Covid-19 trap technology platform

Abstract

Here we describe a two-pronged localized nanomaterial-based approach for limiting SARS-CoV-2 infection in nasal and upper airway epithelial cells that involves lipid nanoparticles (LNPs) that act as receptor decoys to bind viral spike protein (LNP-trap) and LNPs to delivery siRNA to host cells to knock down the viral entry targets (LNP-trim). The LNP-trap formulations consisted of cell- impermeable lipid nanoparticles surface modified with ACE2 peptide, recombinant ACE2 or SARS- CoV-2 spike mAb that can bind to SARS-CoV-2 and potentially neutralize them. The LNP-trim LNP formulations were cell-permeable and capable of delivering siRNA for reducing ACE2 and TMPRSS2 expression in host cells. The in vitro studies demonstrated that both the LNP-trap and LNP-trim formulations were safe and effective at inhibiting virus-host cell interactions and reducing viral infection. Initial assessment of the LNP-trap and LNP-trim LNPs following nasal administration in mice confirmed nasal retention and biocompatibility.

Description

[0001] COVID-19 TRAP TECHNOLOGY PLATFORM

[0002] PRIOR APPLICATION INFORMATION

[0003] The instant application claims the benefit of US Provisional Patent Application US63 / 582,574, filed September 14, 2024, entitled “COVID-19 TRAP TECHNOLOGY PLATFORM”, the entire contents of which are incorporated herein by reference for all purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] The recent emergence of a novel SARS-coronavirus-2 (SARS-CoV-2) and the resulting COVID- 19 pandemic has had crippling effects on public health and the global economy. Statistics from the World Health Organization (WHO) reported over 758 million confirmed cases of COVID-19 and over 6.8 million related deaths. [1] The COVID-19 and earlier SARS (2002) and Middle East respiratory syndrome (MERS; 2012) outbreaks were caused by closely related coronaviruses. [2] The coronaviruses carry surface proteins that resemble spikes on a crown and are hence named coronaviruses (CoV). These viruses use spike proteins to bind and gain entry to specific host cell receptors during infection. While the development of effective vaccines for SARS-CoV-2 have been critical in slowing the spread and limiting mortality, the virus has a high infection rate and can undergo rapid gene mutations resulting in the formation of viral variants. [3] The challenge lies in rapidly developing and updating vaccines to maintain efficacy against viral variants. Hence it is crucial to develop novel therapeutic platforms that would allow rapid modification to target novel viruses and viral variants.

[0006] Traditional vaccines act by inducing the production of viral protein-specific antibodies (IgG and IgA) by priming the body’s immunity machinery. [4] Initially, efforts at identifying antiviral agents effective against SARS-CoV-2 focused on repurposing existing chemical libraries of approved drugs (3, 4). These efforts identified several potential therapeutics and resulted in some “hits” that have been of use such as dexamethasone for ICU and ventilated patients, Paxlovid (orally active) and Remdesivir (iv infusion) antivirals for use in infected patients at risk of morbidity or death from severe infection (5-7). Administration of convalescent plasma from recovered Covid- 19 patients was reported to prevent hospitalizations in a clinical trial of out-patients with recently confirmed SARS-CoV-2 infection (8). Along those same lines, combination therapy with Bamlanivimab and Etesevimab monoclonal antibodies reduced viral load in patients with mild to moderate illness (9). Despite these initial efforts, there is still a need for effective and affordable agents for preventing and treating SARS-CoV-2 infections in the general population.

[0007] The spike protein of SARS-CoV-2 contains two domains (SI and S2) that are separated by a furin cleavage site. [5] For viral infection, the SI receptor binding domain on the viral particle binds to ACE2 receptors on host cells (Figure 1A). Proteolytic cleavage of the SI and S2 domains by Furin and host transmembrane protease serine 2 (TMPRSS2) activates the S2 fusion machinery leading to viral entry and the release of viral genomic RNA into the host cells. [6] The viral RNA hijacks the host cell machinery to express viral mRNA, and non-structural (NSPs) and structural (spike (S), envelop (E), membrane (M), and nucleocapsid (N)) proteins that are vital for viral replication, assembly and release. [6] Once released from the host cells, newly generated virions can infect surrounding cells through the same process.

[0008] Agents acting as decoys for targeting, trapping, and neutralizing viruses are emerging as promising technology to control infections. [3,7] Several monoclonal antibody (mAb) based decoys were developed for neutralizing a wide range of HIV variants. [8] Though the earlier studies with these mAb displayed poor antiviral activity in vivo, the recent development of highly potent new-generation mAbs, and their success in clinical trials, have revived the interest in mAb decoys for HIV.[9,10] Recently, two mAb drugs, bamlanivimab and etesevimab, that bind SARS-CoV-2 S-protein were granted Emergency Use Authorization (EUA) in adult and pediatric (12 years and older) patients with mild to moderate COVID-19 symptoms. [11,12] However, use of mAb therapeutics have the risk of viral variants that can develop drug resistance. Indeed, while bamlanivimab and etesevimab have prevented severe COVID-19 symptoms in patients, these mAb antiviral drugs have been less effective in treating more recent variants of SARS-CoV-2.

[0013] Hence, it is crucial to develop viral decoy and trapping agents with a broad-spectrum affinity against variants of interest to be successful.

[0009] Engineered host cell receptors can act as decoys and competitively bind and neutralize viruses. Several studies have reported the successful use of engineered ACE2 in various forms as a broadspectrum decoy for neutralizing SARS-CoV-2. [13, 14] An advantage of this approach is that host cell receptor-based decoys generally have less chance for resistance, as if such variants were to occur, it naturally would have reduced affinity towards the host cell receptor resulting in impaired infectivity.

[0014] Recombinant soluble ACE2 was developed to treat acute respiratory distress syndrome (ARDS) and progressed successfully through initial clinical trials demonstrating safety.

[0015] However, recombinant ACE2, when administered intravenously, exhibited a short half-life of ~3 hours in humans.

[0016] To improve the pharmacokinetics of ACE2, further modification, for example, fusing with wild-type IgGl- Fc, was performed.

[0017] Similarly, several peptide mimics of ACE2 have been developed as inhibitors for SARS-CoV-2. [18, 19]

[0010] One of the most widely studied host-cell decoy approaches involves encapsulating nanoparticles in cell vesicles isolated from cells expressing the viral host receptor(s) of interest. [18, 19] Wei et al. prepared PLGA nanoparticles that were coated with plasma membrane isolated from CD4+ T cells. These T-cell mimicking nanoparticles were shown to bind and neutralize HIV and prevent infection of human peripheral blood mononuclear cells (PBMCs) in vitro. In regards to SARS-CoV-2, Rao et al. isolated membrane vesicles from THP-1 and T293 / ACE2 expressing cells and used them as decoys for binding viral particles.

[0020] The vesicles with high amounts of ACE2 and cytokine receptors successfully neutralized SARS-CoV-2 in Vero-E6 cells and neutralized inflammatory cytokines, thereby effectively suppressing the immune response and lung injury in the ALI mouse model.

[0011] RNAi using small interfering RNA (siRNA) has been shown to inhibit virus infection by knocking down viral antigen and other accessory genes inside host cells. [21,22] siRNA is small double stranded noncoding RNA of around 20 bp which can be tailored to bind to complimentary mRNA leading to enzymatic degradation.

[0023] Thus siRNA induced RNAi can transiently knock down and silence a specific gene and protein product at posttranscriptional stage. The barriers for systemically administered exogenous siRNA include serum nuclease degradation, poor cell membrane permeability, and endosomal / lysosomal escape into cytoplasm. Hence siRNA require suitable delivery systems to protect, transport, and deliver them to the target cells. Several non-viral vectors such as cationic polymers and lipids that complex with siRNA have been used to transfect in in vitro cell culture studies. However, these cationic polyplexes and lipoplexes suffer from toxicity due to their permanent net cationic charge or low transfection efficiency and has found limited applications in vivo.

[0012] Nanoparticle-based siRNA delivery vehicles, especially lipid nanoparticles, have shown excellent transfection and mRNA / protein knockdown efficiency both in vitro and in vivo. Recently, Patisiran became the first FDA approved LNP siRNA therapeutic used for treating hereditary transthyretin-mediated amyloidosis.

[0024] , A few RNAi based antivirals have crossed into clinical trials. [25-27] For example, aerosolized ALN-RSV01 targeting nucleocapsid gene of respiratory syncytial virus (RSV) was shown to be effective in reducing bronchiolitis obliterans syndrome in phase 2b clinical trials.

[0025] Recently there is growing interest in using RNAi to knockdown SARS-CoV-2 gene inside host cell. As SARS-CoV-2 entry and infection is primarily facilitated by ACE2 and TMPRSS2, knocking down these target proteins in host cells could also provide a method for inhibiting viral infection.

[0013] Nanomaterials have emerged as valuable platforms for both systemic and local drug delivery. [28,29] SUMMARY OF THE INVENTION

[0014] According to a first aspect of the invention, there is provided a method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a pathogen replication inhibitor; and an effective amount of a pathogen binding agent.

[0015] According to another aspect of the invention, there is provided a method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effecti ve amount of a nanoparticle formulation of a pathogen replication inhibitor; and an effective amount of a nanoparticle formulation of a pathogen binding agent.

[0016] According to another aspect of the invention, there is provided a method of tread ng or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a pathogen replication inhibitor comprising a siRNA formulated as a nanoparticle.

[0017] According to another aspect of the invention, there is provided a method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a pathogen binding agent formulated as a nanoparticle.

[0018] According to another aspect of the invention, there is provided a method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a cell-permeable nanoparticle formulation of a pathogen replication inhibitor; and an effective amount of a cell -impermeable nanoparticle formulation of a pathogen binding agent.

[0019] As used herein, an “effective amount” is the amount of the composition that is sufficient to at least reduce the severity associated with one or more symptoms associated with the specific pathogen.

[0020] According to another aspect of the invention, there is provided use of a composition comprising: a cell-permeable nanoparticle formulation of a pathogen replication inhibitor; and a cell-impermeable nanoparticle formulation of a pathogen binding agent for treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen.

[0021] According to another aspect of the invention, there is provided a composition comprising: a cell-permeable nanoparticle formulation of a pathogen replication inhibitor; and a cell-impermeable nanoparticle formulation of a pathogen binding agent for treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Schematic representation of (A) SARS-CoV-2 S pseudotyped lentivirus infection aided by S protein ACE2 binding and TMPRSS2. Knocking down of ACE2 and TMPRSS2 using LNP- siRNA inhibiting lentivirus entry (B). The presence of LNP-decoys with surface ACE2 peptide, rACE2 and mAb bind to the lentivirus thereby inhibiting cell entry and i nfection.

