Methods and compositions for treating respiratory infections

Abstract

Disclosed herein are compositions comprising Oncostatin M, derivatives, variants, and functional fragments thereof. Additionally, methods of using the composition are disclosed for counteracting the immunopathological effects of IFN-I during a viral respiratory infection, are also disclosed. The compositions may be used to maintain or restore alveolar type II composition, maintain or increase epithelial cell proliferation, and maintain or restore lung epithelial barrier integrity.

Description

[0001] PATENT APPLICATION Docket No. HRVY-259-WO1

[0002] METHODS AND COMPOSITIONS FOR TREATING RESPIRATORY INFECTIONS

[0003] RELATED APPLICATION(S)

[0004] This application claims the benefit of and priority to U.S. Provisional Application No.

[0005] 63 / 787,651, filed April 11, 2025, U.S. Provisional Application No. 63 / 763,126, filed on February 25, 2025, and U.S. Provisional Application No. 63 / 751,143, filed on January 29, 2025. The entire teachings of the above applications are incorporated herein by reference.

[0006] GOVERNMENT SUPPORT

[0007] This invention was made with government support under GM150816 and DK043351 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0008] BACKGROUND OF THE INVENTION

[0009] Acute viral respiratory infections caused by viruses such as influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV), place a large burden on healthcare resources and an economic strain on patients and communities. While survival from viral respiratory infections is improving, many of the patients present with postinfectious pulmonary complications. Many of these postinfectious pulmonary complications have been ascribed to the destruction of epithelial cells and the epithelial barrier through mediators of pulmonary inflammation. Thus, there is a need to increase epithelial cell proliferation and restore the epithelial barrier, to thereby reduce or eliminate the incidence of pulmonary complications in patients that have or have had viral respiratory infections.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the disclosure is directed to a method of treating a respiratory infection in a subject comprising administering an agent to the subject prior to, during or after a period of peak infection, wherein the agent is selected from the group consisting of Oncostatin M2 Docket No. HRVY-259-WO1 (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant.

[0012] In another aspect, the disclosure is directed to a method of treating, preventing or reducing lung damage in a subject comprising administering an agent to the subject selected from the group consisting of Oncostatin M (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant.

[0013] In some embodiments, the agent is administered at the onset of a respiratory infection, at a period of peak infection, or during a period of elevated lung damage compared to a baseline level of lung damage. In some embodiments, the period of peak infection is measured from symptom onset. In some embodiments, the period of peak infection or elevated lung damage is determined via a pulmonary function test. In some embodiments, the period of peak infection or elevated lung damage is characterized by increased interferon levels. In some embodiments, the period of peak infection or elevated lung damage is characterized by an inflammatory response.

[0014] In some embodiments, the period of peak infection occurs about 3 to 10 days postinfection. In some embodiments, the period of peak infection occurs about 4 to 6 days postinfection. In some embodiments, the agent is administered for a defined period of time. In some embodiments, the agent is administered for 3 to 10 days.

[0015] In some embodiments, the agent is administered via inhalation, intranasally, intravenously, intraperitoneally, intrathecally, or orally. In some embodiments, the agent is administered via a nebulizer or inhaler. In some embodiments, the agent is administered intravenously.

[0016] In some embodiments, administration of the agent restores epithelial cell proliferation. In some embodiments, administration of the agent restores ATII proliferation. In some embodiments, the agent comprises recombinant OSM (rOSM). In some embodiments, wherein upon completion of administration of OSM to the subject the levels of OSM in the subject decrease over a period of time.

[0017] In some embodiments, the agent is administered in conjunction with at least one additional agent. In some embodiments, the at least one additional agent comprises an antiviral agent, an anti-inflammatory agent, an expectorant, a cough suppressant, an antibiotic, an antifungal agent, or a bronchodilator.

[0018] 4909-9827-7258, v. 13 Docket No. HRVY-259-WO1 In another aspect, the disclosure is directed to compositions comprising at least one pharmaceutically acceptable excipient and an agent selected from the group consisting of Oncostatin M (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant. In some embodiments, the composition takes the form of nanoparticles, microparticles, a liposomal formulation, a micellar formulation, a water-in-oil emulsion, an oil-in-water emulsion, a solution, or a liquid suspension.

[0019] In another aspect, the disclosure is directed to a device configured to deliver an aerosol to a lung of a subject, wherein the device contains the composition as disclosed herein. In some embodiments, the device further contains a propellent. In some embodiments, the device is a dry powder inhaler, and optionally, wherein the at least one pharmaceutically acceptable excipient comprises lactose.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0022] FIGS. 1A-1L demonstrate OSM is required to maintain ATII epithelial cell composition and survive IAV infection. FIG. 1A shows OSM protein levels from homogenized lungs as detected by ELISA (n = 4). FIGS. IB- IE show mice were infected intranasally (i.n.) with 225 - 300 PFU of A / WSN / 1933 (H1N1) and monitored for body weight daily (n = 4) (FIG. IB). Protein from BALF and plasma (FIG. 1C) and RNA from whole lung (FIG. ID) were harvested at indicated time points (n = 3). FIG. IE shows percent initial body weight (left panel) and survival curve (right panel) after infection (n = 3 - 4); representative of at least two independent experiments. FIG. IF provides a schematic of experimental design. FIG. 1G shows uniform manifold approximation and projection (UMAP) clustering and cell cluster annotation of scRNAseq data from the lungs of mock-infected and lAV-infected mice at 2dpi (n = 2). FIG. 1H provides Osm expression in annotated cell clusters. FIG. II shows Osmr expression in annotated cell clusters. FIG. 1 J shows UMAP of re-clustered ATII sub-populations across genotypes. FIG. IK shows UMAP of re-clustered ATII sub-populations in all mice (left panel) and stacked bar plot of ATII subpopulation proportions at baseline or 2dpi across genotypes (right panel). FIG. IL provides a

[0023] 4909-9827-7258, v. 14 Docket No. HRVY-259-WO1 heatmap of the top 7 marker genes for each ATII sub-population. Subclusters were randomly down sampled to 150 cells to populate heatmap. Female mice were used for experiments in this figure. Error bars indicate standard deviation (SD), *p<0.05, ****p<0.0001, Mann-Whitney U test for (FIG. 1A), two-way ANOVA up to day 8 for (FIG. IE, left panel), logrank Mantel-Cox test for (FIG. IE, right panel). EOD = level of detection.

[0024] FIGS. 2A-2J demonstrate OSM-deficiency results in exacerbated IFN-I responses and lung immunopathology during IAV infection and increased susceptibility to viral mimic challenge. FIGS. 2A-2G show mice were infected i.n. with 225 - 450 PFU of A / WSN / 1933 (H1N1) and RNA from whole lung (FIG. 2A) and protein from BAEF (FIGS. 2B and 2C) were harvested at indicated time points (n = 3 - 6). FIG. 2D provides representative histological images of mouse lungs at baseline and various time points throughout the course of IAV infection. FIG. 2E provides histological scoring of the percentage of the lung exhibiting severe damage (n = 3 - 5). FIG. 2F shows BALF was harvested at 2dpi (n = 4 - 6). FIG. 2G shows lung barrier permeability Evans blue dye (EBD) assay of BALF at 6dpi (n = 5 - 7); representative of at least two independent experiments. FIGS. 2H-2J show mice were treated intratracheally (i.t.) with 33.75 - 50 pg of poly(I:C) daily for five consecutive days, and monitored daily. FIG. 2H provides a schematic of experimental design. FIG. 21 shows BALF was collected at indicated time points (n = 4 - 5). FIG. 2J shows mice were monitored for body weight (left panel) and survival (right panel) (n = 3); representative of at least two independent experiments. Male mice were used for (FIG. 2G) and (FIG. 2J), and female mice used for the remaining experiments. Error bars indicate SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant, two-way ANOVA for (FIG. 2A) and (FIG. 2E), Mann-Whitney U test for (FIG. 2B), (FIG. 2C), and (FIG. 2F), unpaired Student’s t test for (FIG.

[0025] 2G), two-way ANOVA up to day 7 for (FIG. 2J, left panel), log-rank Mantel-Cox test for (FIG. 2J, right panel). LOD = level of detection. Relative expression calculated as (2~ACt)*1000.

[0026] FIGS. 3A-3L demonstrate macrophage-derived OSM is required for survival during poly(I:C) challenge. Mice were treated with 33.75 - 50 pg poly(I:C) i.t. daily for five consecutive days and monitored daily. FIG. 3A shows mice were monitored for body weight (left panel) and survival (right panel) (n = 5 - 7). BALF was collected (FIG. 3B) and whole lung RNA harvested (FIG. 3C) at 7 days post- initial poly(I:C) challenge (dpc) (n = 4 - 6); representative of at least two independent experiments for FIGS. 3A-3C. FIG. 3D shows lung barrier permeability Evans blue dye (EBD) assay of BALF at 7dpc (n = 3 - 5). FIG. 3E shows

[0027] 4909-9827-7258, v. 15 Docket No. HRVY-259-WO1 BALF was collected at 7dpc (n = 3 - 5). FIGS. 3F-3G shows lungs were harvested and digested for analysis of total epithelial cell loss (FIG. 3F) and ATII cell loss (FIG. 3G) at 7dpc (n = 11); pooled from three independent experiments. FIG. 3H shows mice were treated intravenously (i.v.) with either 200 pg of anti-mouse IFNAR-1 or isotype control antibody diluted in 100 pl PBS on -1, 0, 1, 3, and 4 dpc. Mice were weighed daily (n = 3 - 4). FIGS.

[0028] 3I-3L shows mice were treated i.t. with Ipg of mouse rOSM in addition to 33.75 - 50pg of poly(I:C) daily for five consecutive days. FIG. 31 shows mice were weighed daily (n = 4 - 5). FIG. 3J shows BALF was harvested at 7dpc (n = 6 - 8). FIGS. 3K-3L show lungs were harvested and digested for analysis of Ki67+ ATII cell numbers (FIG. 3K) and total ATII cell numbers (FIG. 3L) (n = 5); representative of at least two independent experiments for FIGS.

[0029] 3H-3L. A combination of female and male mice was used for experiments in this figure. Error bars indicate SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant, two-way ANOVA up to day 7 for (FIG. 3A, left panel), up to day 6 for (FIG. 3H) and up to day 15 for (FIG. 31), log-rank Mantel-Cox test for (FIG. 3A, right panel), Mann- Whitney U test for (FIG. 3B), (FIG. 3D), unpaired Student’s t test for (FIG. 3C), (FIG. 3E), (FIG. 3F), and (FIG. 3G), nonparametric one-way ANOVA for (FIG. 3J), one-way ANOVA for (FIG.

[0030] 3K) and (FIG. 3L). LOD = level of detection. Relative expression calculated as (2ACt)*1000.

[0031] FIGS. 4A-4H demonstrate exogenous OSM restores epithelial composition in OSM-deficient mice. FIG. 4A shows UMAPs displaying density cluster-specific gene programs and genotype distribution across subpopulations from baseline scRNA seq data in FIG. 1. FIGS.

[0032] 4B-4E shows mice were treated i.t. with either PBS or 1 pg of OSM daily for seven consecutive days. On the day following the final treatment, lungs were harvested and ATII cells sorted for subsequent bulk RNA-seq analysis. FIG. 4B provides a schematic of experimental design. FIG. 4C provides a Volcano plot for cluster 1: OSM-independent gene signature of Osm+I+vs Osm'' mice treated with PBS or a Volcano plot of Osm'' mice treated with rOSM vs untreated Osm'' mice. FIG. 4D provides a volcano plot for cluster 2: OSM-dependent gene signature of Osm+I+vs Osm'' mice treated with PBS or a Volcano plot of Osm'' mice treated with rOSM vs untreated Osm'' mice. FIG. 4E provides a heatmap depicting the expression levels of genes differentially expressed between groups (2-fold differential) and clustered by rows using Pearson correlation. Rows represent genes, and columns represent samples. FIGS. 4F-4H shows mice received daily intratracheal administrations of Ipg mouse rOSM and intraperitoneal injections of 1 pg EdU for three consecutive days. Lungs were harvested 24 hours after the final treatment, and EdU-i- ATII

[0033] 4909-9827-7258, v. 16 Docket No. HRVY-259-WO1 and Ki67+ ATII numbers and proportions evaluated. FIG. 4F provides a schematic of experimental design. FIG. 4G shows quantification of the proportion (left panel) and numbers (right panel) of Ki67+ ATIIs (n = 5). FIG. 4H shows quantification of the proportion (left panel) and numbers (right panel) of EdU-i- ATIIs (n = 5); representative of at least two independent experiments for FIGS. 4G-4H. Female mice were used for experiments in this figure. Error bars indicate SD, **p<0.01, Mann- Whitney U test for (FIG. 4G) and (FIG. 4H).

[0034] FIGS. 5A-5E demonstrate OSM overcomes growth suppressive effects of IFN-I to promote epithelial proliferation. Organoids were cultured in the presence or absence of IFN-I (200 U / ml) supplemented with mouse rOSM at 50 ng / ml, rIL-ip at 20 ng / ml or rIL-6 at 50 ng / ml. FIG. 5A provides an experimental schematic. FIG. 5B provides representative images for organoid cultures. FIG. 5C shows quantification for organoid forming efficiency (OFE). FIG. 5D shows quantification for mean organoid area. FIG. 5E provides a model figure. Experiments in this figure are representative of two independent experiments. Error bars indicate SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant, Brown-Forsythe and Welch ANOVA for (FIG. 5C), and Ordinary one-way ANOVA for (FIG. 5D).

[0035] FIG. 6 demonstrates immune and non-immune cells recovered from lungs of uninfected and lAV-infected mice. A heat map displaying top 7 marker genes (by descending adjusted p-val) of clusters identified in scRNA-seq. Clusters were randomly down sampled to 150 cells to populate heatmap.

[0036] FIGS. 7A-7C demonstrate OSM is expressed by lung myeloid cells and OSMR is expressed by non-immune cells in humans. FIG. 7A shows UMAP clustering and cell cluster annotation of publicly available scRNAseq data from dissociated lung samples of healthy and COVID- 19-infected individuals. Annotations as performed in original manuscript. FIG. 7B shows OSM expression in annotated cell clusters. FIG. 7C shows OSMR expression in annotated cell clusters.

[0037] FIGS. 8A-8E demonstrate Osm is induced by myeloid cells in response to homeostatic signals, and stress and pathogen signals. FIG. 8A shows bone-marrow derived-macrophages (BMDMs) were treated with indicated PAMPs or stressors, supernatant was collected at indicated time points, and protein levels were assessed by ELISA (n = 3).

[0038] Unpaired Student’s t test. Representative of at least two independent experiments. Error bars indicate SD, ****p<0.0001. FIG. 8B shows BMDMs were treated with poly(I:C) at 50 pg / ml and RNA collected at indicated time points (n = 3). Representative of two independent experiments. Error bars indicate SD. Relative expression calculated as (2ACt)*1000. FIG. 8C

[0039] 4909-9827-7258, v. 17 Docket No. HRVY-259-WO1 shows AMs were treated with poly(I:C) at 50 pg / ml, GM-CSF at lOOng / ml, or a combination of both. Supernatant was collected 24 hours after treatment, and protein levels were assessed by ELISA (n = 3). One-way ANOVA. Representative of at least two independent experiments. Error bars indicate SD, ****p<0.0001, ns = not significant. LOD = level of detection. FIG. 8D shows AMs and moAMs were treated with poly(I:C) at 50 pg / ml and GM-CSF at 100 ng / ml. Supernatant was collected 24 hours after treatment, and protein levels were assessed by ELISA (n = 3). One-way ANOVA. Representative of two independent experiments. ***p<0.001, ****p<0.0001, ns = not significant, LOD = level of detection. FIG. 8E provides representative flow cytometry gating for AMs (CD45+ CD64+ F4 / 80+ CD11C+ Siglec F high) and moAMs (CD45+ CD64+ F4 / 80+ CD11C+ Siglec F low), with both populations gated on viable singlets.

