Decoy ICAM-1 receptor proteins and treatment methods

Abstract

Expression vectors, preferably mRNA expression vectors, encoding for soluble ICAM-1 for increasing ICAM-1 levels in a subject in need thereof, as decoy receptor proteins for endogenous transmembrane ICAM-1. 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-1. Uses of mRNA expression vectors to deliver soluble ICAM-1 for both the prevention and treatment of RSV to reduce RSV disease severity and prevent death from viral infection.

Description

DECOY ICAM-1 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 / 495,145, filed April 10, 2023, entitled DECOY ICAM-1 RECEPTOR PROTEINS AND TREATMENT METHODS, incorporated by reference in its entirety herein.SEQUENCE LISTING

[0002] The present application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "SequenceListing_58369.xml," created on April 9, 2024, as 24,195 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-1 for expression as soluble decoy receptor proteins to block RSV infection and also 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.

[0005] The only available preventative treatment for RSV is Palivizumab, which is a monoclonal antibody that needs to be injected monthly during RSV season. Further, although it helps reduce severity of RSV, it is not effective when administered after infection. The same is true for more modern monoclonal antibody therapies currently in clinical trials. There are no current preventative strategies to inhibit RSV infection, and there are no targeted antivirals for use during RSV infection. Thus, developing both preventative and therapeutic tools to prevent RSV infection and severe disease are of urgent need to save lives and reduce the burden of RSV infection.

[0006] Intercellular Adhesion Molecule 1 (ICAM-1) also known as CD54 (Cluster of Differentiation 54) is an endothelial- and leukocyte-associated transmembrane protein. The RSVfusion (F) protein has been shown to interact with several host-cell surface receptor proteins such as ICAM-1, NCL, EGFR, IGF1R, and use these transmembrane proteins to infect respiratory epithelial cells. The RSV F protein is critical for binding host cells and mediating infection and is the target of most candidate vaccines. ICAM-1 expressed on respiratory epithelial cells is also the binding site for rhinovirus, the causative agent of most common colds. ICAM-1 is also up- regulated in response to inflammatory stimulation. ICAM-1 has been of clinical and therapeutic interest for some time now; with the primary focus being on strategies to inhibit endogenous ICAM-1 levels. However, inhibition of ICAM-1 function has not yielded significant clinical impact in reducing symptoms, improving injury resolution, or other conditions.

[0007] Notably, a soluble form of ICAM-1 is also an endogenous protein and one of many soluble proteins also found in human breast milk. Breast feeding has long been associated with reduced RSV severity, although the mechanism is not understood. While maternal antibodies against viruses are commonly assumed to be the drivers of this benefit, there are thousands of immune components in human milk that are an untapped resource of therapeutic potential. Respiratory syncytial virus (RSV) infections result in a significant number of hospitalizations for young children and the elderly each year, and current infection rates continue to rise, particularly after the COVID-19 pandemic, leading to a substantial public health burden. There is an urgent need to find new therapeutics and strategies that reduce severe RSV disease that could improve health and save lives.SUMMARY

[0008] The present disclosure concerns an expression vector, preferably an mRNA expression vector, encoding for soluble ICAM-1 for increasing ICAM-1 levels in a subject in need thereof, wherein the soluble ICAM-1 is translated in vivo from the mRNA and is a decoy receptor protein for transmembrane ICAM-1.

[0009] Also described herein are therapeutic and / or prophylactic formulations comprising an effective amount of an expression vector according to any one of the embodiments described herein, in a delivery vehicle.

[0010] The present disclosure also concerns therapeutic and / or prophylactic treatment methods. The methods generally comprise 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-1.

[0011] Also disclosed herein are various uses of mRNA expression vectors to deliver soluble ICAM-1 for both the prevention and treatment of RSV to reduce RSV disease severity and prevent death from viral infection in subjects in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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.

