interfere or interact with endogenous transmembrane ICAM-5.

5. The expression vector of claim 1, wherein said soluble ICAM-5 is not an anti-ICAM-5 antibody.

6. The expression vector of claim 1, wherein said soluble ICAM-5 is configured for binding with EV-D68 or RSV F protein.

7. The expression vector of claim 6, wherein said soluble ICAM-5 binding blocks the ability of the RSV F protein or EV-D68 to bind with said endogenous transmembrane ICAM-5.

8. The expression vector of claim 1, wherein said soluble ICAM-5 comprises a sequence disclosed herein (SEQ ID NO: 3).

9. The expression vector of claim 1, wherein said mRNA encodes for a fusion protein comprising a sequence disclosed herein (SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3).

10. A therapeutic or prophylactic formulation comprising an expression vector according to any one of claims 1-9 in a delivery vehicle.

11. The therapeutic or prophylactic formulation of claim 10, wherein the delivery vehicle is a lipid nanoparticle.

12. The therapeutic or prophylactic formulation of claim 10, wherein the formulation is a liquid, powder, or gel.

13. The therapeutic or prophylactic formulation of claim 10, wherein the formulation is configured for administration to nasal and airway mucosal surfaces and / or delivery across the blood-brain barrier.

14. The therapeutic or prophylactic formulation of claim 10, for use in treating or inhibiting a viral infection mediated by transmembrane ICAM-5.

15. The therapeutic or prophylactic formulation of claim 10, for use in reducing inflammation of the respiratory tract or CNS of a subject.

16. The therapeutic or prophylactic formulation of claim 10, for use in treating or inhibiting RSV or EV-D68 infection in a subject in need thereof, preferably a child or elderly individual.

17. A therapeutic or prophylactic treatment method, the method comprising: administering to a subject in need thereof a composition comprising an expression vector, preferably an mRNA expression vector, encoding a protein, such that the administration of the composition results in expression, translation, and / or activity of the protein encoded by the expression vector in the subject, wherein the protein is soluble ICAM-5, preferably wherein the subject has or is at risk of a viral infection, preferably RSV or EV-D68 infection.

18. The method of claim 17, wherein the soluble ICAM-5 acts as a decoy receptor protein for transmembrane ICAM-5 in said subject.

19. The method of 17, wherein said expression vector sequence is optimized for translation of a mutated protein having increased affinity for EV-D68 or RSV F protein.

20. The method of 17, wherein said expression vector sequence is optimized for increased secretion of the translated protein.

21. The method of claim 17, wherein said composition is delivered to the nasal and airway mucosa and / or across the blood-brain barrier of the subject.

22. The method of claim 17, wherein said composition is delivered prophylactically to the subject before diagnosis of EV-D68 or RSV infection.

23. The method of claim 17, wherein said composition is delivered therapeutically to the subject after diagnosis of EV-D68 or RSV infection.

24. The method of claim 17, wherein ICAM-5 levels are increased in said subject.

26. Use of mRNA expression vectors to deliver soluble ICAM-5 for both the prevention and treatment of EV-D68 or RSV to reduce EV-D68 or RSV disease severity and prevent death from viral infection.

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