Enhanced lipid nanoparticles

Abstract

The present disclosure provides compositions, methods, and kits for enhanced lipid nanoparticles (LNPs) comprising polynucleotides encoding fusion polypeptides comprising a tag and a cleaved Gasdermin C. In certain embodiments, said polypeptides are capable of increasing payload release.

Description

[0001] 072396.1120

[0002] PATENT

[0003] ENHANCED LIPID NANOPARTICLES CROSS-REFERENCES TO RELATED APPLICATIONS

[0004] The application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 749,852, filed January 27, 2025, and to U.S. Provisional Patent Application No.

[0005] 63 / 767,189, filed March 5, 2025, the contents of each of which are incorporated by reference in their entireties, and to each of which priority is claimed.

[0006] SEQUENCE LISTING A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on January 14, 2026, is entitled 072396.1120_ST26. xml, and is 64,449 bytes in size

[0007] GRANT INFORMATION

[0008] This invention was made with government support under AI163721 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0009] TECHNICAL FIELD

[0010] The present disclosure relates to compositions, methods, and kits for enhanced lipid nanoparticles (LNPs).

[0011] BACKGROUND OF THE INVENTION

[0012] Cell therapies and RNA therapeutics have gained wide attention due to their ability to regulate the expression of therapeutic proteins (e.g., chimeric antigen receptors) and disease-related genes. Delivery vectors play an important role in the development of such therapeutics in preventing nucleic acid degradation and successful delivering to target cells. Compared to viral vectors, nonviral systems have the advantage of being less immunogenic and easier to manufacture. Among the different materials used for nonviral delivery, lipid nanoparticles (LNPs) have been particularly successful, as many LNP-RNA formulations are clinically available or have advanced to clinical trials. Unfortunately, many barriers are still present in realizing the full potential of LNP-RNA and LNP-DNA systems. While most LNP systems are efficiently taken up by the cell via endocytosis, they often remain trapped in endosomal compartments and degrade through the endosome-lysosome acidification pathway. One of the main bottlenecks of LNP-based RNA delivery is poor endosomal escape. Viral delivery

[0013] ACTIVE 131727896.1 072396.1120

[0014] PATENT

[0015] systems have membrane proteins that undergo a conformation change allowing them to easily fuse with the plasma or endosomal membranes to release their payload. Previous studies have shown that for LNP mediated siRNA delivery, less than 2% of the siRNA can successfully escape the endosome and reach the cytosol (Gilleron et al., Nat. Biotechnol. 31, 638-646 (2013)). Thus, there is a need to improve endosomal escape and transfection efficiency of LNPs.

[0016] SUMMARY OF THE INVENTION

[0017] In one aspect, the present disclosure provides a lipid-based carrier comprising a polynucleotide encoding a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide.

[0018] In certain embodiments, the carrier is a lipid nanoparticle, a liposome, a lipoplex, or a nanoliposome. In certain embodiments, the carrier is a lipid nanoparticle. In certain embodiments, the carrier comprises a cation lipid, a non-cationic lipid, a structural lipid, a PEG-modified lipid, or a combination thereof.

[0019] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 9. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 9. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0020] In certain embodiments, the tag polypeptide comprises a FLAG polypeptide, a polyhistidine (His) tag polypeptide, a hemagglutinin (HA) tag polypeptide, a Myc tag polypeptide, a Strep-tag polypeptide, a glutathione S-transferase (GST) tag polypeptide, a calmodulin-binding protein (CBM) polypeptide, a maltose-binding protein (MBP) polypeptide, a T7 tag polypeptide, a V5 tag polypeptide, a green fluorescent protein (GFP) polypeptide, a blue fluorescent protein (BFP) polypeptide, a cyan fluorescent protein (CFP) polypeptide, a yellow fluorescent protein (YFP) polypeptide, a FMN-binding fluorescent protein (FbFP) polypeptide, or a red fluorescent protein (RFP) polypeptide.

[0021] In certain embodiments, the tag polypeptide comprises a FLAG polypeptide. In certain embodiments, the tag polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,

[0022] ACTIVE 131727896.1072396.1120

[0023] PATENT

[0024] about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0025] In certain embodiments, the tag polypeptide comprises an RFP polypeptide. In certain embodiments, the tag polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0026] In certain embodiments, the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are connected by a linker. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0027] In certain embodiments, the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are connected without a linker.