[0013] Fig. 1 shows illustrations of the components of the mRNA constructs, including with reference to their correspondence SEQ ID NOs for the coding sequences and resulting proteins synthesized in the working examples. Signal peptide is important for regulating protein secretion and the transmembrane (TM) domain is important for anchoring the protein to the cell membrane. Alternative versions of the constructs were synthesized with or without TM domain or replacing TM domain with IgG-FC domain to reduce membrane deposition and increase solubility. Alternative versions of the constructs were synthesized with replacing the wild-type signal peptide with the tissue plasminogen activator (tPA) signal peptide that has been shown to increase protein secretion.

[0014] Fig. 2 is a graph showing binding of ICAM-1 soluble protein to RSV F protein using ELISA. RSV prefusion F protein was coated on ELISA plate and increasing concentration of biotin-labeled sICAM-1 added. Streptavidin-HRP used as secondary antibody for visualization.

[0015] Fig. 3 contains graphs showing sICAM-1 neutralization of RSV in A549 and Hep2 target cells. RSV-GFP was mixed with sICAM-1 or PBS (control) for 1 hour then added to target cells. 24hours after infection GFP was quantified to determine percent neutralization relative to negative control.

[0016] Fig. 4 is a graph showing that sICAM-1 blocks neutrophil adhesion to lung A549 cells. Primary human neutrophils were labeled with a dye (calcein AM) and incubated with or without sICAM-1 for 30 minutes. Then neutrophils are added to A549 cells for 30minutes to adhere. Nonadherent neutrophils are washed off and calcein fluorescence is used to determine the level of neutrophil adherence.

[0017] Fig. 5 is a graph showing ICAM-1 and ICAM-5 genes are upregulated after RSV infection. RNA-seq performed 24hours after RSV infection or mock infection control. Number of reads of each ICAM family gene reported. ICAM-1, -3, -4, -5 are expressed but only ICAM-1 and ICAM-5 are significantly increased after infection.

[0018] Fig. 6 shows images from computational modeling of ICAM-1 and RSV F protein interactions to predict interaction sites and select mutants that could modulate interaction affinity. H-DOCK Protein-protein docking based on a hybrid algorithm of template-based modeling and ab-initio free docking. Green is ICAM-1 (PDB:1MQ8); blue is RSV F protein (PDB:5C6B). The docking score of was -234.34 and the confidence score was 0.8464. When the confidence score is higher than 0.7, the two molecules are most likely to bind. On the right are the predicted interacting amino acids shown in pink (the structure is rotated to better see interactions.).

[0019] Fig. 7 is a graph showing ELISA of ICAM-1 detected levels secreted into cell culture supernatant of A549 lung epithelial cells transfected with mRNA expression vectors encoding for ICAM-1.

[0020] Fig. 8 shows imaging from preclinical animal models after various modes of delivering mRNA-LNPs to mice. Expression of luciferase encoding mRNA-LNPs delivered IN, IM or SQ at 6 hours, 1 day, 3 days or 7 days after immunization measured by in vivo whole body bioluminescent imaging (IVIS).

[0021] Fig. 9 shows the results from measurement of soluble human ICAM-1 levels after ICAM- 1 mRNA-LNP delivery to mice. Levels of ICAM-1 measured by ELISA 24 hours after immunization with ICAM-1 encoding mRNA-LNPs in BAL, homogenized lung tissue and peripheral blood.DETAILED DESCRIPTION

[0022] The present disclosure is broadly concerned with an alternative protein-based antiviral to monoclonal antibodies, which counterintuitively seeks to increase soluble ICAM-1 in the treated subject. The secreted ICAM-1 acts as a decoy to compete for receptor-binding sites so that viruses, such as RSV or rhinovirus in an infected subject will bind to the soluble ICAM-1 decoy, instead of the natural or native / endogenous ICAM-1 transmembrane bound receptor on epithelial cells in the subject. In this manner, less virus remains available for binding with host-cell surface receptor proteins meaning less opportunities for virus to use these transmembrane proteins to infect respiratory epithelial cells. Therefore, viral titers will remain low and the infection can be inhibited.