[0040] FIGS. 9A-9B demonstrate Lung epithelial cells respond to OSM. Cells isolated from digested lungs from wild type mice were ex vivo stimulated with rOSM (100 ng / ml) for 30 minutes and stained for surface markers and intracellular pSTAT3. FIG. 9A provides a summary representation of pSTAT3 geometric mean fluorescence intensity (gMFI) in indicated populations following stimulation. pSTAT3-PE fluorescence signal was normalized per population by subtracting the PE signal in pSTAT3 fluorescence-minus one controls (FMO) from the stained sample gMFI values. FIG. 9B provides a representative plot for pSTAT3 staining in epithelial cells following stimulation. Epithelial cells were defined as EpCAM-i- CD45- CD31-, endothelial cells as EpCAM-CD45-CD31+, stromal as EpCAM-CD45-CD31-, all gated on viable singlets (n = 5; representative of two independent experiments). Two-way ANOVA. Error bars indicate SD, ***p<0.001, ****p<0.0001, ns = not significant.

[0041] FIGS. 10A-10E demonstrate Osm- / - mice exhibit an enhanced IFN response during IAV infection. FIG. 10A shows mice were infected i.n. with 225pfu of A / WSN / 1933(H1N1) and whole lung RNA was harvested at 3 or 7 dpi for bulk RNA-sequencing. FIG. 10B provides a transcriptional analysis of whole lung gene expression (n = 2). Pathway enrichment analysis performed using Enrichr on genes that were at least 0.5 log2 fold change upregulated in both Osm+7+and Osm7' mice at 3dpi versus uninfected, and 0.25 log2 fold change more differentially upregulated in lAV-infected Osm7' mice as compared to Osm+7+mice. Gene expression pathways were retrieved from the Reactome Database. FIG. 10C provides FC / FC plots comparing gene expression values at 3dpi versus UI, in Osm'7' versus Osm+7+mice (n = 2). Highlighted genes were induced at least 1.5x fold-change in 3dpi / UI

[0042] 4909-9827-7258, v. 18 Docket No. HRVY-259-WO1 lungs for both Osm+7+and Osm7' mice and had 1.25x higher fold change values in Osm7' compared to Osm+7+mice. FIG. 10D provides volcano plots illustrating expression of MSigDB hallmark interferon alpha response signature genes between Osm+7+and Osm7' mice at 3dpi and 7dpi. FIG. 10E shows expression of MSigDB hallmark IFNa response signature genes in whole lung. Average expression of IFNa response genes (top) and heatmap of selected transcripts from the MSigDB hallmark IFNa response signature gene set (bottom). The top 50 genes that displayed enhanced upregulation in Osm7' mice at 3dpi, as identified in (FIG. 10C), were selected for display. Color scale reflects row Z-score values. UI = uninfected. Female mice were used for experiments in this figure.

[0043] FIGS. 11A-11B demonstrate OSM deficiency leads to elevated levels of IFN during IAV infection. Mice were infected i.n. with 225pfu of A / WSN / 1933(H1N1) and RNA from the whole lung was harvested at indicated time points. FIG. 11 A provides lftia4 and Iftib transcripts (n = 3 - 4). Representative of at least two independent experiments. Two-way ANOVA. FIG. 1 IB provides Isgl5 and Irf7 transcripts (n = 3 - 4). Representative of at least two independent experiments. Two-way ANOVA.

[0044] FIGS. 12A-12C demonstrate OSM deficiency leads to increased immunopathology during IAV infection. Mice were infected i.n. with 225pfu of A / WSN / 1933 (H1N1). FIG. 12A provides representative image showing an example of a region defined as severe lung damage, used to train QuPath for damage classification. FIG. 12B provides histological images of mouse lungs at baseline and at various time points during IAV infection. The blue square indicates the region magnified in the main figure. Error bars indicate SD, ns = not significant. Female mice were used for experiments in this figure. FIG. 12C provides histological scoring of the percentage of lung tissue exhibiting severe damage at baseline was performed. Lung sections from wild type mice at steady state were used to define healthy regions and to train QuPath for damage classification (n = 5 - 6). Unpaired Student’s t test.

[0045] FIGS. 13 provides gating strategies for flow cytometry analysis of lung immune infiltrates during IAV infection. Representative gating includes: representative gating for neutrophils (CD45+CD64-Ly6G+CDllb+); representative gating for monocytes (CD45+CD64-Ly6G-CDllb+Ly6C+); representative gating for IMs / moMacs (CD45+CD64+F4 / 80+SiglecF-CDllb+); representative gating for AMs (CD45+CD64+F4 / 80+SiglecF+CDllc+); representative gating for B cells (CD45+ TCRB-B220+); representative gating for NK cells (CD45+ B220- TCRB- NK1.1+); representative gating for CD4 T cells (CD45+ TCRB+ B220- CD4+ CD8-); representative gating for Treg

[0046] 4909-9827-7258, v. 19 Docket No. HRVY-259-WO1 cells (CD45+ TCRB+ B220- CD4+ CD8- FOXP3+); representative gating for CD8 T cells (CD45+ TCRB+ B220- CD4- CD8+); and representative gating strategy for pDCs (CD45+ CD64-F4 / 80-B220+Ly6C+PDCAl+). All populations gated on viable singlets.

[0047] FIGS. 14A-14J demonstrate flow cytometry analysis of lung immune infiltrates during IAV infection. Mice were infected i.n. with 450 PFU of A / WSN / 1933 (H1N1). Lung immune infiltrates were quantified by flow cytometry at 2 and 6dpi (n = 3 - 6). Two-way ANOVA. Neutrophils (FIG. 14A), monocytes (FIG. 14B), IMs / moMacs (FIG. 14C), AMs (FIG. 14D), CD4+ T cells (FIG. 14E), CD8+ T cells (FIG. 14F), TREGS (FIG. 14G), NK cells (FIG. 14H), B cells (FIG. 141), and pDCs (FIG. 14J). Male mice were used for experiments in this figure. Error bars indicate SD, *p<0.05, **p<0.01, ns = not significant.

[0048] FIGS. 15A-15C demonstrate IAV RNA is not disseminated in Osm- / - mice. Mice were infected i.n. with 225pfu of A / WSN / 1933 (H1N1). FIG. 15A shows plaque forming units detectable in BALF at indicated time points (n = 5 - 6). unpaired Student’s t test or Mann- Whitney. FIG. 15B shows plaque forming units detectable in blood and brain at 6dpi (n = 4 - 6). FIG. 15C provides RT-qPCR analysis of IAV transcripts encoding nucleoprotein (Np) in whole lung or brain homogenate at 6dpi (n = 4 - 6); representative of at least two independent experiments, unpaired Student’s t test. Relative expression calculated as (2_ACt)*1000. LOD = level of detection. Female mice were used for experiments in this figure.

[0049] FIGS. 16A-16F demonstrate OSM is produced locally in response to poly(I:C) by myeloid population. FIGS. 16A-16D show mice were treated i.t. with 33.75 pg - 50 pg of poly(I:C) daily for five consecutive days. FIG. 16A shows plasma was collected for analysis of OSM protein levels at the indicated time points, (n = 4 - 5); representative of two independent experiments. Error bars indicate SD. FIG. 16B shows whole lung RNA was collected at indicated time points (n = 4 - 5). Error bars indicate SD. Relative expression calculated as (2’ACt)*1000. FIG. 16C shows mice were monitored daily for percent initial body weight and survival (n = 3); representative of at least two independent experiments. Error bars indicate SD, **p<0.01, ****p<0.0001, two-way ANOVA up to day 7 for (FIG.

[0050] 16C, left panel), log-rank Mantel-Cox test for (FIG. 16C, right panel). FIG. 16D provides Osm expression in sorted myeloid cells of wild type mice at 7 days post-initial poly(I:C) treatment (n = 5). FIG. 16E provides representative gating for sorted myeloid populations, including Neutrophils (CD45+CD64-F4 / 80-Ly6G+CDllb+), Monocytes (CD45+CD64-CDllb+Ly6C+F4 / 80+), moMacs / IMs (CD45+CD64+F4 / 80+SigF-), and AMs (CD45+CD64+F4 / 80+CDllc+SigF+). All populations gated on viable singlets. FIG. 16F

[0051] 4909-9827-7258, v. 110 Docket No. HRVY-259-WO1 shows mice were treated intraperitoneally with 100 pg of poly(I:C) daily for three consecutive days and monitored daily (n = 6). Error bars indicate SD. Two-way ANOVA up to day 7. Female mice were used for (FIG. 16A), (FIG. 16B), and (FIG. 16D), and male mice used for (FIG. 16F).

[0052] FIGS. 17A-17F demonstrate validation of macrophage-specific OSM-deficient mouse model. FIG. 17A shows mice were treated i.t. with 33.75 pg - 50 pg of poly(I:C) daily for five consecutive days. Osm expression in sorted myeloid cells of mice at 7 days post-initial poly(I:C) challenge (dpc) (n = 4). Representative flow cytometry gating found in FIG. 16. Mann-Whitney U test. Error bars indicate SD, *p<0.05, ns = not significant. FIG. 17B shows blood and lungs of wild type mice at steady state were collected for flow cytometry analysis of monocytes / macrophages (CD45+F4 / 80+CDllb+Ey6C+ viable singlets). Representative gating and CD64 MFI of monocytes / macrophages shown (n = 2). FIGS. 17C-17D show mice were treated i.t. with 33.75 pg - 50 pg of poly(EC) daily for five consecutive days. FIG.

[0053] 17C shows whole lung was collected at baseline and at 7dpc. RT-qPCR quantification of Osm transcripts in whole lung (n = 3 - 5). Two-way ANOVA. Error bars indicate SD, **p<0.01, ****p<0.0001. Relative expression calculated as (2ACt)*1000. FIG. 17D shows BAEF was collected at 7dpc (n = 4). non-parametric one-way ANOVA. Error bars indicate SD, *p<0.05. EOD = level of detection. FIG. 17E shows mice were infected i.n. with 225pfu of A / WSN / 1933 (H1N1). BALF was collected at 5dpi (n = 4 - 7). Mann-Whitney U test. Error bars indicate SD, **p<0.01. FIG. 17F shows mice were treated i.t. with 33.75 pg - 50 pg of poly(EC) daily for five consecutive days. Epithelial cells (CD45-Pdgfra-CD31-EpCAM+), endothelial cells (CD45-Pdgfra-CD31+), and fibroblasts (CD45-CD31-Pdgfra+) were sorted at 7dpc and RT-qPCR was performed for Osmr (n = 3 -5). A combination of female and male mice was used for experiments in this figure.

[0054] FIGS. 18A-18D demonstrate macrophage- specific deletion of Osm during poly(EC) challenge leads to elevated IFN levels and lung immune infiltrates. Mice were treated i.t. with 33.75 pg - 50 pg of poly(EC) daily for 5 consecutive days, and lungs processed for analysis at 7 days post-initial poly(EC) challenge. FIG. 18A provides flow cytometry for neutrophils, monocytes, moMacs / IMs, and AMs (n = 11). Representative gating strategy found in FIG. 8. Whole lung RNA was collected and RT-qPCR was performed for lftia4, Ifnb, and Ifnl2 (FIG.

[0055] 18B) and for lsgl5 and Irf7 (FIG. 18C) (n = 6). FIG. 18D provides representative gating strategy for ATIIs and epithelial cells. Mann-Whitney U test for (FIG. 18B), Unpaired t-test for (FIG. 18A) and (FIG. 18C). Error bars indicate SD, *p<0.05, **p<0.01, ***p<0.001.

[0056] 4909-9827-7258, v. 111 Docket No. HRVY-259-WO1 Relative expression calculated as (2-ACt)*1000. A combination of male and female mice were used for experiments in this figure.

[0057] FIGS. 19A-19F demonstrate anti-IFNAR-1 blockade and local rOSM treatment rescue mice with macrophage- specific OSM depletion during poly(I:C) challenge. AM phagocytic functions are not compromised in FcgrlCre mice. FIG. 19A shows mice were treated i.t. with 33.75 pg - 50 pg of poly(I:C) daily for 5 consecutive days. In addition, mice were treated i.v. with either 200 pg of anti-mouse IFNAR-1 or isotype control antibody diluted in 100 pl PBS on day -1, 1, 3 and 4 after the first poly(I:C) challenge. Mice were monitored daily (n = 3 - 4); representative of at least two independent experiments. FIG. 19B shows mice were treated i.t. with Ipg of mouse rOSM in addition to 33.75 pg - 50 pg of poly(I:C) daily for five consecutive days. Mice were monitored daily for survival (n = 4 - 5); representative of at least two independent experiments. FIG. 19C shows mice were intratracheally administered 50 pg of deep red pHrodo E. coli beads and lungs were harvested for flow cytometry analysis, where neutrophils are gated as CD45+CD64-F4 / 80-Ly6G+CDllb+ and macrophages gated as CD45+CD64+F4 / 80+, with all populations gated on viable singlets (n = 4). FIG. 19D shows lungs from Osmfl / fl or FcgrlCreOsmfl / fl mice at baseline were harvested and flow cytometry performed to quantify total numbers of AMs (CD45+CD64+F4 / 80+SiglecF+CDl lc+) IM / moMacs (CD45+CD64+F4 / 80+SiglecF-CDllb+), neutrophils (CD45+CD64-Ly6G+CDllb+), and monocytes (CD45+CD64-Ly6G-CDllb+Ly6C+) (n = 5 - 6). FIG. 19E and FIG. 19F demonstrate exogenous OSM administration rescues epithelial proliferation. Mice were treated i.t. with rOSM and poly(I:C) daily for five consecutive days. Lungs were collected and Ki67+ ATII cell (FIG.

[0058] 19E) and total ATII cell (FIG. 19F) numbers at 7 dpc were determined by flow cytometry. Log-rank Mantel Cox test for (FIG. 19A) and (FIG. 19B), unpaired Student’s t test for (FIG.

[0059] 19C), Mann- Whitney U test or unpaired Student’s t test for (FIG. 19D). Error bars indicate SD, *p<0.05, ns = not significant. A combination of male and female mice were used for experiments in this figure.

[0060] FIGS. 20A-20C demonstrate Bulk RNAseq analysis for sorted ATII cells. FIG. 20A shows mice were intratracheally treated with PBS or 1 pg of rOSM for seven consecutive days. Lungs were harvested, stained, and ATIIs were sorted as DAPLCD45-Pdgfra-CD31-EpCAM+MHCII+CD104-. FIG. 20B provides representative image of alveolar organoid segmentation analysis. Genes that exhibited at least a 2-fold differential expression in any pairwise comparison between groups were clustered using K-means based on their expression

[0061] 4909-9827-7258, v. 112 Docket No. HRVY-259-WO1 values. The resulting clusters were analyzed for pathway enrichment using MSigDB.

[0062] Enrichment of each cluster across genotypes and experimental conditions. (FIG. 20C).

[0063] FIGS. 21A-21C demonstrate macrophage-derived OSM is required for epithelial integrity during poly(I:C) challenge. FIG. 21 A shows lung barrier permeability assessed using Evans blue dye (EBD) assay on bronchoalveolar lavage fluid (BAEF) at 7 days post initial challenge (dpc). FIGS. 21B-21C show lungs were collected and total epithelial cell (FIG. 2 IB) and ATII cell (FIG. 21C) numbers at 7 dpc were determined by flow cytometry.

[0064] FIG. 22 depicts flow cytometry quantification of total ATIIs left), Sca-lNeg / PosATII proportions (middle) and percent of ATIIs Ki67Pos(right) at 3d post-poly(EC) challenge in FcgrlCreOsnf / flor Osm^ mice.