[0028] In certain embodiments, the fusion protein comprises, from N-end to C-end, the tag polypeptide and Gasdermin C (GSDMC) polypeptide. In certain embodiments, the fusion protein comprises, from N-end to C-end, Gasdermin C (GSDMC) polypeptide and the tag polypeptide.

[0029] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0030] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0031] In certain embodiments, the lipid-based carrier further comprises a second polynucleotide.

[0032] In certain embodiments, the second polynucleotide encodes a payload. In certain embodiments, the second polynucleotide encodes an antigenic polypeptide derived from a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell. ACTIVE 131727896.1 072396.1120

[0033] PATENT

[0034] In certain embodiments, the second polynucleotide encodes a tumor antigen. In certain embodiments, the tumor antigen is selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE- 12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N-acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA); carcinoma- associated mutated mucins (i.e., MUC-1 gene products); EBNA gene products of EBV (i.e., EBNA-I); E7, E6 proteins of human papillomavirus; prostate specific antigen (PSA); prostate specific membrane antigen (PSMA); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes; KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, Kras, Histone, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96.

[0035] In certain embodiments, the second polynucleotide encodes a therapeutic protein. In certain embodiments, the therapeutic protein is selected from transforming growth factor-beta (TGF-beta), interferon-alpha, interferon-beta, interferon-gamma, granulocyte colony stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), interleukin- 1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin- 10, interleukin- 12, interleukin- 13, interleukin- 15, interleukin- 17, interleukin- 18, interleukin-22, interleukin-23, interleukin-35, amylin, anti-Mullerian hormone, calcitonin, cholecystokinin, corticotropin, endothelin, enkephalin, erythropoietin (EPO), follicle-stimulating hormone, gallanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human growth hormone (hGH), inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, luteinizing hormone releasing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, GLP-1, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, vasoactive intestinal peptide, vasopressin, trastuzumab emtansine, brentuximab vedotin, T-DM1, polyvalent IgG2a Fc (M045), SMN1, coagulation factors (e.g., F8 or F9), hemoglobin, ARSA, ABCD1, DDC, ADA, RPE65, ASP A, ARG1, DMD, COL7A1, BCL11A, cas9, WAS, HTT, CTNS, CPS1, OTOF, IDS, TTR, LAMP2, UGT1A1, PAH, KLKB1, ATP7B, GAN, agalsidase beta, imiglucerase, velaglucerase alfa, taliglucerase, alglucosidase alfa, laronidase, idursulfase, galsulfase, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, altumomab pentetate, amatuximab, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, ACTIVE 131727896.1 072396.1120

[0036] PATENT

[0037] belimumab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, cantuzumab ravtansine, capromab pendetide, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, dacetuzumab, demcizumab, detumomab, drozitumab, ecromeximab, eculizumab, elotuzumab, ensituximab, epratuzumab, etaracizumab, farletuzumab, figitumumab, flanvotumab, galiximab, gemtuzumab ozogamicin, girentuximab, ibritumomab tiuxetan, imgatuzumab, ipilimumab, labetuzumab, lexatumumab, lorvotuzumab mertansine, nimotuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, tacatuzumab tetraxetan, tositumomab, trastuzumab, totumumab, and zalutumumab.

[0038] In another aspect, the present disclosure provides a composition comprising the lipid-based carrier disclosed herein.

[0039] In a further aspect, the present disclosure provides a composition comprising a first lipid-based carrier comprising a polynucleotide encoding a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and a second lipid-based carrier comprising a second polynucleotide.

[0040] In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutical acceptable excipient.

[0041] In an additional aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering an effective amount of the lipid-based carrier or the composition disclosed herein.

[0042] In an additional aspect, the present disclosure provides a method of treating a cancer in a subject in need thereof, the method comprising administering an effective amount of the lipid-based carrier or the composition disclosed herein.