[0023] In one or more embodiments, the disclosure concerns synthetic messenger ribonucleic acid (mRNA) encoding an ICAM-1 polypeptide configured for translation in a cell of a subject to yield a functional ICAM-1 polypeptide that acts as a decoy receptor protein for RSV. In one or more embodiments, the mRNA encodes for an ICAM-1 polypeptide comprising SEQ ID NO: 1, ora sequence having at least 95% sequence identity with SEQ ID NO: 1 and retaining binding ability with the virus.QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDS QPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANL TVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSA PYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVT YGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSE GTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIH KNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPI GESVTVTRDLEGTYLCRARSTQGEVTREVTVNVLSPRYE (SEQ ID NO: 1)

[0024] 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-1 (sICAM-1), meaning that the translated ICAM-1 polypeptide is secreted and not transmembrane bound. In particular embodiments, the mRNA is derived from cDNA. 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)-m ethylpseudouridine or 5-methoxyuridine. In particular embodiments, the mRNA may be codon-optimized using standard protocols.

[0025] 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-1 polypeptide (preferably sICAM-1).

[0026] 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.

[0027] In one or more embodiments, the mRNA comprises a translatable region encoding the recombinant ICAM-1 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 thecell from the nucleic acid, and more preferably secreted from the cell as functional, soluble ICAM- 1. In one or more embodiments, the mRNA construct comprises a signal peptide coding sequence that directs the ICAM-1 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 wild type signal peptide comprising SEQ ID NO: 13. 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 NOTO.

[0028] In one or more embodiments, the ICAM-1 coding sequence used in the mRNA construct is modified to remove the native transmembrane domain from the ICAM-1 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-1. 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.

[0029] 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-1 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 comprising SEQ ID NO: 12, or a sequence having at least 95% sequence identity with SEQ ID NO: 12 and retaining Fc functions thereof. In one or more embodiments, the mRNA expression vector comprises a coding region for the linker. In one ormore embodiments, the peptide linker comprises SEQ ID NO: 11. Thus, in such embodiments, the ICAM-1 polypeptide is translated as an ICAM-l-Fc fusion polypeptide. 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 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.

[0030] In one or more embodiments, a particularly preferred ICAM-1 fusion protein comprises SEQ ID NO:8, or a sequence having at least 95% sequence identity with SEQ ID NO:8 and retaining the ability to act as a decoy for binding with the virus.MDAMKRGLCCVLLLCGAVFVSARQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPL PKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPS WQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHH GANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLD GLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQ ETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLK ATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPC QAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTREVTVNVLSPR YEIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 8)

[0031] In the embodiments, after administration, the approach increases the level of circulating ICAM-1 in the treated subject, and this soluble, circulating ICAM-1 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-1 in the subject. In this manner, the soluble ICAM-1 interferes with the ability of the virus to infect the epithelial cell, reducing viral spread as well as associated symptoms of infection, including damage to lung tissue.

[0032] In one or more embodiments, the mRNA comprises an ICAM-1 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-1 polypeptide having one or more mutated amino acid residues for increased affinity binding. For example, any of amino acid substitution identified in the Table in the working examples as “stabilizing” are predicted tostabilize the interaction and could be used alone or in combination to increase binding affinity. In one or more embodiments, the mRNA expression vector is modified to increase translation levels of the ICAM-1 polypeptide, including via increased solubility and secretion of the translated ICAM-1 polypeptide rather than bound to the cellular membrane. For example, the coding sequence for ICAM-1 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 is modified to include a coding region for an IgG-FC domain, wherein said ICAM-1 polypeptide is translated as an ICAM-l-Fc fusion polypeptide.

[0033] In one or more embodiments, provided herein are therapeutic or prophylactic formulations comprising synthetic mRNA encoding ICAM-1, preferably, soluble ICAM-1 in an amount effective to bind and block viral infection normally mediated by endogenous membrane bound ICAM-1 on host 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 of the 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.

[0034] 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 rhinoviruses). 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 toproduce 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 or prophylactic 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.

[0035] 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), 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. Non-limiting 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.

[0036] Embodiments described herein contemplate methods of treating or inhibiting viral infection in a subject in need thereof, such as from a respiratory virus, or viral infection mediatedby ICAM-1 transmembrane protein. Embodiments described herein contemplate methods of increasing levels of ICAM-1 (preferably soluble, circulating ICAM-1) 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) ICAM-1 transmembrane protein, e.g., in epithelial cells.