[0023] Figure 2: Schematic illustration of the preparation of LNP-COOH following microfluidic mixing (A). The lipid mixture DOPC / DSPC / cholesterol / DSPE-PEG-COOH / DiO (46.5 / 10 / 38 / 5 / 0.5%) in ethanol (organic phase) was micromixed with PBS at the flow rate ratio (FRR) of aq:org 3:1 and a total flow rate (TFR) of 12 mL / min to yield LNP-COOH. The LNP-COOH was fluorescently tagged using a green fluorescent dye (DiO). Further, LNP-COOH was surface modified with spike RBD binding ligands following EDC / NHS coupling reaction (B).

[0024] Figure 3: Cytotoxicity of LNP-COOH analyzed using A) cell impedance and B) MTT assays in Calu3 cells. Cytotoxicity MTT of LNP-PEP (C), LNP-rACE2 (D), LNP-mAb (E) and LNP-siACE2 in Calu3 cells. Values are expressed as the mean ± standard error of the mean (n = 3).

[0025] Figure 4: Percentage uptake of (A) LNP-PEP and (B) LNP-rACE2 in Calu-3 cells. Representative confocal microscopy images of Calu3 cells treated with LNP-PEP (C - F) and LNP- siA CE2 (G - J). Cell plasma membrane was stained with WGA Alexa Fluor™ 594 (C and G). The LNPs were tagged with green fluorescent DiO dye (D and H). The nuclei were stained using Hoestch (E and I). Overlay of three channels showing cell membrane, nuclei and LNPs (F and J). Orthogonal projections of Calu-3 cells treated with LNP-si CE2 (K) for 3 hours. Two-dimensional images in z- position where red lines indicate cross-section through y-position and green lines indicate cross-section through x-position. The white dashed box in image K is enlarged. Values are expressed as the mean ± standard error of the mean (n = 3).

[0026] Figure 5: Relative ACE2 (A) and TMPRSS2 (B) mRNA levels in HEK-293-hACE2 cells at different time points after transfection with LNP-si CE2 (40 nM siACE2) and LNP-si' / MPAAS (40 nM siTMPRSS2~), respectively. The control cells in (A) and (B) received LNP-siSCR. Relative ACE2 mRNA level in Calu-3 cells 72 hours after receiving LNP-si CE2 (40 n siACE2') against control cells that received LNP-siSCR (C). Relative ACE2 protein level in Calu-3 cells 72 hours after receiving LNP-siA CE2 (40 nM siACE2) against control cells that received LNP-siSCR (D). Values are expressed as the mean ± standard error of the mean (n = 3).

[0027] Figure 6: Transduction efficiency (luciferase expression) of SARS-CoV-2 S pseudotyped luciferase lentivirus at the given MOIs in HEK-293 and HEK-293-hACE2 cell lines (A). Percentage inhibition of infection in ACE2 KD and TMPRSS2 KD HEK-293-hACE2 cells (B). Percentage inhibition of infection in A CE2 KD (C) and TMPRSS2 KD (D) in airway epithelial Calu3 cell line at different siRNA concentrations (LNP-siRNA: 1, 2, 10, 20 and 40 nM; presented in the graph as log 10 values). The nonlinear regression curve fit analysis of siRNA concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3).

[0028] Figure 7: Percentage inhibition of SARS-CoV-2 S pseudotyped luciferase lentivirus (MOI = 2) infection in Calu-3 cells in the presence of different concentrations (Log C) of PEP (A), rACE2 (B), mAb (C), LNP-PEP (D), LNP-rACE2 (E) and LNP-mAb (F). The nonlinear regression curve fit analysis of concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3).

[0029] Figure 8: Distribution of LNP-PEP (A) and LNP-si CE2 (B) at different time points up to 24 hours after their intranasal administration in nude mice. The LNPs were tagged with IR dye (DiR) to aid visualization. The fluorescent images (Ex / Em 745 / 800) were acquired and analyzed using IVIS imager. The quantitati ve analysis of the fluorescent images showing levels of LNP-PEP (C) and LNP- siA CE2 (D) at different time points. Values are expressed as mean ± SEM (n = 4).

[0030] Figure 9: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase lentivirus infection (MOI = 2) in airway epithelial Calu3 cell line following a combination of conditions including LNP- PEP treatment, and ACE2 and TMPRSS2 knockdown. The extent of infection was measured following a luciferase assay. Values are expressed as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, ** p < 0.01, ***p<0.001, ****p<0.0001; p < 0.05 was considered statistically significant).

[0031] Figure 10: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase LV infection (MOI = 2) in airway epithelial Calu3 cell line following treatment with different concentrations of lactoferrin (A), camostat mesylate (B) and carrageenan (C). Percentage inhibition of infection in Calu3 cells with various combinatory treatments of LNP-PEP (0.1 pg / mL), lactoferrin (10 pM), camostat mesylate (1 pM) and carrageenan (pM). The extent of infection was measured following a luciferase assay. Values are expressed as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, ****p<0.0001; p < 0.05 was considered statistically significant).

[0032] Figure 11. Transduction efficiency of SARS-CoV-2 S (XBB.1.5 Omicron) pseudotyped luciferase lentivirus at the given MOIs in HEK-293 and HEK-293-hACE2 cell lines estimated using luciferase assay. This is the latest Variant of Concern (VOC) and the data shows efficacy with this variant

[0033] Figure 12. Percentage inhibition of SARS-CoV-2 S (XBB.1.5) pseudotyped luciferase lentivirus (MOI = 0.5) infection in HEK293-hACE2 cells in the presence of different concentrations (pM) of LNP-PEP. The nonlinear regression curve fit analysis of concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3). EC50 = 2.4 pg / ml; Emax = 93.3 - 98.8%.

[0034] Figure 13. Percentage inhibition of SARS-CoV-2 S (XBB.1.5) pseudotyped luciferase lentivirus (MOI = 0.5) infection in HEK293-hACE2 cells in the presence of different concentrations (pM) of LNP-CEL. The nonlinear regression curve fit analysis of concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3). EC50 = 11.5 pm; Emax 73.7-118.2%.

[0035] Figure 14. Percentage inhibition of SARS-CoV-2 S (XBB.1.5) pseudotyped luciferase lentivirus (MOI = 0.5) infection in HEK293-hACE2 cells in the presence of different concentrations (Log C) of LNP-GA. The nonlinear regression curve fit analysis of concentration vs i nhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3). EC50 = 6.8 pm; Emax 80.3-103.4%.

[0036] Figure 15. XBB1.5 (0.5MOI) Inhibition by free GA in HEK293-hACE2. As can be seen, in the case of free GA a concentration of 1 mM (840 pg / mL) displayed over 90% inhibition and 0.5 mM displayed over 70% inhibition. In comparison 70% inhibition was observed with around 20-30 mM LNP-GA which is a 20-fold improvement. Free celastrol was toxic to HEK293-hACE2 cells at the concentration of 1 pM and above which impacted the ability to accurately access inhibitory effects on virus uptake. However, no toxicity was observed when the celastrol was attached to the LNP.

[0037] Figure 16. Inhibition of Viral Enzyme (Mpro 3CL protease). Left panel - green fluorescence for 3CL Glow assay (enzyme inhibitory activity); Right panel - Red fluorescence for 3CL Glow assay (cell cytotoxicity).

[0038] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

[0040] Described herein is a method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a pathogen replication inhibitor; and an effective amount of a pathogen binding agent.

[0041] As will be apparent to one of skill in the art, the pathogen may be a fungus, a bacterium or a virus. In some embodiments, the pathogen binding agent is formulated as a nanoparticle, for example, as a cell-impermeable lipid-nanoparticle.

[0042] As discussed herein, the formulations comprising the pathogen binding agent, referred to as LNP-Trap or Nano-trap, neutral lipids can be used, as discussed herein.

[0043] In these embodiments, the pathogen binding agent nanoparticle formulation comprises one or more phosphatidyl choline lipids, for example, but by no means limited to one or more phosphatidyl choline lipids are selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC) hydrogenated soy L-a-phosphatidylcholine (HSPC), hydrogenated Egg L-a- phosphatidylcholine (Egg PC) and combinations thereof.

[0044] Alternatively, the pathogen binding agent nanoparticle formulation may comprise two or more phosphatidyl choline lipids are selected from the group consisting of: l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl- glycero-3 -phosphocholine (POPC) hydrogenated soy L-a-phosphatidylcholine (HSPC) and hydrogenated Egg L-a-phosphatidylcholine (Egg PC).

[0045] In some embodiments, the pathogen binding agent nanoparticle formulation comprises about 10 to about 50% or 10-50% l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and about 10 to about 50% or 10-50% l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0046] As discussed herein, the pathogen binding agent or trapping nanoparticles can be used essentially as decoys which provide non-functional binding receptors or to bind to the pathogen or pathogen products or proteins. For example, the pathogen binding agent nanoparticles may comprise ligands and / or antibodies used to bind to toxins that are released by bacteria or fungi, such as for example but by no means limited to cholera toxin or botulin toxin or bacterial lipopolysaccharide (LPS).

[0047] As discussed below, in these embodiments, the targeting ligands, receptors, protein target and mode of administration are tailored to the pathogen, for example, to the virus of interest.

[0048] For example, SARS-CoV2 is somewhat unique in that it has one dominant receptor for entry into host cells (ACE2), while for other viruses there may be up to 4-6 different receptors on the host that could facilitate viral entry.

[0049] As discussed herein, the pathogen binding agent may be a receptor, an antibody or a ligand.

[0050] In some embodiments, the pathogen binding agent is a host cell receptor.

[0051] In some embodiments, the pathogen is a SARS virus, for example, SARS-CoV-2 virus and the host cell receptor is the ACE2 receptor.

[0052] As will be appreciated by one of skill in the art, these particles effectively act as decoys for incoming virus particles, binding the particles and preventing and / or reducing severity of a viral infection, as discussed herein.

[0053] In other embodiments, the pathogen binding agent is for example acetylneuraminic acid, which is a component of the cell membrane and is involved in binding of viral pathogens to the host cell.

[0054] In some embodiments, the pathogen replication inhibitor comprises an siRNA and when formulated as a nanoparticle is referred to herein as LNP-trim or Nano-trim or comprises a therapeutic and when formulated as a nanoparticle is referred to herein as LNP-treat or Nano-treat.