[0065] FIGS. 23A-23C depict administration of OSM to lAV-infected mice enhances ATII proliferation. FIG. 23A depicts an experimental scheme for IAV infection and OSM administration. Specifically, mice infected intranasally (i.n.) with influenza A virus (IAV; A / PR / 8 / 34) and administered OSM or PBS intratracheally (i.t.) daily on days 9-12 postinfection. The timepoints for administration represent peak epithelial damage after infection. Fungs were harvested 13 days post infection (dpi) and total epithelial cell number (FIG. 23B), ATII cell number, and EdU-i- and Ki67+ ATII cell numbers were quantified using flow cytometry (FIG. 23C). Representative of three independent experiments. Symbols represent individual mice, bars are means, and error bars indicate SD; *P < 0.05; Mann- Whitney U test.

[0066] FIGS. 24A-24C Demonstrate that OSM-deficiency does not impact lung epithelial cell numbers at steady state. FIG. 24A depicts representative flow cytometry gating strategy for total epithelial cells (CD45-, CD31-, Pdgfra-, EpCAM+), ATIIs (CD45-, CD31-, Pdgfra-, EpCAM+, CD104-, MHCII+), and airway cells (CD45-, CD31-, Pdgfra-, EpCAM+, CD 104+, MHCII-). All populations were gated on viable singlets. FIG. 24B show lungs were processed and total epithelial cell, ATII, and airway cell numbers were determined by flow cytometry (n = 4 - 5 mice per group); representative of at least two independent experiments. FIG. 24C provides representative images of CCSP (green) and SP-C (yellow) staining in mouse lungs (n = 4 - 5 mice per group, 1 - 2 fields per mouse). A combination of female and male mice was used for experiments in this figure. Error bars indicate SD, ns = not significant, Unpaired Student's t test for (FIG. 24B). Scale bar represents 100 pm in (FIG.

[0067] 24C).

[0068] 4909-9827-7258, v. 113 Docket No. HRVY-259-WO1 FIGS. 25A-25F demonstrate gene and pathway enrichment analysis of ATII clusters from scRNA-seq data. Mice were infected i.n. with 225 pfu of A / WSN / 1933 (H1N1) or mock-infected with PBS. Lungs were collected from lAV-infected mice at 2 dpi and from mock-infected mice (0 dpi) for scRNA-seq analysis, (n = 2 mice per group). FIG. 25A depict violin plots showing Sftpc (left), Scgblal (middle), and Fox}l (right) expression patterns across annotated cell clusters in scRNA-seq data from lungs at 0 and 2 dpi. FIG. 25B depicts ATII signature expression across annotated cell clusters (UMAP clustering and cell cluster annotation found in FIG. ID). FIG. 25C depicts violin plots showing Sftpc (left), Scgblal (middle), and Foxjl (right) expression across re-clustered ATII subpopulations. Scale bar represents log normalized gene expression. FIG. 25D depict top three pathways identified for each re-clustered ATII subpopulation, identified via Enrichr pathway analysis. Gene expression pathways were retrieved from the Reactome Database. The dotted line represents the significance threshold for the adjusted p value, set at p < 0.05. Female mice were used for experiments in this figure. FIG. 25E depicts OSM is required for survival following local poly(I:C) challenge. Mice were treated intratracheally (i.t.) with 33.75 pg - 50 pg of poly(I:C) or PBS daily for five consecutive days. Lung barrier permeability was assessed using an Evans blue dye (EBD) assay on BALF at 5 days post-initial poly(I:C) challenge (dpc) (n = 4 - 5 mice per group). FIG. 25F depicts pathway enrichment analysis of sorted ATIIs and organoid segmentation. Mice were i.t. treated with PBS or 1 pg of rOSM for seven consecutive days. Lungs were processed, stained, and ATIIs were sorted as DAPI- CD45-Pdgfra- CD31- EpCAM+ MHCII+ CD 104- viable singlets. Genes that exhibited at least a 2- fold differential expression in any pairwise comparison between groups were clustered using K-means based on their expression values. The resulting clusters were analyzed for pathway enrichment using MSigDB. Enrichment of each cluster across genotypes and experimental conditions.

[0069] FIG. 26 depicts why macrophages are integral tissue components.

[0070] FIG. 27 depicts the different populations of lung macrophages.

[0071] FIG. 28 depicts the steady state lung microenvironment.

[0072] FIG. 29 outlines aspects regarding oncostatin M.

[0073] FIG. 30 demonstrates OSM is locally induced and required for survival during IAV infection.

[0074] FIG. 31 depicts the role of OSM at steady state.

[0075] 4909-9827-7258, v. 114 Docket No. HRVY-259-WO1 FIG. 32 depicts diverse immune and non-immune cell populations identified in lungs of uninfected and lAV-infected mice.

[0076] FIG. 33 depicts Osm expression is primarily detected on myeloid cells, while Osmr expression is restricted to non-immune populations.

[0077] FIG. 34 depicts that epithelial cells exhibit the greatest downstream response to OSM. FIG. 35 depicts OSM is required for maintenance of ATII composition at baseline and during early IAV infection.

[0078] FIG. 36 depicts the proposed steady state model of the lung alveolar niche.

[0079] FIG. 37 depicts the role of interferons in immune protection.

[0080] FIG. 38 depicts the role of interferons in pathology.

[0081] FIG. 39 depicts OSM-deficiency results in increased IFN-I levels and more severe lung damage during IAV infection.

[0082] FIG. 40 depicts a model system to avoid confounding effects of viral replication. FIG. 41 depicts OSM is induced and required for survival during poly(I:C) challenge. FIG. 42 depicts macrophage-derived OSM protects mice from alveolar damage.

[0083] FIG. 43 depicts IFN-I signaling blockade or exogenous OSM administration reduces morbidity in macrophage-specific OSM-deficient poly(I:C)-treated mice.

[0084] FIG. 44 depicts exogenous OSM administration did not affect IFN-I levels in response to poly(I:C) challenge.

[0085] FIG. 45 depicts exogenous OSM administration restores ATII proliferation following poly(I:C) challenge.

[0086] FIG. 46 depicts OSM overcomes the growth suppressive effects of IFN-I.

[0087] FIG. 47 depicts the steady state model for the lung alveolar niche which contrasted with viral stimuli and damage model for the lung alveolar niche.

[0088] FIG. 48 depicts OSM-deficiency does not impact lung epithelial cell numbers at steady state.

[0089] FIG. 49 depicts OSM overcomes the growth suppressive effects of IFN-I.

[0090] FIG. 50 depicts exogenous OSM administration induces epithelial proliferation.

[0091] FIG. 51 depicts exogenous OSM administration restores epithelial composition in OSM-deficient mice.

[0092] FIG. 52 depicts macrophages are a major source of lung OSM.

[0093] FIG. 53 depicts validation of FcgrlcreOSMfl / flmice.

[0094] 4909-9827-7258, v. 115 Docket No. HRVY-259-WO1 FIG. 54 depicts a gene list of the top 50 marker genes for each annotated cluster from scRNA- seq. Female mice were infected i.n. with 225 pfu of A / WSN / 1933 (H1N1) or mock- infected with PBS. Lungs were collected from lAV-infected mice at 2 dpi and mock-infected mice (0 dpi) for scRNA-seq analysis (n = 2 mice per group). Markers are ranked by adjusted p-value across all clusters.

[0095] FIGS. 55A-55G depict gene list and Reactome pathway analysis for ATI! subclusters. Female mice were infected i.n. with 225 pfu of A / WSN / 1933 (HIND or mock-infected with PBS. Lungs were collected from lAV-infected mice at 2 dpi and mock-infected mice (0 dpi) for scRNA-seq analysis (n = 2 mice per group). Gene list used for ATII annotation is included, obtained from 30). The ATII cluster was further subclustered into: ATII cluster 1 (OSM-independent (FIG. 55 A)), ATII cluster 2 (OSM-dependent (FIG. 55B)), and ATII cluster 3 (ISGhi (FIG. 55C)). Genes for each subcluster are filtered by adjusted p-value (<1E- 5) and organized in descending order of log2 fold change (log2FC). Reactome pathway enrichment tables (generated using Enrichr) are included for cluster 1 (FIG. 55D), cluster 2 (FIG. 55E), and cluster 3 (FIG. 55F). FIG. 55G outlines the gene list used to annotate ATIIs.

[0096] FIG. 56A-56D depicts sorted ATII bulk RNA-seq gene lists and pathway analysis. Female mice were treated i.t. with PBS or 1 tig of rOSM for seven consecutive days. Lungs were processed, stained, and ATIIs were sorted (representative gating strategy for sorted ATIIs found in Fig 20A) (n = 2- 3 mice per group). Gene list for sorted ATIIs, pre-filtered using a maximum coefficient of variation of 0.2 and a minimum expression threshold of 10. Genes that exhibited at least a 2-fold differential expression in any pairwise comparison between groups were clustered using K-means based on expression values. Gene list from K-means clustering. The resulting clusters were analyzed for pathway enrichment using MSigDB. FIG. 56A outlines MSigDB pathway analysis for Cluster A. FIG. 56B outlines MSigDB pathway analysis for Cluster B. FIG. 56C outlines MSigDB pathway analysis for Cluster C. FIG. 56D outlines MSigDB pathway analysis for Cluster D.

[0097] FIG. 57 demonstrates that macrophages are tissue-resident immune cells with diverse functions.

[0098] FIG. 58 depicts macrophages exhibit high sensory capacity.

[0099] FIG. 59 depicts macrophages are highly plastic and multifunctional cells.

[0100] FIG. 60 depicts macrophages promote tissue-level adaptations to restore homeostasis. FIG. 61 depicts macrophages help balance host defense and tissue repair.

[0101] FIG. 62 depicts infection results in repaired and remodeled tissue.

[0102] 4909-9827-7258, v. 116 Docket No. HRVY-259-WO1 FIG. 63 depicts infection results in tissue damage.

[0103] FIG. 64 provides an overview of Oncostatin M (OSM), its binding partners and relevant pathways.

[0104] FIG. 65 depicts interferons are critical antiviral mediators.

[0105] FIG. 66 depicts OSM-deficiency results in exacerbated IFN-I responses.

[0106] FIG. 67 depicts distinct ATII states emerge following IAV infection.

[0107] FIG. 68 depicts 7 days post infection, a transient ATI! cluster is enriched for ISGs. FIG. 69 depicts ISGhlSca-lposare induced in response to IAV infection.

[0108] FIG. 70 depicts Sca-lposATIIs are the proliferative ATI! population during IAV infection.

[0109] FIG. 71 depicts Sca-lposATIIs are the proliferative ATII population following poly(I:C) challenge.

[0110] FIG. 72 depicts a workflow for determining if Sca-lposATIIs have a higher progenitor function.

[0111] FIG. 73 depicts Sca-lposATIIs exhibit enhanced organoid forming efficiency.

[0112] FIG. 74 depicts macrophage-specific OSM deficiency diminishes ATII proliferation. FIG. 75 depicts IFN-priming makes ATIIs more receptive to signaling via OSM. FIG. 76 depicts Sca-lposATIIs exhibit enhanced OSM signaling capacity.

[0113] FIG. 77 depicts IFN-I signaling primes the lung for epithelial renewal following inflammatory challenge.

[0114] FIG. 78 depicts macrophage-derived OSM promotes epithelial integrity alongside antiviral defenses.

[0115] FIG. 79 depicts different interferon types and associated signaling pathways.

[0116] DETAILED DESCRIPTION OF THE INVENTION

[0117] Methods of treatment

[0118] Acute viral respiratory infections caused by viruses such as influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV), place a large burden on healthcare resources and an economic strain on patients and communities. Despite vaccination efforts and the development of antiviral therapeutics, an estimated 750,000 yearly deaths are attributed to these viral infections throughout the world. While survival from viral respiratory infections is improving, many of the patients present with postinfectious pulmonary complications, resulting in loss of more than

[0119] 4909-9827-7258, v. 117 Docket No. HRVY-259-WO1 105,000,000 disability-adjusted life-years annually. Many of these postinfectious pulmonary complications have been ascribed to the destruction of epithelial cells and the epithelial barrier through mediators of pulmonary inflammation. Thus, there is a need to increase epithelial cell proliferation and restore the epithelial barrier, to thereby reduce or eliminate the incidence of pulmonary complications in patients that have or have had viral respiratory infections. This invention addresses this unmet need by describing compositions comprising macrophage-derived Oncostatin M (OSM) and methods of their use in counteracting the damaging effects of interferon on epithelial cell composition and the epithelial cell barrier during respiratory infection.

[0120] Oncostatin M is a cytokine belonging to the interleukin-6 family that is secreted from macrophages, among other cells of the myeloid lineage. Human OSM is secreted as a 252 amino acid polypeptide having the amino acid sequence of SEQ ID NO: 1:

[0121] MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTD LMQDTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFL QTENATEGCVEHREADEEQREPKAQDEERSGENIEDEEKEQMARPNIE GERNNIYCMAQEEDNSDTAEPTKAGRGASQPPTPTPASDAFQRKEEGC RFEHGYHRFMHSVGRVFSKWGESPNRSRRHSPHQAERKGVRRTRPSR KGKREMTRGQEPR

[0122] The full-length polypeptide may be proteolytic ally cleaved to produce the 227 amino acid pro-OSM protein by removing the signal comprising the first 25 C-terminal amino acids. Removal of an additional 31 residues produces the mature OSM polypeptide having 196 amino acids. OSM has been implicated in regulating gene activation, cell growth, and inflammatory processes. As described more fully below, the inventors recently discovered that OSM may override IFN-I- mediated immunopathology in the lung. In particular, it was discovered that OSM maintains ATII cell composition, promotes epithelial cell proliferation, and restores the epithelial barrier in the lung, even in the face of IFN-I antiproliferative and immunopathological effects during severe infections.

[0123] Thus, in one aspect, the disclosure relates to a method for counteracting the antiproliferative and immunopathological effects of IFN-I by contacting at least one cell with a composition comprising an active agent capable of counteracting at least one antiproliferative or immunopathological effect of IFN-I. In some embodiments, the at least one cell is a cell exposed to inflammatory conditions in a lung during infection. In some embodiments, the cell is an epithelial cell that forms part of an epithelial barrier in the lung.

[0124] 4909-9827-7258, v. 118 Docket No. HRVY-259-WO1 In some embodiments, the epithelial cell is an ATII cell. In some embodiments, the active agent is oncostatin M (OSM). In some embodiments, the active agent is a derivative or variant of OSM (e.g. a homologue, an orthologue or a conservative amino acid substituted variant of OSM). In some embodiments, the active agent is a functional fragment of OSM, capable of counteracting at least one antiproliferative or immunopathological effect of IFN-I. In some embodiments, the active agent is a nucleic acid sequence encoding OSM, an OSM derivative, variant or functional fragment thereof. For ease of reference, the active agent, including the OSM, derivative, variant, and functional fragment thereof, along with nucleic acids encoding OSM, an OSM derivative, variant or functional fragment thereof may be referred to as simply “OSM”, “agent”, or “active agent”. In some embodiments, the OSM is human OSM. In some embodiments, the OSM is derived from rodent (e.g. mouse), rabbit, goat, bovine, pig, horse, guinea pig, or other laboratory or livestock animal.