[0037] The methods comprise administering a therapeutically effective amount of mRNA encoding for ICAM-1 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-1 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-1.

[0038] Advantageously, the therapeutic or prophylactic formulations can be administered via asuitable 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-1 in the treated subject, virus in the subject’s body will more readily interact with and bind to the circulating sICAM-1 reducing the virus that remains free to interact with the subject’s endogenous ICAM-1 transmembrane proteins to infect respiratory epithelial cells. Since ICAM-1 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.

[0039] 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-1 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-1 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).

[0040] Advantageously, ICAM-1 also is a receptor for certain rhinoviruses (common cold viruses), 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-1). 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-1. Non-limiting examples include human rhinoviruses (HRV), some coxsackieviruses, and the major respiratory bacterial opportunistic pathogen, non-typeable Haemophiles influenzae via the type IV pilus.

[0041] The data herein also unexpectedly demonstrates that the soluble ICAM-1 can also blockrecruitment of proinflammatory immune cells to the lung tissue. That is, RSV and other respiratory infections and lung inflammation upregulate transmembrane ICAM-1 on airway epithelial cells. This can recruit immune cells, such as neutrophils and eosinophils (e.g., via ICAM-1 engagement of CD 18 receptors), that are the actual drivers of lung damage. Thus, the mRNA delivered ICAM- 1 may also be effective to inhibit inflammation in the airways, as well as to treat lung tissue damage from a broad spectrum of respiratory infections or other inflammatory condition such as Asthma and COPD, aside from its antiviral properties. Accordingly, in one or more embodiments, methods described herein concern treating, inhibiting, or ameliorating inflammatory conditions of the airways. Embodiments described herein concern methods for reducing inflammation of the respiratory tract of a subject in need thereof. Embodiments described herein concern method of treating, inhibiting, or ameliorating damage to respiratory tissue due to inflammation of the respiratory tract. Methods comprise administering a therapeutically effective amount of mRNA encoding for ICAM-1 to a subject in need thereof. The mRNA can be administered before, during, or after a subject experiences symptoms of respiratory inflammation or distress (e.g., coughing, difficulty breathing, low oxygen levels, and the like). Further, more elevated levels of sICAM-1 have been observed in the blood of individuals with sepsis, cytomegalovirus, severe COVID-19, latent toxoplasmosis, different forms of cancers, atherosclerosis, asthma, COPD, and many other systemic inflammatory diseases. sICAM-1 could also play a role in modulating immune responses. Thus, sIC AM- 1 delivered according to the methods contemplated herein for addressing inflammation (from various conditions), could serve as a biomarker for assessing the extent of inflammation and disease severity. The methods can be carried out using similar modes of administration as described above for viral infection.

[0042] 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.

[0043] 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-1 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-1. However, other expression vectors can be used to express soluble ICAM-1 for subsequent recovery and isolationof a purified protein that can be used or administered therapeutically.

[0044] 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.

[0045] 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.

[0046] 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 are provided, 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

[0047] 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-1

[0048] Prior work has shown that ICAM-1 is the host cell receptor for RSV and rhinoviruses, and shows that using antibodies or soluble recombinant protein to block the interaction can reduce infection. Here, we investigate the use of soluble ICAM-1 as a decoy receptor protein as a potential therapeutic candidate. As shown below, this work demonstrates that soluble ICAM-1 can bind theRSV F protein, and further that soluble ICAM-1 can block RSV infection. Surprisingly, we also identified a role for ICAM-1 in inflammation and have shown that soluble ICAM-1 can reduce neutrophil recruitment. We have identified earlier work with tremacamra, a recombinant, soluble form of the membrane-bound ICAM-1 glycoprotein, as an ICAM-1 receptor blockade for prevention of experimental rhinovirus infection. However, a major drawback of this study was that the recombinant protein had to be administered in multiple repeated dosages, requiring 6 doses per day, at 3-hour intervals for 7 days.