[0055] As will be appreciated by one of skill in the art, any pathogen protein / mRNA essential for pathogen replication or non-essential host protein / mRNA used for viral replication can be targeted by the pathogen replication inhibitor. For example, several non-structural, structural and accessory proteins can be targeted to inhibit pathogen replication, for example, viral replication.

[0056] For Nano-treat, for example, celastrol and glycyrrhizin (FIG. 15) are natural plant derived compounds that have therapeutic actions as does resveratrol (FIG. 16). Compounds like these can be loaded to form the Nano-treat particles and administered so that therapeutic compounds enter directly into the cells.

[0057] Furthermore, several naturally occurring polyphenols and their derivatives with antiviral and anti-inflammatory properties against SARS-CoV-2 and other viruses can be formulated into LNP-Treat or LNP-Trap based on their mechanism of action.

[0056] For example, polyphenols and their derivatives, such as gossypol, neferine, salvianolic acid A, corilagin, hypocrellin A, acteoside, liensinine, and the like are shown to have good potency in inhibiting SARS-CoV-2 in vitro. An extensive list of these polyphenols reviewed elsewhere can be incorporated into LNP-Treat / LNP-Trap to improve efficacy.

[0056] As discussed herein, the pathogen replication inhibitors are formulated as cell-permeable nanoparticles.

[0058] As used herein, the major lipid component is referred to as the “active lipid” and is selected based on the nature of the pathogen replication inhibitor being incorporated. For example, a neutral therapeutic can be loaded using hydrophobic interaction with neutral lipids, anionic therapeutics can be loaded using cationic lipids and cationic drugs can be actively loaded with anionic lipid. This is referred to herein as “pairing” the active lipid with the intended replication inhibitor, which includes siRNAs and therapeutics.

[0059] Specifically, to optimize loading of negatively charged active compounds requires a lipid with a positive charge while a positively charged active compound requires a negatively charged (anionic lipid). In the case of a neutral active compound there is no need for a charged lipid and a neutral lipid like DOPC can be used. A higher percentage of active charged lipid, up to 50%, is desirable to maximize compound encapsulation (ratio of charged lipid to counter-charged drug molecule). In LNP-Trim, since siRNA molecules carry several negatively charged moieties, DLin-MC3, an ionizable cationic lipid, was incorporated to promote electrostatic attraction and active loading. Conversely, since the resveratrol molecule does not possess an ionizable group for charge-mediated active loading, its hydrophobic interaction was utilized for entrapment. Here, the neutral DOPC was used instead of cationic DLin-MC3, as cationic lipids without counterions can exhibit undesirable toxicity. When charge-mediated active loading is feasible, it is preferable over hydrophobic interaction alone, as the former can achieve higher drug encapsulation efficiency.

[0060] In some embodiments, the pathogen replication inhibitor nanoparticle formulation comprises a cationic lipid and a phosphatidyl choline lipid.

[0061] In addition, in some embodiments, the pathogen replication inhibitor nanoparticle formulation may comprise a cationic lipid, a neutral lipid, such as for example but by no means limited to DOPC, DSPC, POPOC and the like; or an anionic lipid such as for example but by no means limited to cardiolipin, linoleic acid, oleic acid, or the like; and a phosphatidyl choline lipid.

[0062] As discussed herein, the phosphatidyl choline lipid may be selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) hydrogenated soy L-a- phosphatidylcholine (HSPC), hydrogenated Egg L-a-phosphatidy I choline (Egg PC) and combinations thereof.

[0063] In some embodiments, the cationic lipid is selected from the group consisting of DLinDMA, DLinDAP, DLin-2-DMAP,DLin-C-DAP, DLin-S-DMA, DLin-K-DMA), DODMA and combinations thereof.

[0064] In some embodiments, the pathogen replication inhibitor nanoparticle formulation comprises about 40 to about 50 molar % cationic lipid and about 10 to about 20 molar % phosphatidyl choline lipid.

[0065] In some embodiments, the pathogen replication inhibitor nanoparticle formulation comprises about 40 to about 50 molar % DLinDMA and about 10 to about 20 molar % DSPC.

[0066] In some embodiments, the pathogen replication inhibitor nanoparticle formulation further comprises a PEG-lipid, for example, functionalized or non-functionalized DMG-PEG, DSPE-PEG, or DOPE-PEG.

[0067] The PEG-lipid may be added at about 1 to about 10 molar %.

[0068] In some embodiments, the PEG-lipid is DMG-PEG.

[0069] In some embodiments, the pathogen replication inhibitor nanoparticle formulation further comprises cholesterol, as discussed herein.

[0070] In some embodiments, the pathogen replication inhibitor nanoparticle formulation further comprises cholesterol added at about 34-= to about 42 molar % or about 38 molar % or 38 molar %. As used herein, “about” or “approximately” refers to the base number plus or minus 10%. For example, “about 100” means “90-110”.

[0071] In some embodiments, a ratio of amine groups in the lipids to the phospho groups in the siRNA is between 3-12 as discussed herein.

[0072] In some embodiments of the invention, the nanoparticle formulation comprises about 40 to about 50% active lipid, about 10 to about 20% phosphatidyl choline lipid, about 34 to about 42% cholesterol and about 1-10% PEG lipid, wherein the active lipid is paired with or compatible with loading of the replication inhibitor based on the nature or charge of the replication inhibitor, as discussed herein.

[0073] As discussed herein, the siRNA reduces translation of a protein involved in or required for viral replication, thereby reducing the rate or efficiency of replication of the pathogen.

[0074] In some embodiments, the siRNA reduces translation of a cellular receptor or a cellular protease.

[0075] In some embodiments, the pathogen is a SARS virus, for example, SARS-CoV-2, and the cellular protease is TMPRSS2 or the cellular receptor is ACE2. in addition to LNP, liposome, nanostructured lipid carrier, niosome, nanoemulsions, polymer (for example but by no means limited to PLGA, chitosan and the like) silica, metal (for example but by no means limited to gold, silver, copper and the like), metal oxide (for example but by no means limited to magnetic iron oxide, zinc oxide, titanium oxide and the like), carbon nanotube, quantum dots (for example but by no means limited to cadmium sulfide / zinc sulfide CdS / ZnS, Zinc selenide ZnSe and the like) nanoparticles and any of these combination nanocomposites (for example but by no means limited to lipid-polymer, lipid-metal / metal oxide and the like) with a protective surface coating (for example but by no means limited to silica, polymer and the like) aiding surface conjugations with virus trapping ligands (for example but by no means limited to peptide, antibody, aptamers, natural compounds (for example but by no means limited to celastrol, glycyrrhizic acid and the like)) could be implemented for inhibiting infection.

[0076] The nanoparticles may be formulated for nasal administration, intravenous administration, oral administration, ocular administration or topical administration, as discussed herein.

[0077] As discussed herein, SARS-CoV-2’ s main entry is via the nose such that the nasal epithelial cells initially become infected and then release more viral particles that can then reach the lung or perhaps even the brain. This mode of entry is used not only by most if not all other respiratory viruses but by other airborne viruses as well, meaning that formulations effective in the nose and upper airway as described herein are suitable for treating most airborne pathogens. For example, the composition may be delivered via an inhaler for lung delivery in a vapor or nebulizer type of delivery platform. Alternatively, intravenous formulations given systemically are used for pathogens circulating in the bloodstream, while vaginal delivery formulations are used for sexually transmitted viruses. Similarly, oral formulations of acid-stable nanoparticle formulations could be used for pathogens of the gastrointestinal track. Topical formulations could be used for surface pathogens or further formulated for ocular administration for treating pathogens of the eye.

[0078] In some embodiments, the cell permeable nanoparticles are administered with an antiviral agent, for example, an antiviral agent is selected from the group consisting of lactoferrin, camostat mesylate and carrageenan. In addition, these cell permeable nanoparticles can be used to deliver natural products like polyphenols that have anti-inflammatory properties to prevent pathogen induced inflammatory cascade.

[0079] Here we describe a two-pronged localized nanomaterial-based approach for limiting SARS- CoV-2 infection in nasal and upper airway epithelial cells that involves lipid nanoparticles (LNPs) that act as receptor decoys to bind viral spike protein (LNP-trap) and LNPs to delivery siRNA to host cells to knock down the viral entry targets (LNP-trim). The LNP-trap formulations consisted of cell- impermeable lipid nanoparticles surface modified with ACE2 peptide, recombinant ACE2 or SARS- CoV-2 spike mAb that can bind to SARS-CoV-2 and potentially neutralize them. The viral trapping ligands were conjugated to the LNPs through simple EDC / NHS coupling reactions.

[0018] Alternatively, trapping ligands may also be attached to nanoparticle surface following other covalent conjugation techniques such as sulfhydryl-maleimide reaction, click chemistry (e.g. alkyne-azide) or non-covalent interactions (e.g. avidin-biotin complex formation). In contrast, the LNP-trim LNP formulations were cell-permeable and capable of delivering siRNA for reducing ACE2 and TMPRSS2 expression in host cells. Target validation of the LNP-trap and LNP-trim LNP formulations were evaluated using viral spike protein and the Calu-3 human airway epithelial cell line and HEK-293 cells transfected with human ACE2 (HEK-hACE2), respectively. Pharmacological effectiveness of LNP-trap and LNP-trim formulations were evaluated in Calu-3 and HEK-hA CE2 using a pseudotyped SARS-CoV-2 lenti virus. The in vitro studies demonstrated that both the LNP-trap and LNP-trim formulations were safe and effective at inhibiting virus-host cell interactions and reducing viral infection. Initial assessment of the LNP-trap and LNP-trim LNPs following nasal administration in mice confirmed nasal retention and biocompatibility.

[0080] The ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an unprecedented threat to public health and has led to significant demand for effective antiviral therapeutics. In recent years, lipid nanoparticles (LNPs) have emerged as versatile and efficient delivery systems for various drugs, including nucleic acids and small molecules. In this study, we prepared two versatile LNP platforms that can inhibit SARS-CoV-2 infection in the nasal cavity. The first one had LNPs conjugated to SARS-CoV-2 spike protein binding ligands such as ACE2 peptide, recombinant ACE2, and a monoclonal antibody that can act as a LNP-trap for viral particles and prevent virus-host cell binding and entry. The second LNP LNP-trim was loaded with siRNA that targets and knocks down ACE2 and TMPRSS2 needed for SARS-CoV-2 host-cell entry. The prepared LNP LNP-trap and LNP-trim were biocompatible based on in vitro MTT assay in airway epithelial Calu3 cells. The LNP LNP-trap and LNP-trim effectively inhibited SARS-CoV-2 S protein pseudotyped virus infection in Calu3 cells. The two platforms were also evaluated in combinations with themselves and other commercially available antiviral agents to see possible synergy. When administered intranasally in mice, the LNP-trap and LNP-trim displayed a reasonable retention time of over three hours in the nasal cavity before being cleared. The presented LNP-LNP-trap and LNP-trim provide a platform for inhibiting viral infection in the nasal cavity.