[0125] In one aspect of the invention, one or more viral vectors are utilized for delivery of the agent into a desired organ, tissue, cell or region of the body. In some embodiments, the one or more vectors can each be an adeno-associated virus (“AAV”) vector. The AAV vector may contain a full-length AAV 5' inverted terminal repeat (ITR) and a full-length 3 ' ITR. A shortened version of the 5' ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intramolecular double- stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, "Self- complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248- 1254. Self-complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7, 125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0126] In some embodiments, a single- stranded AAV viral vector may be used. Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., US Patent 7790449; US Patent 7282199; WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; and US 7588772 B2. In one system, a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s)

[0127] 4909-9827-7258, v. 119 Docket No. HRVY-259-WO1 that encodes rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transfected (transiently or stably) with a construct encoding the transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus. More recently, systems have been developed that do not require infection with helper virus to recover the AAV - the required helper functions (i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system. In these newer systems, the helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level. In yet another system, the transgene flanked by ITRs and rep / cap genes are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al, 2009, "Adenovirus- adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of which is incorporated herein by reference in its entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514;

[0128] 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0129] In one aspect, disclosed herein are methods of treating or preventing moderate to severe respiratory infection. For purposes of this application, “treat”, “treating”, “treatment”, or “treated” are defined to encompass reduction or elimination of one symptom of the disease or disorder, amelioration or resolution of one underlying cause of the disease or disorder, and up to and including cure of the disease or disorder for a period of time. Thus, in some embodiments, the treatment encompasses increasing epithelial cell proliferation in the lung, restoration of lung epithelial barrier integrity, and / or improving lung function. In some embodiments, the treatment increases epithelial cell proliferation by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350% or more compared to a level of cell proliferation observed during a respiratory infection. In some embodiments, treatment restores epithelial barrier integrity by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or about 100%. In some

[0130] 4909-9827-7258, v. 120 Docket No. HRVY-259-WO1 embodiments, the treatment improves lung function by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200% or more compared to lung function assessed during or immediately after a respiratory infection. The terms “prevent”, “preventing”, “prevention”, and “prevented” are defined as encompassing the absence of one or more symptoms associated with a disease or disorder in a subject for a period of time. In some embodiments, preventing is associated with the absence of elevated lung damage or the preservation of lung barrier integrity for a duration of time. The effects of treatment and / or prevention may last for a period of time to include one or more days, weeks, months, and years or may last the rest of the natural lifetime of the subject.

[0131] In some embodiments, the methods disclosed herein are capable of treating or preventing at least one symptom associate with a respiratory infection caused by a pathogen selected from a virus, bacteria or fungus. In some embodiments, the pathogen is influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, MERS-CoV, adenovirus, parainfluenza virus, rhinovirus, enterovirus, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, and measles virus. In some embodiments, the pathogen is Group A B-hemolytic streptococci, Corynebacterium diphtheriae, Neisseria gonorrhoeae, Mycoplasma pneumoniae, Mycoplasma hominis ( type 1 ), Hemophilus influenzae type b, Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Legionella spp, Mycobacterium tuberculosis, Coxiella Burnetii, Chlamydia psittaci, Chlamydia trachomatis, or Chlamydia pneumoniae. In some embodiments, the pathogen is Candida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, or Aspergillus fumigatus.

[0132] In certain embodiments, the methods include administration of at least one additional active agent in combination with the OSM. By way of non-limiting example, the at least one additional active agent is one or more of an anti-viral agent, an anti-inflammatory agent, an expectorant, a cough suppressant, an antibiotic, an antifungal agent, or a bronchodilator. In some embodiments, the anti-viral agent is Molnupiravir, Baloxavir, Zanamivir, Acyclovir, amantadine, Cidofovir, Penciclovir, Famciclovir, Valacyclovir, Vicarabine, Zidovudne, Idoxuridine, Trifluridine, Oseltamivir, rivavirin, or nirsevimab. In some embodiments, the anti-inflammatory agent may be a nonsteroidal anti-inflammatory drug such as aspirin, diflunisal, salsalate, celecoxib, diclofenac, etodolac, fenoprofen, flurbiprofen, ibuprofen,

[0133] 4909-9827-7258, v. 121 Docket No. HRVY-259-WO1 indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, or tolmetin. In some embodiments, the anti-inflammatory agent is a corticosteroid, such as beclomethasone, budesonide, ciclesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, hydrocortisone, prednisone, dexamethasone or prednisolone. In some embodiments, the expectorant is guaifenesin, potassium iodide or ammonium chloride. In some embodiments, the cough suppressant is dextromethorphan, diphenhydramine, codeine or hydrocodone. In some embodiments, the antibiotic is amikacin, gentamicin, kanamycin, neomycin, plazomicin, streptomycin, tobramycin, penicillin, ampicillin, ampicillin / sulbactam, ampicillin / clavulanate, ceftriazone, cefotaxime, ceftazidime, cefepime, aztreonam, ertapenem, imipenem, meropenem, tebipenem, chloramphenicol, daptomycin, ciprofloxacin, norfloxacin, ofloxacin, delafloxacin, Gemifloxacin, levofloxacin, moxifloxacin, a Fosfomycin, Lefamulin, lincosamides, oxazolidinones, streptogrmins, clindamycin, linezolid, tedizolid, quinupristin, dalfopristin, lipoglycopeptides, macrolides, metronidazole, tinidazole, mupirocin, nitrofurantoin, rifamycins, spectinomycin, sulfonamides, tetracyclines, tigecycline, trimethoprim, sulfamethoxazole, or vancomycin. In some embodiments, the antifungal agent is amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, isvuconazonium sulfate, oteseconazole, echinocandins, or flucytosine. In some embodiments, the bronchodilator is albuterol, levalbuterol, arformoterol, formoterol, salmeterol, indacaterol, olodaterol, vilanterol, ipratropium, tiotropium, beclomethasone, budesonide, ciclesonide, fluisolide, fluticasone, mometasone, methylprednisolone, prednisolone, prednisone, cromolyn, nedocromil, montelukast, zafirlukast, zileuton, theophylline, benralizumab, dupilumab, mepolizumab, omalizumab, reslizumab, Tezepelumab, and combinations thereof.

[0134] Methods of Administration / Compositions

[0135] The compositions comprising the active agent according to the invention may be administered by means common in this field. By way of non-limiting example, the compositions comprising the active agent may be administered via inhalation, intranasally, intravenously, intraarterially, intraperitoneally, intrathecally, orally, sublingually, transdermally (e.g. a salve, patch or ointment that may be applied to the skin, such as skin of the chest or neck), rectally, vaginally, intramuscularly, subcutaneously, or buccally. In certain circumstances, it may be beneficial to utilize more than one route of administration during the course of treatment or prevention of a disease, disorder, or one or more symptoms thereof.

[0136] 4909-9827-7258, v. 122 Docket No. HRVY-259-WO1 For example, a composition may be administered intravenously and then administered via inhalation. In some embodiments, the route of administration will be chosen to provide targeted delivery to the lungs. In some embodiments, the composition comprising the active agent is administered systemically.

[0137] In certain embodiments, the composition comprising the active agent is delivered via inhalation. In some embodiments, the composition comprising the active agent may be delivered to the lungs in an aerosolized form. The aerosols may be generated, for example, from liquids, liquid suspensions, or dry powders or agglomerates through use of a device capable of generating aerosols having particles with a mass median aerodynamic diameter (MMAD) of from 20 pm to 0.01 pm. In some embodiments, the particles have a MMAD of about 0.01 pm, about 0.03 pm, about 0.05 pm, about 0.07 pm, about 0.09 pm, about 0.1pm, about 0.25 pm, about 0.5 pm, about 0.75 pm, about 1.0 pm, about 1.25 pm, about 1.5 pm, about 1.75 pm, about 2.0 pm, about 2.25 pm, about 2.5 pm, about 2.75 pm, about 3.0 pm, about 3.25 pm, about 3.5 pm, about 3.75 pm, about 4.0 pm, about 4.25 pm, about 4.5 pm, about 4.75 pm, about 5.0 pm, about 5.25 pm, about 5.5 pm, about 5.75 pm, about 6.0 pm, about 6.25 pm, about 6.5 pm, about 6.75 pm, about 7.0 pm, about 7.25 pm, about 7.5 pm, about 7.75 pm, about 8.0 pm, about 8.25 pm, about 8.5 pm, about 8.75 pm, about 9.0 pm, about 9.25 pm, about 9.5 pm, about 9.75 pm, about 10.0 pm, about 10.25 pm, about 10.5 pm, about 10.75 pm, about 11.0 pm, about 11.25 pm, about 11.5 pm, about 11.75 pm, about 12.0 pm, about 12.25 pm, about 12.5 pm, about 12.75 pm, about 13.0 pm, about 13.25 pm, about 13.5 pm, about 13.75 pm, about 14.0 pm, about 14.25 pm, about 14.5 pm, about 14.75 pm, about 15.0 pm, about 15.25 pm, about 15.5 pm, about 15.75 pm, about 16.0 pm, about 16.25 pm, about 16.5 pm, about 16.75 pm, about 17.0 pm, about 17.25 pm, about 17.5 pm, about 17.75 pm, about 18.0 pm, about 18.25 pm, about 18.5 pm, about 18.75 pm, about 19.0 pm, about 19.25 pm, about 19.5 pm, about 19.75 pm, or about 20.0 pm. In some embodiments, the particles have a MMAD between about 0.5 pm and 2.5 pm, about 1.25 pm and about 3.75 pm, about 2 pm and about 5.0 pm, about 3 pm and about 7.5 pm, about 6.5 pm and about 9.0 pm, or about 8.5 pm and about 12 pm. In some embodiments, at least one of particle size, morphology, size distribution, and bulk density are selected to enhance deposition of the particles into the alveolar region of the lungs.

[0138] Devices useful for delivering the composition comprising the agent to the lungs include a nebulizer, pressurized metered dose inhaler, or a dry powder inhaler, among others. In some embodiments, the nebulizer used may be a jet nebulizer, a vibrating mesh nebulizer,

[0139] 4909-9827-7258, v. 123 Docket No. HRVY-259-WO1 ultrasonic nebulizers, breath-actuated nebulizer, breath-enhanced nebulizers, or other nebulizers commercially available at the time of administration of the agent. The nebulizer may be marketed for home and personal use or may be marketed for the clinic or hospital. It will be appreciated that the dose of agent may be adjusted based upon the efficiency by which an individual nebulizer or type of nebulizer administers the agent to the lung of a patient. For example, it is known that breath-enhanced and breath- actuated nebulizers reduce waste of the active agent and more efficiently deliver an active agent to the lung of a patient. Additionally, vibrating mesh nebulizers have been demonstrated to provide up to three times more lung deposition compared to jet nebulizers. Thus, in certain embodiments, the administered dose may be adjusted downward to account for higher lung deposition and less wastage of the active agent. In contrast, the administered dose may be adjusted upward to account for use of a nebulizer which is less efficient.

[0140] Compositions comprising the active agent (e.g. OSM) for use in a nebulizer or metered dose inhaler may take several forms. In some embodiments, the OSM is provided in a liquid formulation. With an isoelectric point of 9.97 to 10.71, OSM will possess an ionic net charge at biological pH of the blood and lung (e.g. arterial blood (pH 7.35-7.45), bronchoalveolar aspirate (pH —6-8)). Thus, in some embodiments, the OSM may be dispersed / dis solved in a polar solvent having a pH suitable for delivery to a lung. In some embodiments, the polar solvent is an aqueous solution, such as distilled water, water for injection, or a saline solution. The liquid OSM formulation may comprise additional components which are customary or known for inclusion within nebulized compositions, or compositions which comprise a protein active agent. By way of non-limiting example, the components may comprise co-solvents, osmotic agents, pH adjusting agents, antifreeze agents, surface-active agents, preservatives, protease inhibitors, and / or peptidase inhibitors. In some embodiments, the OSM is dissolved in a solvent and is spray-dried, lyophilized, or spray lyophilized to provide a shelf stable solid composition (e.g. powder, agglomerated particles, granules) that may be dispersed / dissolved in a liquid prior to nebulization. Such spray-dried or lyophilized compositions may comprise formulation aids such as sugars (e.g. lactose), sugar alcohols (e.g. mannitol), starches / celluloses (e.g. hydroxypropylmethylcelullose), cryoprotectants (trehalose, propylene glycol), wetting agents, surfactants, and lubricants, among others. The particle size of the solid composition may be reduced by methods known in the art, such as milling, high-pressure homogenization, or supercritical carbon dioxide (SCCChj-based micronization processes.

[0141] 4909-9827-7258, v. 124 Docket No. HRVY-259-WO1 In some embodiments, the OSM is provided in a liquid suspension formulation. In some embodiments, nucleic acids which encode OSM are provided in a liquid suspension formulation. In some embodiments, the liquid suspension formulation may comprise liposomes, micelles, microparticles, nanoparticles, nanolipogel particles, complexes comprising the active agent, or viral particles comprising nucleic acids which encode the active agent. Processes for forming liquid suspension formulations which include liposomes, micelles, microparticles, nanoparticles, nanolipogel particles, or complexes that comprise the active agent will be familiar to those skilled in this field, and include those described in US6,241,969, US9,610,250, US9,415,020, US10,729,786, and US8,900,555, which are incorporated herein by reference in their entireties. When the liquid suspension formulations are included in a metered dose inhaler, they may include one or more propellants. Exemplary propellants for use in MDIs include hydrofluoroalkane propellants 1,1,1,2-tetrafluorethane (HFA 134a), 1,1,1,2,3,3,3-heptafhioropropane (HFA 227), and 1,1 -difluoroethane (HFA 152a), along with alternative propellants propane, n-biitane, isobutane, n-pentane, isopentane, neopentane, dimethylether, and hydrofluoroolefins (e.g. HFO-1234ze).

[0142] In certain embodiments, the composition will be formulated for delivery via a dry powder inhaler. The composition for use in a dry powder inhaler may be prepared as a fine powder or as agglomerated particles which break into smaller particles when exposed to a stream of air to provide a fine aerosol for inhalation. Processes for production of these fine powders and agglomerated particles are known in the art, and include those described in US5,780,014, US5,654,007, and US6,582,728, the teachings of which are incorporated herein in their entireties. For example, a respirable amorphous or crystalline fine powder may be produced by mixing the active agent with a bulking agent in one or more solvents / co-solvents to produce a solution, suspension, or slurry which is atomized and then exposed to air at a temperature sufficiently elevated to remove the solvent from the droplets to provide a dried powder. The dried powder will have a solvent content less than 10%, less than 7.5%, less than 5%, or less than 2.5%. The bulking agents may comprise galactose, D-mannose, sorbose, lactose, trehalose, 2-hydroxypropyl-B-cyclodextrin, raffinose, maltodextrins, dextran, mannitol, xylitol, aspartame, alanine and glycine. Additional excipients that may be mixed with the active agent and bulking agent prior to drying including pH adjusters, salts, and hydrophobic amino acids, including tryptophan, phenylalanine, and tyrosine, which may be used to improve dispersibility of the dried powder. In certain embodiments, the dried powder may be subjected to low shear granulation, high shear granulation, dry granulation,

[0143] 4909-9827-7258, v. 125 Docket No. HRVY-259-WO1 roller compaction, and extrusion to provide agglomerated particles which may be broken down and aerosolized from a dry powder inhaler. In certain embodiments, a non-aqueous binding liquid is utilized in agglomerating the dry powder to improve stability of the protein active ingredient. In some embodiments, the binding liquid is toluene, xylene, benzene, acetone, octane, chloroform, methylene chloride, or a fluorocarbon, such as perfluorodecalin or perfluorooctyl bromide. The agglomerated particle may have a size of 30-500 pm, 50-450 pm, 100-400 pm, or 150-350 pm.

[0144] In some embodiments, the composition comprising the agent is administered via intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intrathecal injection or peritoneal injection. Thus, the composition may be formulated for injection by combining the active agent or nanoparticles, microparticles, complexes, liposomes, or micelles containing the active agent with a liquid diluent recognized as safe for injection. Exemplary liquid diluents may include distilled or ultra-purified water, Ringer’s solution, lactated Ringer’s solution, a saline solution, such as phosphate-buffered saline (PBS), a dilute glucose solution, such as a 5% glucose aqueous solution, or a dilute alcohol or polyol aqueous solution, among others. In certain embodiments, the diluent is Water for Injection. In some embodiments, the diluent is PBS. In some embodiments, the diluent is an isotonic saline solution.