[0049] In this work, we have also developed engineered forms of ICAM-1 for mRNA construction. mRNA constructs were synthesized by GenScript Biotech (New Jersey) using provided sequence information. Each mRNA construct 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 N1 -methylpseudouridine during mRNA synthesis. The signal peptide sequences are cleaved off of the resulting functional protein, but may be important to the expression vector for secretion efficiency and are shown in the sequences. The coding sequences and resultant expressed polypeptide sequences are shown in the sequences (again noting that the signal peptide is likely not part of the final functional protein expressed by each construct).

[0050] We have also used structure-function studies to generate constructs having targeted mutations to increase affinity of ICAM-1 for RSV F protein. Unlike the direct use of soluble protein, the mRNA constructs expressing soluble ICAM-1 will have a more durable effect. We are evaluating this therapeutic approach in preclinical models as a prophylactic or treatment after infection.

[0051] In this study, we confirmed ICAM-1 could bind RSV F protein and inhibit RSV infection via an RSV neutralization assay. We engineered an mRNA-lipid nanoparticle (LNP) expressing soluble ICAM-1. The goal is to utilize the ICAM-1 mRNA-LNP as a passive intranasal vaccine that would result in over-expressed soluble ICAM-1 in respiratory mucosal surfaces to function as a decoy receptor that would reduce RSV infection. The mRNA constructs are shown in Fig. 1. The construct coding sequence (cDNA) including the native signal peptide and transmembrane domain is shown in SEQ ID NO:3, with the resulting expressed protein shown in SEQ ID NO:2. The construct coding sequence (cDNA) including the tPA signal peptide and native transmembrane domain is shown in SEQ ID NO:5, with the resulting expressed protein shown in SEQ ID NO:4. The construct coding sequence (cDNA) including the tPA signal peptide, but wherein the transmembrane domain is removed shown in SEQ ID NO:7, with the resulting expressed protein shown in SEQ ID NO:6. The construct coding sequence (cDNA) for the fusion constructsincluding the tPA signal peptide and IgG Fc domain (connected via a peptide linker) is shown in SEQ ID NO:9, with the resulting expressed fusion protein shown in SEQ ID NO: 8.

[0052] The results of initial studies are shown in Figs. 2-6.

[0053] We have also confirmed that ICAM-1 can be successfully delivered via mRNA and secreted by the transfected cells. We delivered different ICAM-1 mRNAs to A549 lung epithelial cells. 24 hours after delivery, we measured ICAM-1 protein levels in the cell culture supernatant using an anti-ICAM-1 antibody that was tethered to an ELISA plate. Anti-Human-IgG-HRP was used to quantitate the binding levels. The results of this ELISA are shown in Fig. 7. You will see that all formats had expression and secretion into the cell culture supernatant, but the ICAM-1 Fc Chimera (SEQ ID NO:8) had the highest level.

[0054] We have also conducted studies in preclinical animal models and confirmed that ICAM- 1 can be successfully delivered as mRNA using lipid nanoparticle delivery systems and various routes of administration. The results are shown in Figs. 8-9.

[0055] We have also established an RSV neutralization assay that will enable high-throughput screening of soluble factors found in human milk and other biological fluids as well as candidate drugs to identify more candidate molecules that can block RSV infection of cells. This platform technology and our lead candidate (ICAM-1) will serve as a template for discovering and translating therapeutics human milk factors that can block or treat RSV infection.

[0056] ICAM-1 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-1 to be delivered to cells of the airway using suitable expression vectors, such as using mRNA-lipid nanoparticles, so that ICAM-1 will be produced in the airway as a soluble decoy receptor that can bind the RSV virus and thereby block its binding to ICAM-1 transmembrane protein on epithelial cells.

[0057] We have also described modified ICAM-1 sequences with targeted mutated residues to increase affinity for RSV as well as to increase secretion as a soluble protein rather than bound to the cellular membrane.

[0058] Discovery Studio software was used to predict the effect of positional mutations within ICAM-1 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-1 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 Entropycalculation interactions. The lower the more stabilizing for them as well, but these were not used for classification as stabilizing. In the tables below, the results from the structure-function analysis are provided:Table. Details of hydrogen bondsTable. Details of hydrophobic interaction

[0059] In the Table below, all the stabilizing mutations predicted are shown.Table. Predicted mutations with stabilizing interaction between the two proteins.