[0081] The nasal cavities, in addition to being the primary site of contraction, are known to hold a significant amount of SARS-CoV-2 in infected people. For the LNP-trap to effectively inhibit the virus, it must bind the viral particles. Hence the retention time of the LNP-trap in the nasal mucosa will determine the instances of viral encounter and overall efficacy. In the case of LNP-trim, a retention time that would allow for the therapeutic to cross the mucosa and enter the epithelium is crucial for its efficacy.

[0082] In this study, we have demonstrated the versatility of a LNP platform by formulating nanoparticles capable of binding and neutralizing pseudotyped SARS-CoV-2 (LNP-trap) and additional formulations capable of delivery of siRNA to airway epithelial cells to silence ACE2 and TMPRSS2 (LNP-trim). Both LNP-trap and LNP-trim LNPs were shown to be safe and effective, either alone or in combination with other agents, at inhibiting SARS-CoV-2 pseudotyped lentivirus infection in airway epithelial cells.

[0083] The LNPs used in the LNP-trap and the LNP-trim formulations were prepared following microfluidic mixing method as described previously

[0032] , in which the hydrophobic components (lipids) in the organic phase and hydrophilic components in the aqueous phase were perfused through and mixed in micron-sized channels. Alternatively, other LNP formation methods such as high pressure homogenisation, microemulsion, double emulsion, solvent-emulsification diffusion, solvent injection, ultrasonication, membrane contractor technique, electrospray or similar methods may be implemented and are within the scope of the invention. The microfluidic method offers precise control over the aqueous and organic channel flow rate ratio (FRR) and the total flow rate (TFR mL / min.). Manipulation of FRR and TFR, along with the lipid composition and concentrations, allows fine-tuning of the nanoparticle size and size distribution.

[0033] In general, a fast rate of mixing of the organic and the aqueous phase cause a rapid change in polarity and burst nucleation of LNP, resulting in smaller sizes, and also, having low total lipid concentration results in smaller LNP sizes. For the present study, microfluidic parameters and the lipid component concentrations were optimized to yield LNPs that were <100 nm in size and had a narrow size distribution as determined by the low polydispersity index values of 0.2 or lower. This indicates a narrow size distribution of LNPs which is in turn associated with specific physicochemical and biological properties and homogeneity. However, it is important to note that similar therapeutic responses would be obtainable with LNPs of other size ranges, for example, >100 and < 300 nm diameter.

[0084] Microfluidic techniques offer scalability and reproducibility, allowing the production of lipid nanoparticles in large quantities with consistent quality.

[0085] LNPs have been used to encapsulate and deliver a wide range of therapeutics, including small molecules, siRNA, mRNA, etc. Solid core LNPs usually comprise a mixture of lipids chosen for their structural and functional properties. The various lipid components used in the present study included functional lipids o promote the active loading of drug molecules of interest. In some embodiments, ionizing cationic lipids (DODAP, DODMA), helper lipids (POPC, DOPC) and PEG lipids may also be added, as discussed herein For example, helper lipids such as cholesterol, DSPC, DOPC, etc. were included for structural rigidity and PEG-lipids (DSPE-PEG-COOH, DMG-PEG) for surface modification, targeting ligan attachment and stabilization. The ratio of the lipid components determines the physicochemical and functional properties and the overall stability of the resulting LNPs. In this study for LNP-trap formulation, since we didn’t intend to encapsulate any therapeutics, the functional lipid component was excluded in the composition. The LNP-COOH composition included two phosphatidylcholines (PC) - DOPC (46.5 %) and DSPC (10%), cholesterol (38%), DSPE-PEG-COOH (5%) and lipophilic dye (0.5%). These PC lipid molecules with hydrophilic phosphate head and hydrophobic acyl tail groups may have a cylindrical or conical shape. The molecular shape determines their self-assembly and the resulting phase. The DOPC molecule is cylindrical and is known to spontaneously self-assemble to form a stable lamellar phase (bilayer structures). The phase studies of DOPC, DSPC, and cholesterol mixture have been a topic of interest concerning its abundance in cell membranes.

[0034] Based on the ternary phase diagram reported for DOPC / DSPC / cholesterol, the prepared LNP-COOH should have a fluidic liquid-ordered (Lo) phase.

[0034] Hence, the DOPC / DSPC / cholesterol core could offer structural rigidity and stability to LNP-trap over its retention time at the target site. For the LNP-trim formulations, an ionizing cationic MC3 lipid was used as the functional lipid component to promote siRNA loading in the LNP, and the DSPC, cholesterol, and DMG-PEG structure and stabilization. For siRNA based cell delivery, ionizing cationic lipids with an acid dissociation constant (PKa) value below 7 are ideal.

[0035] In the case of MC3, with a PKa value of 6.4, it has a positive charge under acidic (pH < 4) conditions, which can be advantageous during LNP assembly as electrostatic attraction of the negatively charged siRNA enables high encapsulation efficiency with this paired active lipid. In this study, the LNP-siRNA LNP-trim formulations had an encapsulation efficiency of over 95%. Under physiological pH, during exposure to the cells, the MC3 are neutral, and following uptake into the acidic endosome, are once again charged enabling the endosomal escape and delivery of siRNA into the cell cytosol where they can interfere with target protein production. The shorter acyl chain in the DMG-PEG allows for more rapid disassociation from the surface of the LNP promoting enhanced uptake and knockdown efficiency. Hence the combination of MC3 and DMG- PEG has been widely used to deliver siRNA and mRNA in vitro and in vivo with great success ). [36,37] The LNP-trim formulation was shown to be very effective in knocking down ACE2 and TMPRSS2 in airway epithelial cells, with concentrations of siRNA of 20-40 nM incorporated into the LNPs capable of knocking down host cell receptor mRNA expression by ~70 %. Knocking down ACE2 and TMPRSS2 using the LNP-trims 1 and 2 effectively inhibited pseudo virus infection in airway epithelial cells most likely by inhibiting ACE2 / TMPRSS2 dependent cell entry. Hence LNP- trim 1 and 2 on their own or in combination are a safe option for inhibiting infection. In our preliminary mice studies, intranasal administration of the LNP-triml were retained for up to 3 hours in the nasal cavity.

[0086] In the preparation of LNP-trap precursor, DSPE-PEG-COOH was included in the lipid mix to form LNP-COOH with surface carboxylic acid groups for further bioconjugation reactions. For the initial screening of LNP-trap, two precursors (LNP-COOH), with 1.5% and 5% PEG-lipid (DSPE- PEG-COOH), were prepared and conjugated to ACE2 peptide. The LNP-COOH with 5% PEG-lipid was smaller (~ 60 nm) than LNP-COOH with 1.5% PEG-lipid (~80 nm). As will be appreciated by one of skill in the art, in some embodiments, a concentration range of PEG-lipid of between 1.5% to 10% may be used. The size difference was consistent with earlier reports where a higher percentage of PEG-lipid was shown to form smaller LNP sizes.

[0033] Smaller sizes can be advantageous in terms of room available to build upon with bulky targeting ligands. The ability of LNP-COOH (1.5%) and LNP- COOH (5%) conjugated to ACE2 peptide to bind to SARS-CoV-2 spike protein was evaluated using ELISA. From ELISA, it was observed that only the LNP-COOH (5%) conjugated to ACE2 peptide displayed a positive result for binding / inhibiting spike protein. The effectiveness of LNP-COOH(5%)- PEP could be attributed to the denser PEG-lipid capping offering enhanced stabilization of the LNP and accessibility of the ligand for target binding.

[0039] However, it is interesting to note a higher density of lipid-PEG greater than 10% can adversely affect the nanoparticle targeting efficiency.

[0039] All the LNP-trap formulations, including LNP-PEP, LNP-rhACE2 and LNP-mAb were shown to bind and inhibit pseudotyped SARS-CoV-2 infection in airway epithelial cells. Another important feature of these LNPs is the reduced host cell association as demonstrated by the negligible cell uptake in Calu3 cells using both cell imaging and quantitative fluorescence analysis. In contrast to the LNP-trim formulation which displayed uptake into host cells both in vitro and in vivo, there was little evidence of cellular accumulation of the LNP-trap formulations. Further imaging studies of LNP-PEP in mice post intranasal administration showed localization of LNP-PEP in the nasal cavity for up to 3 hours.

[0087] There is growing interest in developing nanomaterials that can bind to and inhibit viral infection of host cells. In a recent report, Papp et al. used a gold nanoparticle platform surface modified with sialic acid-coated glycerol dendrons to bind and inhibit the host cell uptake of influenza virus.

[0040] The influenza virus binds to sialic acid residue in the host cell via viral surface hemagglutinin (HA). In in-vitro studies, AuNPs-HA inhibited infection by 40% compared to control. Similarly, AuNPs modified with ligands for targeting a variety of viruses ranging from influenza, herpes simplex virus type 1, HIV, etc., have been reported. [41-43]

[0088] Several studies have reported using ACE2-containing exosomes for trapping and inhibiting SARS-CoV-2. The exosomes were isolated from the plasma of COVID-19 patients or cell culture media.

[0044] Others have focused on cloaking nanoparticles with host-cell receptor-expressing exosomes or cell membranes. [45 ,46] Another alternative is synthetic nano virus traps from the nano core to surface ligands. Some of the advantages of synthetic nano virus traps include precise control over the formulation and scale-up processes and the choice of materials that are tried and tested for clinical applications. In a recent report, the gold nanoparticle platform surface modified with sialic acid-coated glycerol dendrons was shown to bind and inhibit the influenza virus.

[0040] The influenza virus binds to sialic acid residue in the host cell via viral surface hemagglutinin (HA). In in-vitro studies, AuNPs-HA inhibited infection by 40 % compared to control. In another study, AuNPs were conjugated to truncated ACE2 peptide, and its binding to SARS-CoV-2 spike receptor binding domain was assessed based on size growth observed in dynamic light scattering.