[0145] The composition may further comprise pharmaceutically acceptable excipients, such as those listed in the Handbook of Pharmaceutical Excipients (Handbook of Pharmaceutical Excipients, (2006) United Kingdom: Pharmaceutical Press). In some embodiments, selection of pharmaceutically acceptable excipients will be done with the goal to increase stability and effectiveness of the agent included in the composition. For example, buffers or pH adjusting agents may be selected to adjust the pH to less than 9, 8.5, 8, 7.8, 7.5. In some embodiments, the buffer is selected to increase the pH to greater than 3, 3.5, 4, 4.5, 4.8, 5, 5.5, 5.8, or 6.0. In some embodiments, the buffer and / or pH adjusting agent may be present at a concentration of 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or more. Buffers and pH adjusting agents include those which are commercially available and those disclosed herein. In some embodiments, the buffer and / or pH adjusting agent may comprise one or more of PBS, potassium phosphate, mono- or dibasic potassium phosphate, potassium chloride, potassium gluconate, potassium acetate, phosphoric acid or mixtures thereof, calcium chloride, calcium gluconate, calcium citrate, calcium carbonate, calcium lactate, calcium levulinate, calcium lactobionate, sodium acetate, sodium bicarbonate, sodium

[0146] 4909-9827-7258, v. 126 Docket No. HRVY-259-WO1 citrate, sodium lactate, sodium chloride, mono- or dibasic sodium phosphate, and mixtures thereof, HEPES, alkali earth hydroxides, alkali hydroxides, aluminum hydroxide, citric acid, and acetic acid. In some embodiments, the concentration of buffers and / or pH adjusting agents is selected to prevent aggregation of the viral vectors in solution. In some embodiments, a mixture of two, three, four, five or six of the above buffers are selected. In some embodiments, the composition comprises PBS in combination with magnesium chloride in a concentration of at least 300 mM, at least 400 mM, at least 500 mM, or at least 600 mM. In some embodiments, specific buffers and their concentrations will be selected to reduce pH shift during freezing, and / or will be combined with excipients which reduce the crystallization of the buffer on cooling / freezing. Such excipients include sucrose, fibrous disaccharides, seaweed sugar, cellobiose, mannitol or ionic cryoprotectants (e.g., TMAC1).

[0147] In some embodiments, the composition comprises one or more surface active agents and / or emulsifiers to enhance stability and efficacy of the composition comprising the agent, enhance penetration of cellular membranes, and to reduce adsorption of the agent onto surfaces used to prepare and administer the composition. In some embodiments, the surfactants are included in a concentration of 0.00001 w / v% to 3.0 w / v%., 0.0001 w / % to 1 w / v%, or 0.001 w / v% to 0.5 w / v%. The surface active agents are not particularly limited and include non-ionic, cationic, anionic and amphiphilic surfactants which are commercially available and those described herein. In some embodiments, the surface active agent may be sorbitan fatty acid esters, polyoxyethylene esters, sucrose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene ethers, nonionic triblock copolymers, such as poloxamers (e.g. P188) or pluronic copolymers, carbomers, high molecular weight alcohols, colloidal clays, natural emulsifiers (e.g. lecithin, cholesterol), diethylene glycol monolaurate, potassium oleate, sodium lauryl sulfate, benzalkonium chloride, cetylpyridinium chloride, among others. In some embodiments, the surface-active agent is combined with a buffer.

[0148] It will be appreciated by the ordinarily skilled artisan that the composition may comprise other pharmaceutically acceptable excipients to aid in formulating, preserving and administering the composition. For example, if the composition is to be frozen or lyophilized, cryoprotectants may be used to protect the stability and efficacy of the composition. The cryoprotectants utilized are not particularly limited and include those which are commercially available and those described herein. In some embodiments, the cryoprotectant is one or more of sucrose, lactose, glucose, mannitol, sorbitol, alginate, polyvinylpyrrolidone, ethylene glycol, propylene glycol, glycerol, hydroxyethyl starch, and one or more salts. These

[0149] 4909-9827-7258, v. 127 Docket No. HRVY-259-WO1 cryoprotectants may include polyols, sugars, and salts, at a concentration between 0.5-55wt% of the composition. In addition, preservatives may be added to inhibit microbial growth and prevent oxidation of contents of the composition. Pharmaceutically acceptable excipients may also comprise osmolarity adjusting agents, solvents, vehicles, dispersion aids, suspension aids, as well as other excipients listed in Remington: the science and practice of pharmacy (Remington, Joseph Price, Vol. 1. Lippincott Williams & Wilkins, 2006).

[0150] Dose and Timing

[0151] When formulating the composition according to the invention, a dose of the agent will be selected that is effective to treat or prevent a disease or disorder or ameliorate a symptom of the disease or disorder in a subject in need thereof. In some embodiments, an effective dose of OSM comprises from about 0.05 pg to about 5 mg, about 0.1 pg to about 4.5 mg, about 0.25 pg to about 4 mg, about 0.5 pg to about 3.5 mg, about 0.75 pg to about 3 mg, about 1 pg to about 250 pg, about 10 pg to about 400 pg, about 300 pg to about 750 pg, about 450 pg to about 1 mg, about 500 pg to about 1.5 mg, about 750 pg to about 2 mg, about 1 mg to about 2.5 mg, about 1.25 mg to about 3 mg, about 1.5 mg to about 5 mg, about 2 mg to about 4.25 mg, or about 2.5 mg to about 4.5 mg. In some embodiments, the effective dose is about 0.05 pg, about 0.1 pg, about 0.15 pg, about 0.2 pg, about 0.25 pg, about 0.5 pg, about 1 pg, about 1.5 pg, about 5 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, about 100 pg, about 125 pg, about 150 pg, about 175 pg, about 200 pg, about 225 pg, about 250 pg, about 275 pg, about 300 pg, about 325 pg, about 350 pg, about 375 pg, about 400 pg, about 425 pg, about 450 pg, about 475 pg, about 500 pg, about 525 pg, about 550 pg, about 575 pg, about 600 pg, about 625 pg, about 650 pg, about 675 pg, about 700 pg, about 725 pg, about 750 pg, about 775 pg, about 800 pg, about 825 pg, about 850 pg, about 875 pg, about 900 pg, about 925 pg, about 950 pg, about 975 pg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 4.75 mg, or about 5 mg.

[0152] When the method or composition comprises a non-retroviral vector, the effective dose falls within the range of 1 x 107viral genomes (vg) / kg to 1.0 x 1015vg / kg. In some embodiments, the effective dose is approximately 1.0 x 107vg / kg, 5.0 x 107vg / kg, 1.0 x 108

[0153] 4909-9827-7258, v. 128 Docket No. HRVY-259-WO1 vg / kg, 5.0 X 108vg / kg, 1.0 X 109vg / kg, 5.0 x 109vg / kg, 1.0 x 1010vg / kg, 5.0 x 1010vg / kg, 1.0 x IO11vg / kg, 2.5 x IO11vg / kg, 5.0 x IO11vg / kg, 7.5 x IO11vg / kg, 1.0 x 1012vg / kg, 2.5 x 1012vg / kg, 5.0 x 1012vg / kg, 7.5 x 1012vg / kg, 1.0 x 1013vg / kg, 2.5 x 1013vg / kg, 5.0 x 1013vg / kg, 7.5 x 1013vg / kg, 1.0 x 1014vg / kg, or 1.0 x IO15vg / kg. In some embodiments, the effective dose is approximately 1.0 x 107vg / kg to approximately 1.0 x 108vg / kg, approximately 5.0 x 107vg / kg to approximately 5.0 x 108vg / kg, approximately 1.0 x 109vg / kg to approximately I.0 x IO10vg / kg approximately 5.0 x 109vg / kg to approximately 5.0 x IO10vg / kg, approximately 1.0 x 1011vg / kg to approximately 1.0 x 1012vg / kg, approximately 2.5 x 1011vg / kg to approximately 2.5 x 1012vg / kg, approximately 5.0 x 1011vg / kg to approximately 5.0 x 1012vg / kg, approximately 7.5 x 1011vg / kg to approximately 7.5 x 1012vg / kg, approximately 1.0 x 107vg / kg to approximately 5.0 x 109vg / kg, approximately 1.0 x IO10vg / kg to approximately 5.0 x 1012vg / kg, approximately 5.0 x 107vg / kg to approximately 2.5 x 1013vg / kg, approximately 1.0 x 108vg / kg to approximately 1.0 x 1014vg / kg, approximately 5.0 x 108vg / kg to approximately 7.5 x 1012vg / kg, or approximately 1.0 x 109vg / kg to approximately 1.0 x 1015vg / kg. In some embodiments, the composition is in the form of a unit dose.

[0154] In some embodiments, an effective dose may be administered 1, 2, 3, 4, 5, 6 or more times a day. In some embodiments, an effective dose may be administered 2-5 times a day, 1-4 times a day, 3-10 times a day, 4-8 times a day, or 2-6 times a day. In some embodiments, an effective dose may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times during a week or two-week period. In some embodiments, an effective dose may be administered 1-30 times. 3-15 times, 4-20 times, 5-25 times, 6-18 times, or 8-26 times during a week or two-week period. In some embodiments, an effective dose may be administered once or twice a month. In some embodiments, an effective dose may be administered 1-6 times, 2-10 times, 4-20 times, 6-25 times, or 10-35 times a month. The duration of treatment may be continued for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. In some embodiments, the duration of treatment may be continued for between 2-36 hours, 2-24 hours, 2-12 hours, 2-8 hours, 4-32 hours, 4-24 hours, 4-12 hours, 4-8 hours, 6-24 hours, 8-36 hours, 6-12 hours, 8-36 hours, 8-24 hours, 8-12 hours, 12-24 hours, 12-36 hours, or 12-18 hours. In some embodiments, the treatment may be continued for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days. In some embodiments, the treatment may be continued for 1-365 days, 7-365 days, 14-365 days, 30-365 days, 45-365 days, 60-365 days, 90-365 days, 120-365 days, 150-365 days, 180-365

[0155] 4909-9827-7258, v. 129 Docket No. HRVY-259-WO1 days, 210-365 days, 240-365 days, 270-365 days, 300-365 days, or 330-365 days. In some embodiments, the treatment may be continued for 1-20 years, 2-15 years, 3-10 years, or 4-8 years. In certain embodiments, the composition may be administered continuously or intermittently for months, years, or for a duration of time during which amelioration or prevention of symptoms are desired or needed.

[0156] Initiation of treatment or prophylactic administration of the composition may correspond with a period of maximum impact or need. In some embodiments, the composition may be administered at the outset of symptoms of a viral respiratory infection, or upon confirmed diagnosis thereof. In some embodiments, initiation of treatment commences during a period of peak viral load / peak infection. In some embodiments, the period of peak infection is measured from symptom onset. In some embodiments, the agent is administered during a period of elevated lung damage compared to a baseline level of lung damage, such as a healthy lung before infection. In some embodiments, the period of peak infection occurs about 3 to 10 days post- infection. In some embodiments, the period of peak infection or elevated lung damage is determined via a pulmonary function test. Assessing pulmonary function can be performed using techniques and devices known in the art, including spirometry or plethysmography. In some embodiments, the period of peak infection or elevated lung damage is characterized by increased interferon levels. Interferon levels may be assessed from biological fluids, such as blood, saliva, sputum, urine or broncho-alveolar lavage fluid. In some embodiments, the period of peak infection or elevated lung damage is characterized by an inflammatory response. In other embodiments, the initiation of treatment occurs during a period of increased lung barrier permeability. Lung barrier integrity may be assessed by methods known in the art, such as those described in Herrero et al. (Curr Protoc Toxicol. 63 (2015):24.3.1-24.3.15, PMID: 25645245) and Pell et al. (SLAS Discovery 26.7 (2021): 909-921). In some embodiments, initiation of treatment commences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days post-infection or after the onset of symptoms. In certain embodiments, treatment commences 7, 6, 5, 4, 3, 2, or 1 day post-infection or after onset of symptoms. The period of peak infection may vary based on the patient and / or based on the type of underlying infection.

[0157] 4909-9827-7258, v. 130 Docket No. HRVY-259-WO1

[0158] EXAMPLE 1: MACROPHAGE-DERIVED ONCOSTATIN M REPAIRS THE LUNG EPITHELIAL BARRIER DURING INFLAMMATORY DAMAGE

[0159] Tissue repair programs must function alongside antiviral immunity to restore the lung epithelial barrier following infection. We found that macrophage-derived oncostatin M (OSM) counteracted the pathological effects of type I interferon (IFN-I) during infection and damage in mice. At baseline, OSM-deficient mice exhibited altered alveolar type II (ATII) epithelial cell states. In response to influenza or viral mimic challenge, mice lacking OSM exhibited heightened IFN-I responses and increased mortality. OSM delivery to the lung induced ATII proliferation and was sufficient to protect deficient mice against morbidity. Furthermore, OSM promoted organoid formation despite the growth-inhibitory effects of IFN-I. These findings identify OSM as an indispensable macrophage-derived growth factor that maintains the homeostasis of lung epithelial cells and promotes their proliferation to overcome IFN-I- mediated immunopathology.

[0160] Oncostatin M production by macrophages is required to regulate lung epithelial cell states and restore the epithelial barrier following viral-induced immunopathology.

[0161] Tissue damage is an inevitable consequence of infection, requiring repair programs to function alongside strategies to restrict pathogen spread. Mucosal tissues such as the respiratory tract are exposed to the external environment, necessitating robust inflammatory responses to fight infections. However, inflammation can also cause tissue damage, compromising epithelial barrier integrity and function. For example, type I interferons (IFN-I), which are essential antiviral mediators, can exacerbate lung pathology by driving immune cell infiltration, inducing epithelial cell death, and suppressing epithelial proliferation (1-4). Despite virus-mediated cell death and the inhibitory effects of IFN-I on proliferation, the epithelial barrier is rapidly restored after viral respiratory infections, suggesting the existence of mechanisms that counteract the impacts of IFN-I to maintain lung function.

[0162] We predicted that macrophages — innate immune cells involved in pathogen defense, tissue repair, and homeostasis (5-9) — may provide crucial signals that limit the detrimental effects of antiviral responses and facilitate epithelial repair in the damaged lung. Alveolar macrophages (AMs) reside within lung alveoli, where they interact with alveolar type I (ATI) and type II (ATII) epithelial cells (10). ATIs facilitate gas exchange, and ATIIs produce surfactants and serve as progenitors for both epithelial cell types (77). Given their strategic positioning and immunomodulatory functions, we hypothesized that a macrophage-derived

[0163] 4909-9827-7258, v. 131 Docket No. HRVY-259-WO1 factor may contribute to epithelial homeostasis and repair after viral infection. We focused on the cytokine oncostatin M (OSM), which is implicated in the pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease, pulmonary fibrosis, rheumatoid arthritis, and cancer {12-14). However, its functions at steady state and in response to acute inflammatory challenges remain poorly understod.

[0164] Results

[0165] OSM is required for maintenance of ATII composition and host survival during UAV infection.

[0166] At baseline, OSM was present in the lungs of wild-type {Osm+ +) mice but was undetectable in the lungs of OSM-deficient Osm7') mice (FIG. 1A). To investigate the role of OSM in acute inflammation and tissue damage, we utilized a sublethal model of influenza A virus (IAV) infection (A / WSN / 1933; H1N1) (FIG. IB). We measured OSM levels in bronchoalveolar lavage fluid (BALF) and plasma of infected wild-type mice and detected only local production (Fig. 1A). OSM levels in BALF and transcript expression in the lung both peaked at 5 days post-infection (dpi) (FIGS. 1A-1B).

[0167] Because OSM levels increased during IAV infection, we hypothesized that OSM may contribute to antiviral host defense. We infected Osm+I+and Osm7mice and found that Osm / _mice succumbed to sublethal IAV infection (FIG. 1C). Given that OSM was detectable at baseline and that weight curves diverged early during infection, we investigated the role of OSM in the lung at steady state and 2 dpi by performing single-cell RNA sequencing (scRNA-seq) on cells isolated from the lungs of Osm+ / +and Osm'7' mice (FIG. ID, FIG. 6C). Osm expression was detected primarily in myeloid subsets including AMs, monocytes, and neutrophils in wild-type mice (FIG. IE). We reanalyzed publicly available datasets to examine the expression of the gene encoding OSM OSM) and its coreceptor OSMR {OSMR) (FIGS. 7A-7C). OSM was found to be robustly expressed in myeloid subsets from dissociated lung samples of COVID- 19 patients (FIGS. 7A-7B). These data suggested that Osm expression in myeloid cells was conserved across mice and humans during viral respiratory infections.