[0060] In the Table below, we selected the amino acid change with the lowest mutational energy value at each stabilizing position.Table. Data fdtered for higher predicted stabilizing mutations.

[0061] In the Table below, the data is fdtered again for only positions that were predicted to have direct contact between ICAM-1 and RSVF by the earlier modeling analysis.Table. Highest predicted mutations for increased affinity.Example 2 In vivo studies

[0062] In the first part of the study, ICAM-1 mRNA-LNP are delivered via various routes (intranasal (IN), intramuscular (IM), subcutaneous (SQ)) 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.

[0063] First, BALB / c mice were immunized via IN, IM or SQ injection routes with 0.04mg / kg of mRNA-LNPs encoding firefly luciferase. At the indicated timepoints, mice were injected with D-luciferin via the intraperitoneal route and rested for 15 minutes. Whole-body imaging was performed using the IVIS Lumina X5 instrument to measure bioluminescent luciferase intensity. The results from the live animal imaging are shown in Fig. 8. As can be seen, the IN route of administration initially resulted in the highest concentration in the lungs, but was quickly cleared. The IM route of administration appeared to perpetuate the longest in the body.

[0064] In the second part of the study, BALB / c mice (n=4-5 mice per group) were immunized with 0.04 mg / kg ICAM-1 mRNA-LNPs IN and Img / kg ICAM-1 mRNA-LNPs IM or SQ. As control, mice were immunized with saline (n=3). After 24 hours, mice were euthanized and bronchoalveolar lavage (BAL) was performed along with lung tissue and peripheral blood collection. Mouse tissue was homogenized, and blood separated into serum. Commercial ELISA assays (Abeam) were used to detect the levels of human sICAM-1 were used to quantify the amount of ICAM-1 in each tissue generated through each immunization route. The results are shown in Fig. 9. As can be seen, the IM injection generated the highest levels of sICAM-1 in lung and blood tissues at Day 1 after administration.

[0065] In the third part of the study, the prophylactic effect of ICAM-1 mRNA-LNP on RSV infection is tested. The mice are grouped as follows:

[0066] On day 0, the mice are administered the ICAM-1 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 and nasal tissues are analyzed for RSV viral loads, pathology, and immune cell characterization.

[0067] Mice receiving the ICAM-1 mRNA-LNP exhibit improved body weight, reduced RSV viral loads, and reduced lung pathology score.

[0068] In the third part of the study, the therapeutic effect of ICAM-1 mRNA-LNP on RSV infection is tested. The mice are grouped as follows:

[0069] On day 0, the mice are inoculated with RSV or mock RSV via intranasal swab. On day 1, the mice are administered the ICAM-1 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 and nasal tissues are analyzed for RSV viral loads, pathology, and immune cell characterization.

[0070] Mice receiving the ICAM-1 mRNA-LNP exhibit improved body weight, reduced RSV viral loads, and reduced lung pathology score.

Claims

CLAIMS:

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

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

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

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

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

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

7. The expression vector of claim 6, wherein said soluble ICAM-1 binding blocks the ability of the RSV F protein to bind with said endogenous transmembrane ICAM-1.

8. The expression vector of claim 1, wherein said soluble ICAM-1 comprises SEQ ID NO: 1.

9. The expression vector of claim 1, wherein said mRNA encodes for a fusion protein comprising SEQ ID NO: 8.

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 alipid 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.

14. The therapeutic or prophylactic formulation of claim 10, for use in treating or inhibiting a viral infection mediated by transmembrane ICAM1.

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

16. The therapeutic or prophylactic formulation of claim 10, for use in treating or inhibiting RSV 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-1.

18. The method of claim 17, wherein the soluble ICAM-1 acts as a decoy receptor protein for transmembrane ICAM-1 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 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 of the subject.

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

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

24. The method of claim 17, wherein ICAM-1 levels are increased in said subject.

26. Use of mRNA expression vectors to deliver soluble ICAM-1 for both the prevention and treatment of RSV to reduce RSV disease severity and prevent death from viral infection.

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