[0047] Among the various virus trapping platforms, the LNP based LNP-trap presented here is a versatile and safe option that displayed high efficacy in trapping and inhibiting (>90 %) pseudovirus infection in airway epithelial cells. It was also observed that conjugating ligand to the LNP-trap platform especially in the case of ACE2 peptide improved inhibition of infection compared to free peptide. While not wishing to be bound to a particular theory or hypothesis, it is believed that it is the effect of PEGylation and steric stabilization of peptide molecules that improves its availability and ability to bind spike protein. In the case of free peptide the inhibition of infection plateaued at around 60% at concentrations above Ipg / mL (Figure 8A).

[0089] SARS-CoV-2 is primarily transmitted by inhaling virus-containing aerosols expelled by infected people. After entering the nasal cavity, the viral particles come in contact with the nasal mucosa lining the airway epithelium. The nasal and bronchial epithelial cells are known to express both ACE2 and TMPRSS2 required for SARS-CoV-2 cell entry and infection. [48,49] The nasal cavity is estimated to have a surface area of 160 cm2in adult humans which is substantial space for lodging airborne viruses.

[0050] Moreover, it is unclear if vaccinated people have immune protection in the nasal mucosa.

[0051] Hence, nanoparticle virus traps localized in the nasal cavity could be an effective first line of defense in binding and neutralizing incoming viruses. The intranasal delivery route has been successfully used for drug delivery. However, nanotherapeutics intended for the nasal cavity has to overcome the mucous barrier. The mucous lining the airway epithelial cells present a semi-permeable barrier to inhaled therapeutics which may undergo rapid clearance by the motile cilia. Hence nanotherapeutics should be designed to overcome these barriers to produce a therapeutic effect. For example, Nanotherapeutics intended to enter the airway epithelial to deliver siRNA / mRNA must cross the mucous barrier. On the other hand, nanotrap for virus should exhibit mucoadhesion and reasonable retendon time.

[0090] Having PEG chain on the nanoparticle surface can promote mucoadhesion properties.

[0052] While not wishing to be bound to a particular theory or hypothesis, it is believed that PEG helps mucoadhesion by means of entanglement between PEG polymer chain and mucus protein molecules Polyacrylic hydrogels and PLGA nanoparticles attached to PEG, showed mucoadhesion in ex vivo and in vivo studies. [53,54] In recent reports, LNP-siRNA with a similar lipid-PEG composition as in LNP- siACE2 and LNP-siTMPRSS2 LNP-trim was shown to deliver their siRNA payload inside the nasal cavity of mice. LNP encapsulated with luciferase mRNA and a mixture of luciferase mRNA and siRNA was shown to express luciferase in the nasal cavity and lungs four hours after intranasal administration in mice.

[0055] Thus the LNP-trim LNP-siRNA is able to penetrate the mucin layer and knock down the target gene in the nasal cavity. In the mice that were intranasally administered with LNP-siACE2, a significant drop in fluorescence intensity was not observed after three hours. This indicates that the accumulation of LNP-si CE2 inside the airway epithelial cells, thereby escaping mucous clearing. In the case of LNP-PEP, most of the nanoparticles appear to have cleared the nasal cavity at three hours. This could be attributed to the majority of nanoparticles being localized in the mucous layer and undergoing gradual clearing from the mucosa. While not wishing to be bound to a particular theory or hypothesis, it is believed that the elimination of the trapped virus through normal mucous deposition and expiration in the expired air is important and significant.

[0091] The versatile LNP-trap and LNP-trim were shown to be effective in inhibiting infection. Moreover, both LNP-trap and LNP-trims can be combined in combination therapy to achieve lower dosage and improved virus inhibition activity. The combination of LNP-trap 1 (0.1 ug / mL ACE2 PEP) and LNP-triml (10 nM si / 1 CE2) displayed 80% inhibition of infection at lower concentrations that showed no inhibition on their own. We also explored the possibility of achieving favorable combination effect with other commercially available antiviral agents such as lactoferrin, camostat mesylate and carrageenan. Combination of LNP-trap with carrageenan displayed a synergistic effect in inhibiting pseudovirus infection. Hence both these platforms are a safe option for combination therapy for topical nasal cavity administration.

[0092] In conclusion, we have developed two versatile and biocompatible LNP based antiviral agents namely LNP-trap and LNP-trim for effective inhibition of SARS-CoV-2 virus. The LNP-trap had an LNP core with surface SARS-CoV-2 spike protein binding ligands. The LNP-trap were shown to effectively inhibit pseudotyped SARS-CoV-2 lentivirus in airway epithelial cells. The LNP-trim had siACE2 or sTMPRSS2 encapsulated in the LNP core. The LNP-trims effectively knocked down ACE2 and TMPRSS2 in airway epithelial cells and as a result inhibited pseudotyled-SARS-CoV-2 lentivirus entry and infection. Both LNP-trap and LNP-trim formulations administered intranasally in mice were shown to have a reasonable retention time in the nasal cavity.

[0093] The LNPs were loaded with resveratrol (25 and 50 uM) and the ability to inhibit the Mpro 3CL protease (the main viral enzyme that is targeted to prevent virus replication) was determined using a 3CL transfection assay 3CLglow, as shown in Figure 16. Furthermore, using HEK293 cells transfected with the 3CL glow assay, we can monitor reductions in enzyme activity as well as assess any toxicity to the cells due to the inhibitor itself. In this way, we were able to show that the Resveratrol-LNP formulations had improved enzyme inhibition compared to free resveratrol.

[0094] Furthermore, the data included herein and summarized in Table 1 demonstrates that the Nanotrap formulations do not accumulate in host cell, due to higher PEG content that prevents interactions of the LNP with the host cell and the type of PEG-lipid (Eg. DSPE-PEG) with longer alkyl chain which maintains the integrity of LNP for longer time period than short alkyl chain PEG-Lipid. The Nano-trap formulations can inhibit from 85-95% of SARS-CoV-2 pseudoviral particle uptake in Calu 3 cells and is a versatile platform that can be surface decorated with peptide, protein and natural products that bind to spike protein and prevent uptake of viral particles into host cell. Consequently, Nano-trap formulations can be used alone or with other agents, such as for example, Nano-Trim and / or NanoTreat as well as other natural products with antiviral activity, as discussed herein.

[0095] Furthermore, while directed at local nasal delivery applications initial ly, the ability to use natural products like glyzerinic acid and celestrol means these formulations may be effective as oral agents to prevent viral particle uptake in intestines.

[0096] Regarding the Nano-Trim formulations, the incorporation of ionizable lipids allows high encapsulation efficiency for siRNA. This also allows for the delivery of siRNA without toxicity that is usually observed with other delivery systems. Finally, it is of note that the Nano-Trim formulations can be used alone or in combination with the other LNP formulations.

[0097] Regarding the Nano-Treat formulations, modification of the % of PEG in the LNP (1.5%) and PEG-Lipid with short alkyl chain (PEG-DMG) results in a cell penetrable formulation that can deliver incorporated viral protease inhibitors to the cell. This is shown by the activity demonstrated with resveratrol as the active therapeutic at concentrations that were ineffective when used without the LNP encapsulation.

[0098] Commercial applications for this technology include but are by no means limited to a localized preventative and / or treatment for airborne pathogens. In these embodiments, this includes formulation into a nasal spray for direct application to the nose, or formulation in a nebulizers or other devices to allow inhalation of the particles into upper airway.

[0099] The LNP-Trap formulations using celestrol and GA, can also be administered as an oral preventative or treatment for reducing severity of viral infection via the gastro-intestinal route.

[0100] For the LNP-trap and LNP-trim formulations, we have demonstrated the technology against SARS-CoV2 but these LNP formulations are readily adaptable to any pathogen for which the pathogen protein(s) or the host receptor(s) involved viral uptake are known. This will have applications in both the livestock industry for preventing spread of infections to livestock as well as preventing transfer of infections from the livestock to the farm workers. Accordingly, in some embodiments, the individual is an animal, for example, a livestock animal or a mammal or a human.

[0101] The invention will now be explained and / or elucidated by way of examples; however, the invention is not necessarily limited to or by the examples.

[0102] EXAMPLE 1

[0103] In this study we have explored a two-pronged approach in inhibiting SARS-CoV-2 pseudovirus. The LNP-trap that can bind to the SARS-CoV-2 S protein via surface ligand and thereby neutralize the virus. The second approach using LNP-trim looks at the effectiveness of RNAi induced host cell receptor ACE2 and transmembrane serine protease 2 (TMPRSS2) in inhibiting SARS-CoV-2 pseudovirus. The LNP-trap core platform consisted of DOPC lipid with a neutral charge, along with DSPC helper lipid and structural cholesterol and a lipid-PEG component, DSPE-PEG2K-COOH, to stabilize the LNP in an aqueous media and to facilitate ligand bioconjugations via the surface carboxylic acid group. In addition, lipophilic fluorescent dye (DiO or DiR) was also incorporated into the LNP-trap LNPs for visualization and tracking in vitro and in vivo. The DOPC / DSPC / cholesterol / DiO / DSPE-PEG-COOH LNPs were prepared following the microfluidic mixing method (Figure 2A). The LNP-trap base formulation was surface modified with either SARS- CoV-2 spike RBD targeting ACE2 peptide (LNP-trapl), rhACE2 protein (LNP-trap2) and mAb (LNP- trap3) at molar ratios of PEG: ligand 1:0.3 (Figure 2B). The resulting hydrodynamic sizes for LNP- trapl-3 were ~60 nm, -130 nm and -120 nm, respectively. All the LNP-trap formulations had a neutral zeta potential and a polydispersity index of less than 0.2.

[0104] The ability of LNP-trap 1 with 1.5 % and 5 % surface PEG conjugated with 0.1 and 0.3 molar equivalent ACE2 peptide to bind to SARS-CoV-2 spike protein was evaluated using enzyme-linked immunosorbent assay (ELISA). From ELISA the formulation with 5 % PEG and 0.3 molar equivalent ACE2 peptide (10 pg / mL) displayed a positive result with highest binding and inhibition (-31 %) of SARS-CoV-2 spike protein. Moving forward, the LNP-traps were formulated to have 5 % PEG-lipid and 0.3 molar equivalent of conjugated ligands. The biocompatibility of the LNP-trap LNPs was evaluated in airway epithelial Calu-3 cells using both cell impedance and MTT assays (Figure 3). For the cell impedance studies, the cell index (CI) in control and LNP-trap treated cells was monitored over a 24-hour period (Figure 3A). The LNP-trap LNPs, at concentrations as high as 1 mg / ml, displayed comparable baseline normalized CI values to those of the control group receiving only media. As a decrease in cell attachment or proliferation would result in a decrease in CI values over time, these studies indicated that the LNP-trap base formulation was biocompatible. The cytotoxicity of LNP-trap base formulation and LNP-trap 1, 2 and 3 were evaluated using MTT assay (Figure 3B-F). No change in cell viability was observed in the all the LNP-trap treated cells further confirming the safety of the LNP-trap platform. The uptake of LNP-trap 1 and 2 was evaluated in airway epithelial cells, a key site for SARS-CoV-2 infection. Both LNP formulations showed negligible uptake (<1%) in Calu-3 cells (Figure 4). The low cell uptake observed for the LNP-trap was likely the result of the 5% PEG-lipid on the LNP surface. PEGylation of therapeutics including nanoparticles are widely used for imparting desirable stability, non-immunogenic properties and long circulation half life.