[0168] To evaluate macrophage production of OSM in response to diverse stimuli, we treated bone marrow-derived macrophages (BMDMs) with various stressors and pathogen-associated molecular patterns (PAMPs) in vitro and found heightened OSM levels after treatment (FIGS. 8A-8B). We next stimulated AMs with the PAMP poly(I:C) ex vivo.

[0169] 4909-9827-7258, v. 132 Docket No. HRVY-259-WO1 Poly(I:C) mimics a double- stranded RNA viral replication product and stimulates TLR3 and RIG-I / MDA5 pathways (75). Unlike what we observed in BMDMs, poly(I:C) alone was insufficient to induce OSM production in AMs (FIG. 8C). GM-CSF, a growth factor and ATII-derived signal essential for AM maintenance (16, 17), was required for OSM production, and its combination with poly(I:C) enhanced OSM levels (FIG. 8C). To determine whether ontogeny influences OSM production, we stimulated fetal-derived AMs and monocyte-derived AMs (moAMs) ex vivo. AM origin was determined by using Siglec-F expression (18). We found that moAMs produced higher levels of OSM compared with fetal-derived AMs (FIGS. 8D-8E). Therefore, although both populations produce OSM, moAMs may serve as a major source of OSM during challenge. Altogether, these data demonstrated that OSM was produced by myeloid cells at steady state and elicited in response to infection and environmental stressors.

[0170] Expression of Osmr was restricted to non-immune populations including epithelial cells, fibroblasts, and endothelial cells in wild-type mice (FIG. IF). Analysis of OSMR expression in healthy and COVID-19 patients (19, 20) showed similar expression patterns (FIGS. 7A-7C). Ex vivo stimulation of mouse lung cells with OSM, followed by analysis of downstream STAT3 phosphorylation, revealed robust responses in epithelial and stromal cells, with no substantial response detected in endothelial or immune cells (FIGS. 9A-9B). Given that the lung epithelium exhibited the greatest responsiveness to OSM, we assessed airway epithelial cells and ATIIs but found no defects in distribution or numbers in Osm ' mice (FIGS. 24A-24C). Considering the susceptibility of ATIIs to IAV infection (27), their critical role in maintaining and replenishing the alveolar epithelium (77), and their close proximity to OSM-producing AMs, we next examined ATII transcriptional profiles in the absence of OSM. Gene expression analysis validated our ATII annotation, showing high Sftpc expression compared with airway epithelial markers (Scgblal and Foxjl), together with ATII signature enrichment (FIGS. 25A-25B). Subclustering of ATIIs (FIG. 25C) revealed three populations: a cluster predominant in

[0171]

[0172] mice (cluster 1; OSM-independent), a cluster enriched in Osm+7+mice (cluster 2; OSM-dependent), and a cluster characterized by expression of interferon stimulated genes (ISGs) (cluster 3; ISG111) (FIGS.

[0173] 1G-1H, and 55A-55G). Although cluster 1 showed no specific pathway enrichment, cluster 2 was enriched in protein translation pathways and cluster 3 in interferon signaling (FIG. 25D and 55A-55G). These data indicated that OSM shaped the expression profile of epithelial cells at baseline and during early IAV infection. Notably, at 2 dpi, cluster 3 (ISG111) was

[0174] 4909-9827-7258, v. 133 Docket No. HRVY-259-WO1 enriched in Osm ' mice, suggesting an exacerbated inflammatory response to IAV infection in the absence of OSM (FIG. 1H).

[0175] OSM-deficiency results in exacerbated IFN-I responses and lung immunopathology during IAV infection and increased susceptibility to viral mimic challenge.

[0176] As increased morbidity and mortality occurred at later time points after IAV infection, we performed bulk RNA-seq on whole lungs from Osm+I+and Osm7' mice at 3 and 7 dpi (FIGS. 10A-10E). At 3 dpi, pathway enrichment analyses (22, 23) indicated a more prominent IFN-I signature in Osm7' mice as compared with Osm+ / +mice (FIGS. 10A-10E). These findings were corroborated by an increase in lung ISG expression, including Ifitl, Isgl5, and Irf7, and an increase in IFN-I transcripts, including Iftib and lftia4 (FIG. 2 A and FIGS. 11A-1 IB). Osm1' mice also showed elevated IFN-o. and IFN-P in BALF at 2 dpi compared with Osm+ / +mice (FIGS. 2B-2C). We investigated histological differences at 2, 4, and 6 dpi (FIGS. 2D-2E, and FIGS. 12A-12B) and found that Osm7' mice displayed more severe damage at 4 dpi (FIG. 2E). lAV-infected Osm'7' mice also exhibited increased BALF total protein levels and barrier permeability compared with Osm+I+mice (FIGS. 2F-2G), indicating a loss of lung barrier integrity, a known hallmark of IFN-mediated immunopathology (2). Elevated immune infiltration was also observed in Osm'7' mice, with increased numbers of neutrophils at both 2 and 6 dpi, monocytes at 2 dpi, and interstitial macrophages (IMs) / monocyte-derived macrophages (moMacs) — which are indistinguishable using conventional gating schemes — at 6 dpi (FIG. 13 and FIGS. 14A-14C). However, we found no differences in AM, plasmacytoid dendritic cell, or lymphocyte numbers between Osm+I+and Osm'7' mice at either early- or late-infection time points (FIG. 13 and FIGS. 14D-14J). Notably, we observed no differences in lung damage or IFN-I levels at baseline (FIG.

[0177] 2B-2C, and FIGS. 11A-1 IB, and FIG. 12C), indicating that although OSM regulated ATII gene expression, this did not lead to overt lung pathology under steady-state conditions.

[0178] To evaluate whether the increases in IFN-I and tissue damage correlated with differences in viral load, we performed viral plaque assays on BALF collected from mice at 1, 2, and 6 dpi. We did not observe differences in viral load at 1 or 6 dpi and detected no viremia in either Osm+I+or Osm'7' mice (FIGS. 15A-15C). However, at 2 dpi, we detected an increase in viral load in Osm'7' mice compared with wild type (FIG. 15A). Although equivalent viral loads at 6 dpi suggested that viral burden was not the driver of the IAV survival phenotype, we sought to avoid this confounding factor. To do so, we utilized an

[0179] 4909-9827-7258, v. 134 Docket No. HRVY-259-WO1 additional in vivo model using intratracheal (i.t.) delivery of poly(I:C) (FIG. 2H), which is a potent inducer of IFN-I (24) and tissue damage (FIG. 16A).

[0180] We found that OSM levels increased locally in BALF and lung in response to i.t. poly(I:C) treatment, but not systemically in plasma (FIG. 21 and FIGS. 16B-16C).

[0181] Furthermore, Osm7' mice succumbed to i.t. poly(I:C) treatment (FIG. 2J), an outcome conserved across sexes (FIG. 16D). We detected Osm expression throughout the myeloid compartment in sorted AMs, IMs / moMacs, neutrophils, and monocytes from the lungs of wild-type mice at steady state and at 7 days post-initial challenge (dpc) (FIGS. 16E-16F). Given the localized presence of OSM in the lung after viral stimuli, we investigated whether OSM provided protection in mice treated intraperitoneally (i.p.) with poly(I:C) for 3 consecutive days. Osm+I+and Osm7mice both exhibited high levels of morbidity in response to systemic poly(I:C) (FIG. 16G). These results suggested that OSM provided local protection against viral stimuli in the lung but did not confer protection against systemic viral stimuli.

[0182] Macrophage-derived OSM is required for survival during poly(I:C) challenge.

[0183] To determine the importance of macrophage-derived OSM upon i.t. poly(I:C) challenge, we developed a macrophage- specific conditional knockout mouse model by crossing Osm111’ and FcgrlCr&(CD64Cre) (25) mouse lines. We validated the loss of Osm in sorted AMs and IMs / moMacs at 7 dpc (FIG. 17A). Additionally, we found CD64 expression to be exclusive to F4 / 8CFCD1 lb+Ly6C+cells in the lung and not in the blood, indicating that circulating monocytes do not express CD64 at the protein level (FIG. 17B). To confirm that macrophages were the major source of OSM in the context of viral stimuli, we measured lung OSM levels after poly(I:C) challenge and observed diminished RNA and protein expression in OSM-deficient mice (FIGS. 17C-17D). Macrophage- specific Osm deletion also reduced OSM levels during peak production after IAV infection (FIG. 1A and FIG. 17E). We assessed myeloid cell phagocytic capacity and baseline numbers in Fcgrl^Osmf1^ mice and found no defects in macrophage or neutrophil phagocytosis (FIG. 19C and Supplementary Text 1 (see below)) and no differences in AM, IM / moMac, or monocyte numbers, with a reduction in neutrophils (FIG. 19D). Additionally, we examined Osmr expression in various cell types in poly(I:C)-treated and -untreated FcgrlCreOsm11711' and Osm11711mice and did not observe differences in receptor expression across genotypes (FIG. 17F).

[0184] 4909-9827-7258, v. 135 Docket No. HRVY-259-WO1 Macrophage- specific deletion of Osm during poly(I:C) challenge led to increased morbidity and mortality (FIG. 3A), increased BALF IFN-I levels and infiltration of IMs / moMacs, monocytes, and neutrophils into the lung, and a decrease in AMs (FIG. 3B and FIG. 18A). This was accompanied by increased IFN and ISG transcript expression, enhanced barrier permeability, and elevated levels of total protein in BALF (FIGS. 3C-3E, and FIGS.

[0185] 18B-18C).

[0186] Previous studies demonstrated IFN-driven disruption of lung epithelial barrier function (2, 24). We tested whether, after challenge, mice lacking macrophage-derived OSM exhibited a loss of epithelial cells, which might explain their compromised barrier integrity. FcgrlCr&Osnf / flmice showed a reduction in total lung epithelial cells and specifically ATIIs at 7 dpc (FIGS. 3F-3G), suggesting alveolar damage.

[0187] To test the hypothesis that OSM protected mice from succumbing to poly(I:C) treatment through an IFN-I-mediated pathway, we administered intravenous (i.v.) IFN-o / p receptor antibodies [anti-interferon a / p receptor subunit 1

[0188]

[0189] &Osm'l / '1m\ during treatment with poly(I:C) (FIG. 19A). Upon IFNAR1 neutralization, FcgrlCr&Osnfi / flva c& exhibited no differences in morbidity or mortality compared with anti-IFNAR1 treated Osnf / flmice (FIG. 3H, FIG. 19A, and Supplementary Text 2 (see below)). Moreover, reconstitution of OSM in the respiratory tract by administration of recombinant mouse OSM, lessened morbidity in FcgrlCr&Osnf / flmice after poly(I:C) treatment (FIG. 31 and FIG. 19B). Local administration of OSM did not change BALF IFN-I levels in FcgrlCr&Osnf / flmice after poly(I:C) treatment (FIG. 3J) but led to elevated numbers of Ki67+ATIIs, total ATIIs, and an increase in Ki67+ATII proportions (FIGS. 3K-3L, and FIG. 19C). This suggested that although IFN-I signaling was necessary for morbidity in the absence of OSM, it was not sufficient; moreover, OSM signaling restored ATII proliferation after epithelial damage.

[0190] OSM administration restores ATII composition in OSM-deficient mice.

[0191] We hypothesized that in addition to promoting epithelial cell proliferation, local OSM treatment could restore the OSM-dependent ATII cell state (cluster 2) (FIGS. 1G-1H). We administered i.t. OSM to Osm+I+and Osm'' mice, harvested ATIIs, and performed bulk RNA-seq (FIG. 4A, FIG. 20A, and 56A-56D). On the basis of the clusters identified from scRNA-seq data (FIG. 1H), ATII cluster 1 (OSM-independent)-enriched genes were increased in ATIIs from Osm'' mice compared with Osm+I+mice, and ATII cluster 2 (OSM-

[0192] 4909-9827-7258, v. 136 Docket No. HRVY-259-WO1 dependent) -enriched genes were increased in Osm+I+mice (FIGS. 4B-4C). ATII cells from Osm'' mice treated with i.t. OSM, when compared to those from Osm'' mice, recapitulated the differences observed between Osm+I+and Osm'' ATIIs, suggesting restored gene expression in response to OSM treatment (FIGS. 4B-4C).

[0193] We identified all genes that were at least twofold differentially expressed in any pairwise comparison between groups and performed k-means clustering based on expression values (FIG. 4D and 56A-56D). Cluster A was associated with nuclear factor KB signaling and cluster D was associated with IFN signaling (FIG. 4E). Cluster B, which was enriched for genes associated with cholesterol homeostasis, showed lower expression in Osm'' mice compared to controls, but increased expression after the addition of OSM (FIG. 4E and FIG.

[0194] 20B). The expression of cluster C, which reflected cell-cycle progression, did not vary between Osm+I+and Osm'' mice, but was increased upon administration of OSM in Osm'' mice (FIG. 4E and FIG. 20B). These data suggested that at steady-state levels, OSM regulated cholesterol homeostasis, a functional hallmark of mature ATIIs (26), whereas elevated OSM levels induced cell-cycle progression.

[0195] OSM promotes epithelial proliferation by overriding IFN-I-driven growth inhibition Informed by our findings that OSM increased the number of total and Ki67+ ATIIs in FcgrlCr&Osnf / flmice after poly(EC) treatment, we investigated whether, even in the absence of damage and OSM deficiency, elevated OSM levels induced ATII proliferation. We administered i.t. OSM to wild-type mice at steady state for 3 consecutive days (FIG. 4F). Mice treated with OSM displayed elevated ATII Ki67 positivity and 5-ethynyl-2'-deoxyuridine (EdU) incorporation compared to with phosphate-buffered saline (PBS)-treated controls (FIGS. 4G-4H).

[0196] We next hypothesized that OSM may induce epithelial proliferation under inflammatory conditions. We grew alveolar organoids (27, 28) in the presence or absence of IFN-I supplemented with interleukin- ip (IL-ip) or STAT3 activators, OSM or IL-6 (FIG. 5A and FIG. 20C). IL-ip has been found to promote both epithelial proliferation and differentiation of ATII cells into ATI cells (29, 30). As previously reported (29-31), IFN-I limited organoid forming efficiency (OFE) and mean organoid area, whereas IL-ip increased the area of organoids (FIGS. 5B-5D). IL-6 had no effect on either metric. IL-ip was unable to overcome the growth suppressive effects of IFN-I, and IL-6 showed a marginal effect on OFE. By contrast, OSM modestly increased OFE at baseline and completely restored both

[0197] 4909-9827-7258, v. 137 Docket No. HRVY-259-WO1 organoid size and OFE under IFN-I-mediated growth suppression to the levels observed in untreated organoids (FIGS. 5B-5D). These data revealed that the growth-promoting function of OSM was dominant over IFN-I. Collectively, this study demonstrated that OSM regulated transcriptional programs in epithelial cells at steady state and promoted host survival by driving ATII proliferation after IFN-I signaling (Fig 5E).

[0198] Discussion

[0199] Our study establishes that macrophage production of OSM is critical for maintaining and restoring lung epithelial homeostasis. Studies have shown that the protective or pathological roles of interferons depend on their temporal expression and spatial activity within the respiratory tract (32, 33). We conclude that OSM mitigates the pathological effects of IFN by promoting epithelial proliferation, even in conditions typically associated with growth suppression. This underscores the integral role of epithelial-immune communication in tissue maintenance and repair (34), which is akin to the role of other macrophage- stromal circuits identified in our previous work 35).

[0200] Prior research has described both pro- and anti-inflammatory roles for OSM (36-38). Our findings demonstrate that OSM deficiency alters the transcriptional profiles of epithelial cells, potentially explaining these pleiotropic effects. The regulation of lung epithelial cell states by OSM may also explain previous reports that OSM overexpression in the lung exacerbates inflammation and disease (13, 14). Notably, several studies have shown that increased OSM signaling enhances allergic inflammation (38-42). Our work hints that these prior observations could be due to disruptions in epithelial homeostasis.

[0201] Recent studies of OSM across various tissues highlight its emerging role in regulating stem and progenitor cell states (43-47). In muscle (44) and hair follicles (43), OSM was found to regulate stem cell quiescence. OSM was also found to inhibit adipocyte differentiation from mouse embryonic fibroblasts (45) and mesenchymal stromal cells in adipose tissue (46), demonstrating a role in regulating cell differentiation. OSM has also been implicated in tissue repair (48, 49), perhaps through effects on cell differentiation and proliferation, although these mechanisms were not specifically addressed. Our findings extend this theme, identifying an OSM-dependent ATII cell state in the lung and supporting a role for OSM in cell proliferation and / or differentiation. Future investigations will define the precise role of OSM on stem and progenitor cell fate in the respiratory tract and other mucosal tissues.

[0202] 4909-9827-7258, v. 138 Docket No. HRVY-259-WO1 In the lung, epithelial repair operates concurrently with a robust antiviral response to prevent prolonged disruption of the epithelial barrier and vulnerability to secondary infections. Our study identifies a mechanism of disease tolerance in which macrophage-derived OSM plays a crucial role in stimulating the proliferation of lung epithelial cells, even under the antiproliferative effects of IFN-I. Furthermore, our work suggests that OSM may function as a “counter-inflammatory” signal (50), opposing the harmful effects of inflammation on target tissues rather than simply reducing the overall inflammatory response. These findings also highlight the potential of OSM as a therapeutic agent to activate repair pathways, particularly in severe infections in which expedient restoration of the epithelial barrier is critical.

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[0263] 4909-9827-7258, v. 143 Docket No. HRVY-259-WO1 Koziol- White, W. F. JesterJr., B. J. Jenkins, Y. Cao, C. Clarke, C. Austin, D. Lafkas, M. Xu, P. J. Wolters, J. R. Arron, N. R. West, M. S. Wilson, Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma. Sci Transl Med 14, eabf8188 (2022).

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[0278] Tanaka, H. Lorchner, S. Schimanski, M. Szibor, H. Warnecke, T. Braun, Oncostatin M Is a Major Mediator of Cardiomyocyte Dedifferentiation and Remodeling. Cell Stem Cell 9, 420-432 (2011).

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[0287] Beppu, H. J. Deeg, C. McFarland, K. R. Loeb, W. J. Valente, N. G. Ericson, E. A. Stevens, J. P. Radich, T. S. Mikkelsen, B. J. Hindson, J. H. Bielas, Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).

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[0292] 4909-9827-7258, v. 145 Docket No. HRVY-259-WO1 56. C. Hafemeister, R. Satija, Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019).

[0293] 57. E. Pemet, J. Downey, D. C. Vinh, W. S. Powell, M. Divangahi, Leukotriene B4-type I interferon axis regulates macrophage-mediated disease tolerance to influenza infection. Nat. Microbiol. 4, 1389-1400 (2019).

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[0295] Supplementary Tables

[0296] Table SI: Antibodies

[0297]

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[0299]

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[0301]

[0302] 4909-9827-7258, v. 149 Docket No. HRVY-259-WO1

[0303]

[0304] Table S2: Oligonucleotides

[0305]

[0306] 4909-9827-7258, v. 150 Docket No. HRVY-259-WO1

[0307]

[0308] Materials and Methods

[0309] Mice

[0310] All animal experiments were performed in accordance with institutional regulations after protocol review and approval by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School (protocol IS00003152). Mice were bred and housed in a specific pathogen-free facility, with room temperature set to 71 °F (+ / -30), humidity set to 50% (+ / -15%), with a 12:12-hour light / dark cycle, and ad libitum access to food (LabDiet 5053) and water. C57BL / 6J mice (RRID:IMSR_JAX:000664) and DsRed.T3 (RRID:IMSR_JAX:006051) were obtained from Jackson Laboratories (J AX), FcgrlCxe. mice were provided by Dr. Ming Li (MSKCC) (27), and gene targeted Osm “Knockout-first” mice were obtained from the KOMP repository (RRID:MMRRC_059829-UCD). To generate conditional Osm alleles, mice were bred to the FLPo deleter strain (RRID:IMSR_JAX:012930). Osm conditional mice were then bred to FcgrlCxe. to achieve conditional deletion. Animals were euthanized by administration of a lethal dose of ketamine / xylazine mixture. Female and male mice aged 8 to 12 weeks were used for all experiments, as specified.

[0311] Infection and in vivo treatments

[0312] Mice were acclimated for at least one week following their transfer to the BSL-2 facility before infection. For influenza virus or mock infection, mice were anesthetized with a ketamine / xylazine mixture, then administered 225 - 450 plaque forming units (pfu) of virus or PBS intranasally (i.n.) in a 30 pl volume. For intratracheal (i.t.) treatments, mice were anesthetized by inhalation of isoflurane / propanediol and given 33.75 - 50 pg of HMW poly(I:C) (InvivoGen) in 50 pl PBS, or PBS alone, using a pipette. For IFNAR1 -blockade treatments, mice were injected intravenously (i.v.) with 200 pg of InVivoPlus anti-mouse IFNAR1 or rat IgG2a isotype control (Bio X Cell) in 100 pl volume. For rOSM rescue

[0313] 4909-9827-7258, v. 151 Docket No. HRVY-259-WO1 experiments in the context of poly(I:C) challenge, mice were administered 1 pg of murine rOSM (Bio-Techne) or PBS in addition to daily treatment with 33.75 - 50 pg of HMW poly(I:C). For systemic poly(I:C) challenge, mice were treated intraperitoneally (i.p.) with 100 pg of poly(I:C) daily for three consecutive days. For steady state rOSM rescue experiments, mice received daily i.t. administrations of 1 pg of rOSM in a 50 pl volume for seven consecutive days. For phagocytosis assays, mice were i.t. administered 50 pg pHrodo E.Coli BioParticles (Thermo Fisher Scientific) 3 hours before euthanasia. For steady state lung epithelium proliferation assays, mice received daily i.t. administrations of 1 pg rOSM in a 50 pl volume and i.p. injections of 1 pg EdU (Thermo Fisher Scientific) in a 200 pl volume for three consecutive days. Mice were euthanized and tissues collected 24 hours after the final treatment. For the Evans blue dye (EBD) lung barrier permeability assay, mice were injected i.p. with 10 mg / kg of EBD (Sigma) 16 hours before euthanasia.

[0314] Plaque assay and virus propagation

[0315] Influenza virus strain A / WSN / 33 was originally obtained from Dr. Peter Cresswell (Yale University School of Medicine). The virus was propagated and titered by plaque assay using Madin-Darby Canine Kidney (MDCK) cells (ATCC, CCL-34, NBL-2). To determine viral titer, a 10-fold serial dilution of virus was prepared in 0.1% BSA in PBS in 200 pl volume. The cells were infected at 37 °C for 1 hour and shaken every 15 minutes. Cells were washed twice with PBS and an agarose overlay of 1% agarose gel mixture including TPCK Trypsin (1:2000), 0.21% BSA, 0.225% NaHCO3, 100 U / mL penicillin / streptomycin in MEM was added to each well and the plate was incubated at 37°C for 48-72 hours upside down. Wells were fixed with 10% formalin for at least two hours. Gels were removed, 1 mL of crystal violet solution was added to each well, and the plate was incubated at room temperature for 30 minutes. For viral propagation, MDCK cells were infected at an MOI of 0.002 in 100 pl of DMEM + 1% BSA per well and incubated for 10 minutes at 37°C. 1.5 mL of DMEM + 1% BSA were added to each well and cells were left to grow for 48 hours. Cells were spun down at 850G for 10 minutes and supernatant collected.

[0316] Histological analysis

[0317] For histological analysis, lung lobes were fixed in 10% formalin for 48 hours, then embedded, sectioned, and H&E stained by the HMS Rodent Histopathology core. Histology images were acquired using an Olympus VS200 Slide scanner with a color camera, using the

[0318] 4909-9827-7258, v. 152 Docket No. HRVY-259-WO1 UPlan X Apo 10x / 0.4 Air objective. Severe damage quantification was performed using QuPath software. Representative training regions were selected and annotated for background, healthy tissue, and severe damage. These regions were utilized to train the pixel classifier, which was subsequently optimized and applied to all slides to obtain classification areas. Severe damage was defined as regions characterized by thickened alveolar walls and immune infiltrates within the alveolar walls and alveoli. The percentage of lung severely damaged was calculated as the area classified as severe damage divided by the total lung area, normalizing for background differences. Representative images were obtained using ImageJ software.

[0319] Tissue collection and cell isolation

[0320] Lungs were harvested and processed as previously described (51). Briefly, mice were euthanized and perfused with 2 mM EDTA in PBS. For flow cytometry analysis of myeloid and epithelial cells, the trachea was exposed, nicked, and a 22-gauge catheter was inserted. A 1 mL-PBS-filled syringe was attached to the catheter and used to inflate then deflate lungs to collect cells present in BALF. The syringe was replaced with a 1 mL-dispase-filled syringe and lungs were inflated. Inflated lungs were removed, and lung lobes were chopped and immersed in enzyme solution (100 pg / mL DNase and 83 pg / mL Liberase in RPMI) and placed in a 37 °C shaker for 40 minutes. BALF and lung digest cells were filtered through a 70 pm cell strainer to obtain a single cell suspension and exposed to hypotonic lysis (ACK lysing buffer, ThermoFisher) to remove red blood cells. For flow cytometry analysis of lymphocytes and pDCs, lungs were removed, and lung lobes were chopped and immersed in enzyme solution (100 pg / mL DNAse and 2 mg / mL Collagenase IV (Worthington Biochemical) in PBS). Lungs were placed in a 37°C shaker for 30 minutes. Lung digest cells were filtered through a 70 pm cell strainer to obtain a single cell suspension and exposed to hypotonic lysis to remove red blood cells.

[0321] Flow cytometry and cell sorting

[0322] Antibodies used for flow cytometry are listed in table SI. Cell viability was determined using Zombie Aqua Fixable Viability Kit (BioLegend) following the manufacturer’s protocol for flow cytometry and DAPI at 10.9 pM (BioLegend) for cell sorting. Samples were Fc-blocked with purified anti-mouse CD16 / 32 antibodies (1:250). For surface staining analysis, cells were fixed in 2% PFA for 20 minutes at room temperature,

[0323] 4909-9827-7258, v. 153 Docket No. HRVY-259-WO1 then washed. For intracellular staining of Ki67 or Foxp3, following surface staining and washes, cells were fixed and permeabilized overnight using Foxp3 Fix / Perm (eBioscience), and staining was performed for 1 hour at room temperature. Analysis of EdU uptake by flowcytometry was performed using the Click-iT® Plus EdU assay kit (ThermoFisher) following manufacturer instructions. To calculate total number quantifications, 123 eBead counting beads (ThermoFisher) were used to normalize for acquisition volume across samples and calculations were performed as per manufacturer instructions. For pSTAT3 staining, following fixation, samples were washed and permeabilized overnight in 90% methanol at 20°C. After permeabilization, samples were washed with ice-cold FACS buffer (1% FBS in PBS) and stained for pSTAT3 and any surface antigens that were marked with methanolsensitive fluorophores. Flow cytometry samples were acquired on a Symphony A5 or Al (BD) and analyzed using FlowJo (Tree Star) software. Sorting samples were acquired in an Aria 561 (BD) or MoFLo Astrios (Beckman Coulter Life Sciences).

[0324] Immunofluorescence staining and imaging

[0325] Following euthanasia, the trachea was exposed, nicked, and a 22-gauge catheter was inserted. A 1 mL syringe filled with a 50% OCT and 50% PBS + 4% PFA solution was attached to the catheter and used to inflate the lungs. Inflated lungs were removed and fixed in 2% PFA on ice for 2 hours. Fixed lungs were incubated in 30% sucrose at 4°C for 24 hours, followed by an additional 24-hour incubation in 15% sucrose and 50% OCT under the same conditions. Lungs were embedded in OCT and cryosectioned at 14 pm and mounted on slides. Sectioned samples were blocked in 3% BSA and 10% donkey serum with 0.1% Triton-X in PBS for 1 hour at room temperature. Sections were incubated overnight at 4°C with primary antibodies (rabbit anti-pro-SP-C, 1:1000, Abeam and rabbit anti-uteroglobin, 1:200, Abeam) diluted in blocking solution. Sections were washed with 0.1% Triton-X in PBS and stained with secondary antibody (donkey anti-rabbit AF647, 1:200, Biolegend) for 1 hour at room temperature. After washing, sections were stained with DAPI (1:10,000) for 5 minutes at room temperature, washed again, and mounted with Diamond Antifade (ThermoFisher). Images were taken on an Olympus VS200 Slide scanner equipped with a Hamamatsu Orca fusion BTsCMOS monochrome camera, using the UPlan X Apo 10x / 0.4 Air objective. Representative images were obtained using ImageJ software.

[0326] 4909-9827-7258, v. 154 Docket No. HRVY-259-WO1 Collection and analysis of blood, whole lung, and BALF

[0327] Blood was collected and plasma isolated using lithium heparin coated plasma separator tubes (BD). Following euthanasia, the trachea was exposed, nicked, and a 22-gauge catheter was inserted. A 1 ml-PBS-filled syringe was attached to the catheter and used to inflate and deflate the lungs twice. The lungs were collected into PBS-filled bead microtubes with ceramic beads and homogenized using a Bead Mill Homogenizer (OMNI International). The homogenized lungs were centrifuged for 10 minutes at 4°C and a speed of 10,000G. The BALF was centrifuged for 5 minutes at 4°C and a speed of 300G. Supernatants were aliquoted and frozen down at -80°C for protein analysis. Total BALF protein levels were quantified using the Bio-Rad Protein Assay. Mouse IFN-a2 was quantified using the LumiKine Xpress mIFN-a 2.0 kit (Invivogen) and mouse IFNP using the LumiKine Xpress mIFN-P 2.0 kit (Invivogen) according to manufacturer instructions. Mouse OSM was quantified using the Mouse OSM Quantikine ELISA kit (R&D Systems) according to manufacturer instructions. For EBD barrier permeability assay, 200 pL of collected BALF was read at 620 nm absorbance in a 96-well plate. All plates were read using a BioTek Synergy HTX plate reader. For flow cytometry analysis of circulating monocytes, blood was collected into 2 mM EDTA, and red blood cells were removed using ACK lysing buffer.

[0328] RNA extraction and quantification

[0329] Tissue samples were collected into TRIzol reagent (Invitrogen) and homogenized in microtubes with ceramic beads and RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research). RNA from in vitro samples was extracted using phenol-chloroform isolation. For sorted cells, samples were collected into TRIzol LS reagent (Invitrogen) and RNA was harvested via phenol-chloroform isolation and co-precipitated with GlycoBlue (Invitrogen). RNA samples used for Ifnb, Ifna4, and Ifnl2 RT-qPCR analysis were treated with DNA-free DNA Removal Kit (Invitrogen) before cDNA synthesis. cDNA synthesis was performed using MMLV reverse transcriptase (Takara Bio) and oligo(dT) primers. RT-qPCR reactions were performed on the Applied Biosystems QuantStudio5 Real-Time PCR System (ThermoFisher) using PowerUp SYBR Green Master Mix (ThermoFisher). Relative expression in RT-qPCR analysis was calculated as (2-ACt)*1000. Fold induction in RT-qPCR analysis was calculated as (2-AACt), where ACt values were normalized to wild-type controls. ACt was calculated by subtracting Rpll3 Ct values from Ct values of genes of

[0330] 4909-9827-7258, v. 155 Docket No. HRVY-259-WO1 interest. Values displayed as -Ct were normalized by RNA input. All oligonucleotides are listed in Table S2.