[0031] A low cell internalization profile for the LNP-trap was desired to maximize the effectiveness for neutralizing incoming SARS-CoV-2. The LNP-trim nanoparticles (MC3 / DSPC / cholesterol / DiO / DMG-PEG) were loaded with siA CE2 (LNP-trim 1) or siTMPRSS2 (LNP-trim2) using the microfluidic mixing method. Both the LNP-trim formulations resulted in LNPs with a hydrodynamic size of ~90 nm and a neutral zeta potential. The transfection and gene knockdown efficiency of the LNP-trim formulations was evaluated in HEK-293-hACE2 and Calu-3 cells. In HEK-293-hACE2 cells, both LNP-triml and LNP-trim2 at 40 nM siRNA concentration produced maximum knockdown (>70%) of the target genes (ACE2 and TMPRSS2') at the mRNA level 72 hours after transfection (Figure 4A and B). In Calu-3 cells transfected with LNP-siA CE2 (40 nM si / l CE2), the cell internalization / localization of LNPs was visualized using fluorescence microscopy (Figure 4C). A uniform distribution of the LNP-siA CE2 in cells was observed six hours after transfection (Figure 4C). A maximum knockdown of ACE2 at the mRNA (-70%) and protein (-50%) levels was observed 72 hours after transfection with LNP-si CE2 (Figure 4D, E and F).

[0105] The transduction efficiency of the SARS-CoV-2 S pseudotyped luciferase lenti virus over a series of MOI was evaluated in HEK293, HEK-293-hACE2 and Calu-3 cells (Figure 7). In the nontransfected HEK-293 control cells, which do not express ACE2, there was no significant luminescence with any MOI examined, indicating a lack of infection in these cells (Figure 7A). On the contrary, the HEK-293-hACE2 cells showed a concentration-dependent increase in luminescence following exposure to the SARS-CoV-2 pseudotyped lentivirus (MOI of 1 - 3). Similarly, the Calu-3 human airway epithelial cell line which naturally express human ACE2 receptors also exhibited a concentration-dependent luminescence increase at all the MOIs examined (1 - 20). SARS-CoV-2 cell entry / infection is dependent on ACE2 and TMPRSS2. The infectivity of the SARS-CoV-2 pseudotyped lentivirus was examined following treatment with LNP-siA CE2 and LNP-si' / MPAASC in HEK-293- hACE2 cells (Figure 7B). Both ACE2 and TMPRSS2 knockdown in HEK-293-hACE2 resulted in over 87% inhibition of infection compared to the control (Figure 7B). Similarly, in airway epithelial Calu3 cells, knocking down ACE2 and TMPRSS2 resulted in over 90% inhibition of SARS-CoV-2 pseudoviral infection with concentrations of siRNA in the LNPs as low as 20 nM. Significant reductions in psuedoviral infection were observed with even lower concentrations of siRNA (Figure 7C and D). The IC50 values based on non-linear regression fit for LNP-siA CE2 and LNP-siTAfPRSS2 were determined to be 21.9 nM and 1.7 nM, respectively. These results confirmed that pseudotyped SARS- CoV-2 lentivirus infected cells through an ACE2 and TMPRSS2 dependent process and that the extent of infection was reduced with LNP-siA CE2 and LNP-siTAfPRSS2 treatments.

[0106] The viral neutralizing property of the LNP-trap formulations towards SARS-CoV-2 pseudotyped luciferase lend virus was also evaluated in Calu-3 human airway epithelial cells (Figure 8). In the case of free ACE2 peptide, the maximum achievable inhibition of 60% was achieved at a concentration of 1 pg / mL or above with an estimated IC50 concentration of 0.1 pg / mL (Figure 8A). In the case of rhACE2, the IC50 concentration for pseudovirus inhibition was determined to be 0.17 pg / mL with over 70% inhibition achievable at concentrations > 1 pg / mL (Figure 8B). The free mAb displayed an IC50 concentration of 0.2 pg / mL and had the greatest efficacy with over 95% inhibition of infection at 1 pg / mL concentration (Figure 8C).

[0107] Conjugation of the trapping ligands to the ENP-trap LNPs improved activity in the pseudovirus infeed vity assay (Figure 8D-F). For ENP-trap 1, maximal efficacy at inhibiting pseudo virus infectivity was increased to > 90% compared to the approximately 60% inhibition observed with free peptide. For the LNP-trap2 both efficacy and potency were considerably better than the free rhACE2 (Figure 8D). While the LNP-trap3 formulation had a similar > 90% efficacy as free mAb, there was a significant increase in potency observed.

[0108] As the intended use of the LNP-trap and LNP-trim formulations is for local administration via the nose, the retention of representative formulations of each were examined in the nasal cavity following intranasal administration (Figure 9). The localization of LNPs were fluorescently tracked over different time points as shown in figure 9 A and B. From the fluorescent images obtained, both the LNP-trapl (LNP-PEP) and LNP-triml (LNP-si CE2) were retained in the nasal cavity for up to 3 hours. In the case of LNP-trapl, fluorescent signals representing about 25% of the administered dose present even after 24 hours. The quantitative analysis of the fluorescent signals with ROI encircling the head region showed a gradual drop in signal for LNP-PEP over three hours (Figure 9C) likely due to clearing of LNPs. In the case of LNP-si CE2 the fluorescent signal remained stable over three hours indicating the retention of the LNPs in the mucosa or the airway epithelial cells. Hence it can be concluded that both LNP-PEP and LNP-siRNA with its retention in upper respiratory tract could bind and inhibit viral particles and prevent infection.

[0109] Figure 10 shows the LV inhibitory effect of combinations of LNP-trapl along with either of the LNP-trim formulations. Using a suboptimal concentration of LNP-trapl (0.1 pg / mL), LNP-triml (10 nM) and LNP-trim2 (2 nM) were used to identify the additive or synergistic effect on LV inhibition between different groups. It was observed that the combination of LNP-trapl and LNP-triml produced significantly higher levels of inhibition compared to their individual responses displaying synergism as indicated by a combination therapy index score less than 1 (CTI < 0.7). The combination of these two LNP formulations is desirable to achieve maximal inhibition of infection with the lowest effective doses. We further investigated combinations of LNP-trapl (0.1 pg / mL) with commercial agents (lactoferrin, camostat mesylate and carrageenan) with antiviral properties (Figure 11). Figure 11 A, B and C show the concentration dependent inhibition of infection in Calu3 cells with lactoferrin, camostat mesylate and carrageenan, respectively. Based on these data, we identified the concentrations of compounds that produced suboptimal inhibition of infection for further combination studies with LNP- PEP (Figure 11D). Among the combination of LNP-trapl (0.1 pg / mL), lactoferrin (10 pM), camostat mesylate (1 pM) and carrageenan (1 pM) studied, the combination of LNP-trap with carrageenan displayed a synergistic effect in inhibiting infection. (CTI < 0.7).

[0110] Materials and methods:

[0111] 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) and cholesterol was obtained from Avanti / Sigma-Aldrich (St. Louis, MO). 4- (dimethylamino)-butanoic acid, ( 10Z, 13Z)- 1 -(9Z, 12Z)-9 , 12-octadecadien- 1 -yl- 10, 13-nonadecadien- 1 - yl ester (DLin-MC3-DMA or MC3) was obtained from Cayman Chemicals (Ann Arbor, MI). SARS- CoV-2 S Pseudotyped Luciferase Lenti virus was purchased from Creative Biogene (Shirley, NY). N- (3-Dimethylaminopropyl)-M-ethylcarbodiimide hydrochloride (EDC), N-hydroxy succinimide (NHS) and Pierce™ Firefly Luciferase Glow Assay Kit was obtained from Thermo Fisher (Rockford, IL). Benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-l-propenyl]-, perchlorate (DiO) was obtained from Invitrogen (Carlsbad, CA). The ACE2 peptide (Sequence: H- EEQAKTFLDKFNHEAEDLFYQSS-OH) was custom synthesized by CanPeptide (St-Laurent, QC). The Human ACE2 Protein, His Tag and Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgGl were purchased form ACROBiosystems (Newark, DE). The SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA kit was purchased from Cell Signalling Technology (Danvers, MA). Silencer Select pre-designed siRNA targeting ACE2 (siACE2 sense 5'— >3' GGAUCCUUAUGUGCACAAAtt and antisense 5'^3' UUUGUGCACAUAAGGAUCCtg), TMPRSS2 (siTMPRSS2 sense 5'^3' GCAGAUAUGUCUAUGACAAtt and antisense 5'^3' UUGUCAUAGACAUAUCUGCtg ) and negative siRNA control siSCR were purchased from Ambion by Life Technologies (Carlsbad, CA). Lipid nanoparticles were prepared following microfluidic mixing method using NanoAssemblr Benchtop instrument by Precision NanoSystem (Vancouver, BC).

[0112] Cell culture: Human lung epithelial - Calu-3 (CVCL_0609) and human embryonic kidney epithelial - HEK-293 cell lines were obtained from ATCC (Manassas, VA). HEK-293 / human ACE2 cell line (HEK-293-hACE2) was obtained from ACRO Biosystems (Newark, DE). Calu-3 cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) (Gibco, Carlsbad, CA) with 10% fetal bovine serum (FBS; Gibco), GlutaMAX (Gibco, Carlsbad, CA) and 1% penicillin- streptomycin (Gibco, Carlsbad, CA). HEK-293 and HEK-293-hACE2 cells were cultured in DMEM (Gibco, Carlsbad, CA) supplemented with 10% FBS, non-essential amino acids (Gibco, Carlsbad, CA) and 1% penicillin-streptomycin. Cells were grown in cell culture flasks at 37 °C in a humidified CO2 (5 %) incubator for routine cell culture.