[0331] Cell culture

[0332] To generate bone-marrow derived macrophages (BMDMs), female C57BL / 6J mice were euthanized and their femurs and tibias were isolated and cleansed with ethanol. Bone marrow was flushed with RPMI 1640 and cells were treated with ACK lysis buffer. Cells were cultured in complete RPMI (cRPMI) (RPMI 1640 + 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 200 U / mL penicillin / streptomycin, 10% FBS, and 2-mercaptoethanol (BME)). The following day (day 1), non-adherent cells were harvested, and 10 x 106 cells were resuspended in a mixture of 70% cRPMI and 30% L929-conditioned media and plated on a petri-dish. On day 4, growth media was supplemented. Cells were used for experiments on day 6. All cell cultures were maintained in a 37°C incubator at 5% CO2. Treatment conditions were as follows: ER stress was induced by 5 pM thapsigargin, heat shock performed at 42°C, and cells stimulated with 10 ng / mL LPS or 50 pg / mL poly(I:C). MDCK cells were cultured in complete DMEM (2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 200 U / mL penicillin / streptomycin, and 10% FBS). For AM stimulation experiments, cells collected from BALF of female C57BL / 6J mice were plated in tissue culture-treated 24- well plates and cultured in cRPMI under the following conditions: 50 pg / mL poly(I:C), 100 ng / mL GM-CSF (Bio-Techne), or a combination of both. For moAM stimulation experiments, lungs from female C57BL / 6J mice infected with IAV for 28 - 31 days were collected. Cells were isolated, sorted, plated in tissue culture-treated 96-well plates, and cultured in cRPMI with stimulation using a combination of 50 pg / mL poly(I:C) and 100 ng / mL GM-CSF. After 24 hours, the supernatants were collected and frozen for subsequent protein analysis.

[0333] Organoid culture

[0334] DsRed.T3 mice were anesthetized with avertin, perfused with 10 mL PBS, and intratracheally instilled with 2 mL Dispase (Coming). Lungs were iced, minced, and incubated in enzyme solution (0.0025% DNase and 100 mg / mL Collagenase / Dispase (Roche) in PBS) for 45 minutes at 37°C. Lung digest cells were filtered through 100 pm and 40 pm cell strainers, and centrifuged for 5 minutes at 4°C and a speed of 215G. Cells were resuspended in red blood cell lysis buffer (0.15 M NH4C1, lOmM KHCO3, 0.1 mM EDTA)

[0335] 4909-9827-7258, v. 156 Docket No. HRVY-259-WO1 for 1.5 minutes, washed with Advanced DMEM, and resuspended in PF10 (10% FBS in PBS) at 1 million cells / 100 pF. Cells were stained for 15 minutes on ice before cell sorting. FACS isolated murine lung CD31- CD45- EpCAM-i- SCA1- viable cells were resuspended in 3D media (DMEM / F12 supplemented with 10% FBS, penicillin / streptomycin, 1 mM HEPES, and insulin / transferrin / selenium (Corning)) at a concentration of 5,000 live cells (trypan blue negative) per 50 pL. As supporting cells, a mix of neonatal stromal cells was isolated as described elsewhere (28, 29). The stromal cells were pelleted and resuspended in growth factor reduced (GFR) Matrigel (Corning) at a concentration of 50,000 cells per 50 pL. Equal volumes of cells in 3D media and supporting cells in GFR Matrigel were mixed and 100 pL were pipetted into a Transwell (Coming). Plates were incubated for 20 minutes at 37 °C, 5% CO2 until Matrigel solidified. Finally, 500 pL of 3D media was added to the bottom of the well. 3D media was changed every other day.

[0336] Organoid treatment, imaging, and analysis

[0337] Organoid cultures were treated with murine rOSM (50 ng / mE, Bio-Techne), rIE-ip (20 ng / mE, Bio-Techne), or rIL-6 (50 ng / mL, Bio-Techne or PeproTech), either alone or in combination with IFN-I (200 U / mL, a 1:1 mixture of rIFN-a and rIFN-pi, BioLegend).

[0338] Organoid media were replenished every 2 - 3 days, with IFN-I treatment spanning a total of four days. Plates were imaged at 14 days post-seeding. Images were captured using an In Cell Analyzer 6000 with a sCMOS monochrome camera from PCO with rolling shutter, using a Plan Apo 4X objective with a 0.20 aperture. 488 laser and 605 / 52 filter used to capture dsRed organoids. Images captured as max intensity projection of 57 Z slices. Images were stitched together and enhanced using non-local mean filtering, followed by threshold binarization and morphological closing of the binary image. Organoids were counted by standard image segmentation. Organoids of area under 5000 pm2 (corresponding to 80 pm diameter) were excluded from analysis. All analysis was done using in-lab MATLAB scripts. The MATLAB code developed for this study is available at github.com / louisonthorens / organoidDetection under a Creative Commons Attribution 4.0 International License (CC-BY 4.0).

[0339] Bulk RNA sequencing

[0340] For tissue samples, RNA was extracted as previously described. For individual cell populations, cells were sorted and RNA extracted using the Direct-zol RNA Miniprep kit. Purified RNA samples were diluted to 2 ng / pL, and 1 pL of the sample was resuspended in 5

[0341] 4909-9827-7258, v. 157 Docket No. HRVY-259-WO1 pL of TCL buffer with 1% BME. Data was processed for sequencing and normalization according to the Immgen protocol (immgen.org / img / Protocols / ImmGenULI_RNAseq_methods.pdf). Whole lung bulk RNA sequencing data are available from the NCBI Gene Expression Omnibus (GEO) under accession number GSE249668 and sorted ATII bulk RNA sequencing data under GSE287742.

[0342] Transcriptional Analysis

[0343] Normalized reads were quality filtered based on minimum expression and coefficient of variation and then visualized with volcano plots and FC / FC plots using Multiplot Studio (GenePattem; Broad Institute). Pathway analysis was performed using Enrichr (25), incorporating data from the MsigDB (24) and Reactome (52) databases. Heat map visualizations and clustering were performed using Morpheus (Broad Institute, software.broadinstitute.org / morpheus).

[0344] scRNA sequencing

[0345] Female mice were infected as described above. Two days post-infection, lungs were collected and digested using the dispase-based digest as described above. Hashing was performed by staining eight percent of the whole lung single cell suspension with TotalSeq-B0301 (Biolegend, Catalog #155831) or TotalSeq-B0302 (Biolegend, Catalog #155833) before encapsulation.

[0346] Upstream processing of IQx single cell data

[0347] Reference files for alignment and counting of lOx single cell reads were prepared using Gencode Release M33 (GRCm39; gencodegenes.org / ) according to build notes provided by lOx genomics (10xgenomics.com / support / software / cell-ranger / downloads / cr-ref-build-steps) (53). Prior to running cellranger mkref, complete H1N1 segment cRNA sequences from LC333182, LC333183, LC333184, LC333185, LC333186, LC333187, LC333188, and LC333189 (ncbi.nlm.nih.gov / nuccore / ) were added to the primary assembly GRCm39 fasta file. Matching GTF file entries were also prepared for H1N1 segment reference files and added to the filtered GRCm39 GTF file. Modified GRCm39 reference files containing H1N1 segment information were then passed to cellranger mkref to complete the lOx genomics reference generation pipeline. Gzipped FASTQ files containing gene and

[0348] 4909-9827-7258, v. 158 Docket No. HRVY-259-WO1 antibody hashtag data were aligned to the H1N1 modified GRCm39 reference files using lOx Genomics Cell Ranger v8.0.1.

[0349] Downstream processing of lOx single cell data

[0350] Data were analyzed using the Seurat package in R (54, 55). Filtered feature matrix.mtx, barcode.tsv and feature.tsv files generated by Cell Ranger were loaded into R using Seurat < v5.1.0. The random seed was set to 9999 using the R set.seed function prior to starting downstream analyses of lOx data. Antibody hashtag reads were normalized using centered log ratio transformation. Hashtag data was demultiplexed using the Seurat HTODemux function with a positive.quantile setting of 0.9. Non-singlet cells were removed from the analysis. Dead and apoptotic cells were filtered based on mitochondrial RNA enrichment using the SeuratWrappers vO.3.5 and flexmix v.2.3-19 RunMiQC function with posterior.cutoff = 0.75, model.slot = "flexmix_model" settings. Cells were further filtered to remove low quality cells and doublets by excluding cells expressing < 500 unique genes and > 9500 unique molecular identifiers (UMIs) and using scDbleFinder vl.18, respectively. Filtered cells were normalized for dimensionality reduction and clustering using the Seurat SCTransform function (56). Principal component analysis (PC A) was run using the Seurat RunPCA with n = 100 npcs, and the final uniform manifold approximation (UMAP) was generated using n = 20 dimensions of the previously calculated PCA reduction as input. Nearest neighbor graph construction and cell clustering were applied using the Seurat FindNeighbours with dims = 1:20 and the FindClusters functions with resolution = 0.5, algorithm = 1. Resulting clusters were then manually collapsed to segment broad cellular populations. Log-normalized gene counts for differential expression analysis were produced using the Seurat NormalizeData function using the settings normalization.method = "LogNormalize" and scale.factor = 10000. The following additional R packages were used for data analysis: Seurat v5.1.0, dplyr < vl.1.4, and Nebulosa vl.14.0. ATII subclustering was performed via Seurat via using subset and reprocessed as described above except for resolution = 0.4 for FindClusters and the genotype-dependent subclusters were collapsed. Subcluster gene modules were generated using FindAllMarkers and taking the top 100 genes with an adjusted p-value < le-5 from just WT mice. Subcluster gene module enrichment was generated using AddModuleScore and displayed using plotdensity from Nebulosa. All analyses were run on a Pop!_OS 22.04 LTS GNU Linux 64-bit system using Anacoda Python version 24.9.2, R version 4.4.2 (2024-10-31) and RStudio 2024.09.1 Build 394 or a MacBook

[0351] 4909-9827-7258, v. 159 Docket No. HRVY-259-WO1 Pro Apple M3 Max Sonoma 14.536GB system and RStudio 2024.09.1 Build 394. The scRNA-seq data generated in this study is available in the Gene Expression Omnibus (GEO) under accession number GSE291698.

[0352] Reanalysis of public human scRNA-seq data

[0353] Data and metadata obtained from published analyses of human lung samples from COVID- 19 patients (21, 22) were obtained from the Broad Institute Single Cell Portal.

[0354] Original UMAP dimensions and cell type annotation from deposited data were used for visualization. Plots reflecting these UMAP coordinates, gene expression levels, and cell annotations were generated using the R package ggplot2.

[0355] Data analysis and statistics

[0356] Statistical tests were performed using GraphPad Prism, with significance defined as p<0.05. The log-rank Mantel-Cox test was used to compare survival curves between groups. Two-way ANOVA was conducted to assess the combined effects of two independent variables on a dependent variable. One-way ANOVA was applied to compare the means across more than two independent groups, and a non-parametric one-way Welch’s ANOVA was used when data did not meet normality or equal variance assumptions. To perform post hoc comparison of selected group means we performed Tukey’s test on parametric ANOVA data sets and Dunnett’s test on non-parametric ANOVA datasets. For-two group comparisons, an unpaired Student’s t-test was used when assumptions of normality and equal variance were met; otherwise, a Mann- Whitney U test was applied. If all control values were identical, a one sample t and Wilcoxon test was used.

[0357]

[0358] Text

[0359] Supplementary text 1

[0360] ATII-derived GM-CSF is required for AM maintenance in the lung (12, 18, 19). Given our findings that OSM is necessary for maintaining ATII composition, we sought to determine whether perturbations in the ATII population at baseline in FcgrlCreOsmfl / fl mice might indirectly influence the primary function of macrophages, phagocytosis. We administered E. coli pHrodo beads i.t. to Osmfl / fl and FcgrlCreOsmfl / fl mice and did not detect any differences in phagocytic capacity in macrophages or neutrophils (FIG. 19C).

[0361] 4909-9827-7258, v. 160 Docket No. HRVY-259-WO1

[0362] Supplementary text 2

[0363] While studies have suggested that IFN-a and IFN-P exhibit different kinetics and functions (57), we did not observe different kinetics between these two interferons in our influenza time course (FIGS. 2B-C). We used IFNAR1 blockade, which indiscriminately inhibits both IFN-a and IFN-P signaling, and observed decreased morbidity and mortality in FcgrlCreOsmfl / fl mice (FIG. 3H, FIG. 19A). It remains possible that there are different roles for IFN-a and IFN-P in mediating the increased damage and immune infiltration in poly(I:C)-induced damage in the absence of OSM, however this experimental system cannot discriminate between these two interferons.

[0364] 4909-9827-7258, v. 1

Claims

1. 61 Docket No. HRVY-259-WO1CLAIMSWhat is claimed is:

1. A method of treating a respiratory infection in a subject comprising administering an agent to the subject prior to, during or after a period of peak infection, wherein the agent is selected from the group consisting of Oncostatin M (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant.

2. A method of treating, preventing or reducing lung damage in a subject comprising administering an agent to the subject selected from the group consisting of Oncostatin M (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant.

3. The method of claim 2, wherein the agent is administered at the onset of a respiratory infection, at a period of peak infection, or during a period of elevated lung damage compared to a baseline level of lung damage.

4. The method of claim 1, wherein the period of peak infection is measured from symptom onset.

5. The method of any one of claims 1-4, wherein the period of peak infection or elevated lung damage is determined via a pulmonary function test.

6. The method of claim 1, wherein the period of peak infection or elevated lung damage is characterized by increased interferon levels.

7. The method of claim 1, wherein the period of peak infection or elevated lung damage is characterized by an inflammatory response.

8. The method of claim 1, wherein the period of peak infection occurs about 3 to 10 days post-infection.

9. The method of any one of claims 1-8, wherein the period of peak infection occurs about 4 to 6 days post-infection.4909-9827-7258, v. 162 Docket No. HRVY-259-WO1 10. The method of any one of claims 1-9, wherein the agent is administered for a defined period of time.

11. The method of any one of claims 1-10, wherein the agent is administered for 3 to 10 days.

12. The method of any one of claims 1-11, wherein the agent is administered via inhalation, intranasally, intravenously, intraperitoneally, intrathecally, or orally.

13. The method of claim 12, wherein the agent is administered via a nebulizer or inhaler.

14. The method of any one of claims 1-13, wherein the agent is administered intravenously.

15. The method of any one of claims 1-14, wherein administration of the agent restores epithelial cell proliferation.

16. The method of any one of claims 1-15, wherein administration of the agent restores ATII proliferation.

17. The method of any one of claims 1-16, wherein the agent comprises recombinant OSM (rOSM).

18. The method of claim 1, wherein upon completion of administration of OSM to the subject the levels of OSM in the subject decrease over a period of time.

19. The method of any one of claims 1-18, wherein the agent is administered in conjunction with at least one additional agent.

20. The method of claim 19, wherein the at least one additional agent comprises an antiviral agent, an anti-inflammatory agent, an expectorant, a cough suppressant, an antibiotic, an antifungal agent, or a bronchodilator.

21. A composition comprising at least one pharmaceutically acceptable excipient and an agent selected from the group consisting of Oncostatin M (OSM), an OSM derivative, an OSM variant, a nucleic acid encoding OSM, a nucleic acid encoding an OSM derivative, and a nucleic acid encoding an OSM variant.4909-9827-7258, v. 163 Docket No. HRVY-259-WO1 22. The composition of claim 21, wherein the composition takes the form of nanoparticles, microparticles, liposomal formulation, micellar formulation, a water-in- oil emulsion, an oil-in-water emulsion, a solution, or a liquid suspension.

23. A device configured to deliver an aerosol to a lung of a subject, wherein the device contains the composition of claim 21.

24. The device of claim 22, further containing a propellent.

25. The device of claim 23, wherein the device is a dry powder inhaler, and optionally, wherein the at least one pharmaceutically acceptable excipient comprises lactose.4909-9827-7258, v. 1

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