[0113] Preparation of LNP-COOH: The lipid mix containing DOPC / DSPC / cholesterol / DSPE-PEG- COOH / DiO at the molar ratio (46.5 / 10 / 38 / 5 / 0.5) and a total lipid concentration of 10 mg / mL was prepared in ethanol. The LNP-COOH was prepared following the micromixing method with the lipid mix in ethanol as the organic phase (Org.) and PBS (pH 7.4) as the aqueous phase (Aq.). A flow rate ratio (FRR) of 1:3 (organic phase: aqueous phase) and a total flow rate (TFR) of 12 mL / minute were used for the LNP-COOH preparation. The crude LNP-COOH was washed using PBS (pH 7.4) following centrifugal filtration (2000g, MWCO 10,000). The purified product in PBS was stored at 4 °C until further use.

[0114] Preparation of LNP-trap formulations: The base LNP-COOH in PBS was exchanged to 0.1 M MES buffer (pH = 6). To the LNP-COOH in MES buffer, molar excess of EDC (lOx) and NHS (25 x) with respect to DSPE-PEG-COOH was added. The contents were mixed at ambient condition for one hour. Later mercaptoethanol (0.6pL / mL) was added and the pH of the reaction mixture was adjusted to 7.4 using 1 M NaOH. Later, 0.3% molar equivalent of peptide, ACE2 or mAb compared to DSPE-PEG- COOH was added to the activated LNP-COOH and the mixture was mixed for two hours. After two hours the pH of the reaction mixture was raised to 9.0 using 1 M NaOH. Finally, the crude product was washed (PBS pH= 7.4) and concentrated using centrifugal filtration (2000g; MWCO 100 kDa). The concentration (mg / mL) of conjugated ligands was quantified based absorbance at 205 nm (A205) and Scopes method option using Thermo Scientific NanoDrop spectrophotometer.

[0030] The as-prepared LNP-PEP (LNP-trapl), LNP-rACE2 (LNP-trap2) and LNP-mAb (LNP-trap3) in PBS were stored at 4 °C for up to a month until further studies.

[0115] Preparation of LNP-trim LNP: The lipid mix containing MC3 / DSPC / cholesterol / DSPE-PEG- COOH / DiO at the molar ratio (50 / 10 / 38 / 1.5 / 0.5) and a total lipid concentration of 10 mg / mL was prepared in ethanol. The siRNA (siACE2 or siTMPRSS2') was dissolved in sodium acetate buffer (25 mmol, pH = 4) to yield 0.33 mg / mL concentration. The organic lipid mix and the aqueous siRNA solution were micromixed at a flow rate ratio (FRR) of 1 :3 (organic: aqueous) and a total flow rate (TFR) of 12 mL / min. The as prepared LNP-siRNA was diluted (50x) in PBS (pH = 7.4) and concentrated using a centrifugal filter (2000xg, MWCO 10,000) to its original volume. The concentration of the encapsulated siRNA in the LNP-siRNA formulation was measured using a Thermo Scientific NanoDrop spectrophotometer (Madison, WI). The LNP-siRNA was stored at 4 °C for up to three months without loss of stability and activity.

[0116] Size and charge determination of LNPs: The particle size (hydrodynamic) and the net surface charge (zeta potential) were measured using ZetaPALS (Brookhaven Instruments, NY, USA) dynamic light scattering instrument. LNPs were diluted to 20pg / mL lipid concentration in PBS (pH 7.4) before analysis. The polydispersity index (PDI) obtained from the dynamic light scattering instrument was used to determine the size distribution of the LNPs (lower PDI meaning monodisperse LNPs).

[0117] Cell impedance studies: The real-time cell cytotoxicity assay was performed using the Agilent xCELLigence RTCA DP instrument (San Diego, CA). Calu-3 cells were seeded (14000 cells / well) in collagen-coated xCELLigence 16 well E-plates. The E-plates were background corrected prior to seeding the cells. The E-plates were transferred to the RTCA DP instrument maintained inside the 5% CO2 incubator at 37 °C. The cell growth and attachment were monitored in real-time using cellular impedance presented as a unitless parameter termed cell index (CI). The CI values increased with cell growth / attachment. The following day different concentrations of LNP-COOH (10 - 1000 pg / mL) were added to the wells. The CI values of control and the treatment groups were recorded at 15 min intervals for 24 hours. Data analysis was performed using the xCELLigence software package to obtain the baseline normalized CI values. The control group was used to set the baseline, and the CI values were normalized to a time point just before the addition of treatments to obtain the baseline normalized CI values.

[0118] Cytotoxicity studies of LNPs: For cytotoxicity studies, Calu-3 cells were seeded (187,500 cells / cm2) in 96-well plates and grown for 24 hours. The following day desired concentrations (0.01, 0.032, 0.1, 0.32, 1.00 mg / mL) of LNPs was added to the cells and incubated for 24 hours. After 24-hours, the media and treatments were removed and replaced with fresh media and the cells were further cultured for 48 hours before MTT assay. Briefly, the MTT reagent was prepared by dissolving 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide in PBS (pH 7.4) at a concentration of 5 mg / mL. For the assay, 25 pL of the MTT reagent along with 100 pL of complete media was added to each well and the cells were incubated in 5% CO2 incubator for two hours at 37 °C. The viable cells convert water soluble MTT (yellow color) to water insoluble formazan (purple color) crystals via NAD(P)H-dependent oxidoreductase enzymes. After two hours, media with MTT reagent was carefully removed without disturbing the cells and DMSO was added to dissolve the purple formazan crystals. Later, the absorbance at 570 nm was measured using a plate reader to quantify the formazan. The percentage cell viability was calculated as follows: [(A570 Treatment / A570 control) xl00]. Cell uptake studies: Calu-3 cells were seeded (100,000 cells / cm2) in 24 well plates and grown to confluence in a 5% CO2 incubator at 37 °C. For uptake studies, 100 pg / mL of LNP-PEP and LNP- rACE2 were added to the cells and the uptake was monitored over different time points (1 - 5 hours). At each time point, the LNP treatment was removed, and the cells were washed three times with PBS (pH = 7.4). After washing, the PBS was completely aspirated, and the cells were lysed by adding 0.1 % Triton-X-100 (in PBS). The cell lysates were analyzed using Thermo Scientific™ Varioskan™ LUX multimode microplate reader at Ex / Em: 484 / 510. The amount of LNPs in lysates was quantified using a standard curve obtained using corresponding LNPs. The percentage cell uptake was calculated as described in equation (1)

[0119] (1) (Amount of LNP in cell lysate / Total amount of LNP added to cells) xlOO Transfection using LNP-siRNA: Cells were seeded (20,000 cells / cm2) in a 6-well plate. The following day, LNP-siRNA (siACE2, siTMPRSS2 or siSCR) equivalent to 40 nM siRNA concentration was added to the wells and incubated for 24 hours in a 5% CO2 incubator at 37 °C. After 24 hours, the transfection media was replaced with fresh complete media, and the cells were placed in the CO2 incubator. The transfected cells were further incubated for the desired length of time for before mRNA and protein isolation for quantification. For pseudovirus infection studies, cells were seeded (20,000 cells / cm2) in T-25 flasks and transfected with LNP-siRNA, as discussed above. Forty-eight hours after transfection, the cells were seeded in 96 well plates (20,000 cells / well) for further pseudovirus infection studies.

[0120] Real-Time One-Step RT-PCR: Total mRNA was isolated from cells using TRIzol reagent (Invitrogen, Burlington, ON), following the manufacturer’s protocol. The concentration of isolated mRNA in the solution was estimated spectrophotometrically. The one-step qPCR reactions were performed using iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad) following the manufacturer’s protocol. For a 20 pL reaction, 0.5 pg mRNA was used. The primers (Invitrogen) specific for ACE2 (sense 5'-GGTGGGAGATGAAGCGAGAG-3' and antisense, 5'- ACATGGAACAGAGATGCGGG- 3'), TMPRSS2 (sense 5'-AGGTGAAAGCGGGTGTGAGG-3' and antisense, 5'- ATAGCTGGTGGTGACCCTGAG-3'), and the internal control 18S (sense 5'- AAACGGCTACCACATCCAAG-3' and antisense, 5'-CCTCCAATGGATCCTCGTTA-3') was used. Thermocycling [Reverse transcription: 50 °C (10 min.), Polymerase activation and DNA denaturation: 95 °C (1 min.), 40 cycles (Denaturation: 95 °C (15 sec.), Annealing: 60 °C (60sec.) and readout)] was carried out using AppliedBiosystems AB7500 instrument. The relative mRNA levels compared to controls were calculated following the 2-AACTmethod. Western Blot: Cells were washed with PBS (3x) and lysed using RIPA buffer. Lysates in ice were sonicated for 10 seconds and centrifuged at 15000g for fifteen minutes. The supernatant was collected, and the total protein concentration was measured using the Pierce® BCA Protein Assay Kit (Fisher Scientific, Waltham, MA). The lysates (40 pg protein / well) were mixed with 5x loading buffer and separated on 10% polyacrylamide gel, and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was incubated for an hour in blocking buffer [5% (w / v) skimmed milk in TBST - tris-buffered saline (pH 7.4) with 0.1% (v / v) Tween®20] for an hour at room temperature. The membrane was incubated overnight with anti -A CE2 (SN0754) in 5% (w / v) skimmed milk in TBST - tris-buffered saline (pH 7.4) at 4 °C overnight. The membrane was washed (4x 15 minutes) in TBST buffer and incubated with HRP-conjugated secondary antibody for one hour at room temperature. Later, the membranes were washed (4x 15 minutes) in TBST buffer, and blots were visualized by enhanced chemoluminescence (Biorad) as per the manufacturer’s protocol.

[0121] Staining and Confocal Imaging: Calu3 cells were seeded (10000 cells / chamber) in 8-well glass bottom chamber slides (Thermo, 155382) and grown to -50% confluency. Cells were treated with 200 pg / mL of LNP-siA CE2 and LNP-PEP and incubated for three hours at 37 °C. Following the incubation period, the treatments were removed, and the cells were washed three times with PBS (pH = 7.4). Cell membranes were stained with Wheat Germ Agglutinin Alexa Fluor™ 594 (WGA 5940: Thermo, W11262) to visualize cell boundaries. WGA 594 conjugate was prepared according to manufacturer’s recommendations. Briefly, 5mg of WGA 594 conjugate was resuspended in 5mL of lx PBS to make a 1.0 mg / mL stock solution. Protein conjugate was centrifuged to eliminate protein aggregates and reduce non-specific binding and supernatant was used for the experiment. A working concentration of 5 pg / mL was made up in Hanks Buffered Salt Solution (HBSS) for staining. Cells were stained for 10 mins at 37 °C and fixed with 4% paraformaldehyde for 15 mins. Cells were washed three times with lx PBS and nuclei were stained with Hoechst dye (Thermo, 33342) at a final concentration of 20 pM for 30-mins at room temperature and washed with lx PBS prior to mounting. Slides were allowed to dry and were mounted with Fluoromount-G® Mounting Medium (SouthernBiotech, 0100-01). A 1.5mm thick microscope cover glass was used (Fischer, 12544G). Standard 2D and z-stack images were acquired using CarlZeiss LSM700 laser scanning confocal microscope and visualized using the ZenPro software. All images were taken using a 40x / 1.4 oil -immersion objective. Imaging parameters for z- stack acquisition were selected to optimize resolution and are as follows: x-, y-scaling = 0.156pm, z- scaling =lpm, pixel dwell=3.5ps, average = line 2, master gain = 550, digital gain = 1, pinhole=34pm, 30pm, and 39pm, beam splitters = 585nm, 587nm and 492nm, laser power 2%. SARS-CoV-2 spike protein binding studies: The ability of LNP-trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following manufacturer’s protocol. Briefly 65 pL of LNP-PEP of desired concentrations and controls were pre-incubated with 65 pL SARS-CoV-2 Spike RBD Protein for one hour at 37 °C. Later 100 pL of the preincubated sample mixtures were added to the ACE2 protein coated microwell plate. The wells were sealed with the sealing tape and incubated for 1 hour at 37 °C. The contents in the wells were removed and washed four times with supplied ELISA wash buffer. To the washed wells, 100 pL of supplied 3, 3’, 5, 5’ tetramethylbenzidine (TMB) was added, sealed with the tape and incubated for ten minutes in dark. Later 100 pL of the supplied stop solution was added to the assay wells and the absorbance at 450 nm was measured using a plate reader. The percentage inhibition was calculated using the following equation:

[0122] 100 - [(OD value of Sample 4- OD Value of RBD-HRP only control) x 100%]

[0123] The inhibition > 20% indicated a positive result for SARS-CoV-2 spike protein binding and inhibition. Infection using SARS-CoV-2 S pseudotyped luciferase lentivirus (LV): Cells were seeded (62,500 cells / cm2) in 96 well -plate and incubated overnight at 37 °C with 5% CO2. The following day desired amount of LV transduction unit [TU = MOI x cell number] corresponding to the desired mul tipi icity of infection (MOI) was added to the cells and incubated for 6 hours. Six hours later, the media containing LV was removed and replaced with fresh media. Forty-eight hours after infection, the transduction efficiency was estimated using a firefly luciferase assay kit following the manufacturer’s protocol.

[0124] Pseudotyped SARS-CoV-2 virus inhibition assay: Virus inhibition assays using LNP-trap and LNP- trim formulations were performed the cells were seeded and cultured as described above in the virus infection studies. For studies using the LNP-trap formulations, various amounts of LNP-PEP, LNP- rACE2 or LNP-mAb were added to the cells along with LV for cell infection as described above. In the case of studies with LNP-trim formulations, the cells were first treated with various concentrations of LNP-siA CE2 or LNP-siTMPRSS2, as described earlier. Forty-eight hours after transfection, the cells were seeded for the LV infection studies, as described above. Forty-eight hours after LV infection, the transduction efficiency was estimated using a firefly luciferase assay kit following manufacturer’s protocol. The percentage inhibition of the virus was calculated as follows:

[0125] Intranasal administration and distribution of LNP-PEP and LNP-siA CE2-. Adult male and female nude mice were purchased from Charles River. The mice were maintained in the University of Manitoba Central Animal Care Facility under a temperature-controlled environment with a 12-hour dark-light cycle and unlimited access to food and water. All experiments complied with the Canadian Council on Animal Care guidelines and the protocol (#22-040) approved by the University of Manitoba Animal Care Committee. Before intranasal administration, the mice were anesthetized with 2.5% isofluorane. The LNP formulation (LNP-PEP and LNP-si CE2) in PBS was administered with a Hamilton microsyringe with blunt needle tip inserted 3 mm into the nostril (5 pL per nostril; 4 mg / Kg final dose). Serial images of the fluorescent LNPs were taken using a Perkin Elmer / Caliper IVIS Spectrum optical imaging system and Ex / Em wavelengths of 745 / 800 nm and total fluorescence activity in nasal passage monitored from 0-240 minutes following nasal administration. Blood and tissue samples were collected at 24-hours following LNP administration.

[0126] Analysis of coefficient of therapy interaction (CTI): Synergistic activity was evaluated using a coefficient of therapy interaction (CTI). The CTI values were calculated using the following equation: CTI = AB / (A x B), where AB is the ratio of the RLU (luminescence assay) of the combination of two therapies (A and B) to the control, and A and B are the ratios of the individual treatment groups to the control.38-39A CTI < 1 indicates synergism, a CTI = 1 indicates an additive response, and CTI > 1 indicates antagonism of the combined therapies.

[0127] Statistical analysis: All data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way or two-way ANOVA, followed by Tukey’s test. In all studies, p < 0.05 was considered statistically significant.

[0128] While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

[0129] Table 1: Summary of LNP Formulations REFERENCES

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Claims

CLAIMS1. A method of treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen: an effective amount of a cell-permeable nanoparticle formulation of a pathogen replication inhibitor; and an effective amount of a cell-impermeable nanoparticle formulation of a pathogen binding agent.

2. The method according to claim 1 wherein the pathogen is a fungus, a bacterium or a virus.

3. The method according to claim 1 wherein the cell -impermeable pathogen binding agent nanoparticle formulation comprises one or more phosphatidyl choline lipids.

4. The method according to claim 1 wherein the one or more phosphatidyl choline lipids are selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 -palmitoyl -2-oleoyl-glycero-3-phosphocholine (POPC) hydrogenated soy L-a-phosphatidylcholine (HSPC), hydrogenated Egg L-a- phosphatidylcholine (Egg PC) and combinations thereof.

5. The method according to claim 1 wherein the pathogen binding agent nanoparticle formulation comprises two or more phosphatidyl choline lipids are selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) hydrogenated soy L- a-phosphatidylcholine (HSPC) and hydrogenated Egg L-a-phosphatidylcholine (Egg PC).

6. The method according to claim 1 wherein the pathogen binding agent nanoparticle formulation comprises about 10 to about 50 molar % l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and about 10 to about 50 molar % l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

7. The method according to claim 1 wherein the pathogen binding agent is a receptor, an antibody or a ligand.

8. The method according to claim 1 wherein the pathogen binding agent is a host cell receptor.

9. The method according to claim 8 wherein the host cell receptor is the ACE2 receptor.

10. The method according to claim 1 wherein the cell-permeable nanoparticleformulation comprises about 40 to about 50 molar % active lipid, about 10 to about 20% phosphatidyl choline lipid, about 34 to about 42% cholesterol and about 1 to about 10% PEG-lipid.

11. The method according to claim 1 wherein the pathogen replication inhibitor comprises a siRNA.

12. The method according to claim 10 wherein the the active lipid is a cationic lipid.13 The method according to claim 12 wherein the cationic lipid is selected from the group consisting of DLinDMA, DLinDAP, DLin-2-DMAP,DLin-C-DAP, DLin-S-DMA, DLin-K- DMA), DODMA and combinations thereof.

14. The method according to claim 10 wherein the phosphatidyl choline lipid is selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) hydrogenated soy L-a-phosphatidylcholine (HSPC), hydrogenated Egg L-a- phosphatidylcholine (Egg PC) and combinations thereof.

15. The method according to any one of claims 12-14 wherein the pathogen replication inhibitor nanoparticle formulation comprises about 40 to about 50 molar % DLinDMA and about 10 to about 20 molar % DSPC.

16. The method according to claim 10 wherein the PEG-lipid is functionalized or non-functionalized DMG-PEG, DSPE-PEG, or DOPE-PEG.

17. The method according to claim 10 wherein the PEG-lipid is DMG-PEG.

18. The method according to claim 12 wherein a ratio of amine groups in the lipids to the phospho groups in the siRNA is between 3-12.

19. The method according to claim 12 wherein the siRNA reduces translation of a protein involved in or required for viral replication.

20. The method according to claim 19 wherein the siRNA reduces translation of a cellular receptor or a cellular protease.

21. The method according to claim 20 wherein the cellular protease is TMPRSS2.

22. The method according to claim 20 wherein the cellular receptor is ACE2.

23. The method according to claim 1 wherein the nanoparticles are formulated for nasal administration.

24. The method according to claim 1 wherein the nanoparticles are formulated for intravenous administration.

25. The method according to claim 1 wherein the nanoparticles are formulated for oral administration.

26. The method according to claim 1 wherein the nanoparticles are formulated for ocular administration.

27. The method according to claim 29 wherein the nanoparticles are formulated for topical administration.

28. The method according to claim 1 wherein the nanoparticles are administered with an antiviral agent.

29. The method according to claim 28 wherein the and viral agent is selected from the group consisting of lactoferrin, camostat mesylate and carrageenan.

30. The method according to claim 1 wherein the cell-impermeable nanoparticle formulation of the pathogen binding agent comprises 10-50 molar % of at least two phosphatidyl choline lipids selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) hydrogenated soy L-a-phosphatidylcholine (HSPC) and hydrogenated Egg L-a- phosphatidylcholine (Egg PC); and the cell-permeable nanoparticle formulation of the pathogen replication inhibitor comprises about 40 to about 50 molar % active lipid, about 10 to about 20% phosphatidyl choline lipid, about 34 to about 42% cholesterol and about 1 to about 10% PEG-lipid.

31. The method according to claim 30 wherein the pathogen binding agent is the ACE2 receptor.

32. The method according to claim 30 wherein the pathogen replication inhibitor is an siRNA and the active lipid is a cationic lipid.

33. The method according to claim 32 wherein the siRNA targets transmembrane serine protease 2.

34. The method according to claim 30 wherein the pathogen replication inhibitor is a pathogen treatment agent.

35. Use of a composition comprising: a cell-permeable nanoparticle formulation of a pathogen replication inhibitor; and a cell-impermeable nanoparticle formulation of a pathogen binding agent for treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen.

36. A composition comprising: a cell-impermeable nanoparticle formulation of a pathogen replication inhibitor; anda cell-permeable nanoparticle formulation of a pathogen binding agent for treating or preventing or reducing the severity of symptoms associated with exposure to a pathogen comprising administering to an individual who is at risk of being exposed to or who has been exposed to said pathogen.

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