[0043] In certain embodiments, the cancer is selected from an adrenal cancer, a breast cancer, a colon cancer, a leukemia, a bile duct cancer, a bone cancer, a lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor), a bladder cancer, a brain cancer, a bronchial cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, an ependymoma, a retinoblastoma, a gallbladder cancer, a gastric cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, a heart cancer, a liver cancer, a pancreatic cancer, a melanoma, a kidney cancer, a laryngeal cancer, a lip or oral cancer, a lymphoma, a mesothelioma, a mouth cancer, a myeloma, a nasopharyngeal cancer, a neuroblastoma, an oropharyngeal cancer, an ovarian cancer, a thyroid cancer, a penile cancer, a pituitary cancer, a prostate cancer, a rectal cancer, a renal cancer, a salivary gland cancer, a sarcoma, a skin

[0044] ACTIVE 131727896.1 072396.1120

[0045] PATENT

[0046] cancer, a stomach cancer, a testicular cancer, a throat cancer, a uterine cancer, a vaginal cancer, and a vulvar cancer. In certain embodiments, the subject is human.

[0047] In a further aspect, the present disclosure provides the lipid-based carrier or the composition disclosed herein for use in treating a disease. In certain embodiments, the disease is a cancer. In certain embodiments, the cancer is selected from an adrenal cancer, a breast cancer, a colon cancer, a leukemia, a bile duct cancer, a bone cancer, a lung cancer (e.g., nonsmall cell lung cancer, small cell lung cancer, and lung carcinoid tumor), a bladder cancer, a brain cancer, a bronchial cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, an ependymoma, a retinoblastoma, a gallbladder cancer, a gastric cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, a heart cancer, a liver cancer, a pancreatic cancer, a melanoma, a kidney cancer, a laryngeal cancer, a lip or oral cancer, a lymphoma, a mesothelioma, a mouth cancer, a myeloma, a nasopharyngeal cancer, a neuroblastoma, an oropharyngeal cancer, an ovarian cancer, a thyroid cancer, a penile cancer, a pituitary cancer, a prostate cancer, a rectal cancer, a renal cancer, a salivary gland cancer, a sarcoma, a skin cancer, a stomach cancer, a testicular cancer, a throat cancer, a uterine cancer, a vaginal cancer, and a vulvar cancer.

[0048] In another aspect, the present disclosure provides a kit comprising the lipid-based carrier or the composition disclosed herein. In certain embodiments, the kit is for use in culturing and transfecting a cell.

[0049] In a further aspect, the present disclosure provides a method of delivering a payload to a cell. In certain embodiments, the method comprises contacting the cell with an effective amount of the lipid-based carrier, the composition, or the kit disclosed herein.

[0050] In another aspect, the present disclosure provides a method of increasing the transfection efficiency of a payload into a cell. In certain embodiments, the method comprises contacting the cell with an effective amount of the lipid-based carrier, the composition, or the kit disclosed herein.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1 A-1D illustrate cleaving properties of Cathepsin S (CTSS) on both human and murine Gasdermin C (GSDMC) proteins. Figure 1A shows cleaving of human GSDMC by CTSS. Figures 1B-1D shows cleaving of murine GSDMC by CTSS.

[0053] Figures 2A-2C illustrate identification of cleavage site of CTSS on human GSDMC. Figures 2A and 2B show mass spectrometry analysis of GSMNC sequence and identification of cleavage sites. Figure 2C shows detection of cleaved GSDMC by immunoblotting. In

[0054] ACTIVE 131727896.1072396.1120

[0055] PATENT

[0056] Figure 2A, SEQ ID Nos: 21-25 are presented in order from top to bottom. In Figure 2B, SEQ ID Nos: 26-29 are presented in order from top to bottom.

[0057] Figures 3A and 3B illustrate localization of CTSS-generated GSDMCN ter. Figure 3A shows localization of CTSS-generated GSDMCN terin HeLa cells. Figure 3B shows localization of CTSS-generated GSDMCN terin C. elegans.

[0058] Figures 4A and 4B illustrate effects of GSDMCN terin payload release. Figure 4A shows microscopy images analysis of mCherry-tagged GSDMCN terin HEK293T cells. Figure 4B shows expression of mCherry-tagged GSDMCN ter.

[0059] Figure 5 shows colony counts of cells transfected with GSDMCN ter.

[0060] Figures 6A and 6B illustrate ability of GSDMCN terin transfecting NIH3T3 cell lines. Figure 6A shows fluorescent microscopy and FACS analysis. Figure 6B shows immunoblotting analysis.

[0061] Figure 7 shows protective effects of tagged GSDMCN terfrom pyroptosis. TO-PRO-3 staining and percentage value of cell death observed in HEK293T cells transfected with the tagged GSDMCN terare depicted.

[0062] Figures 8A-8F illustrate boosting effects of tagged GSDMCN teron transfection efficiency. Figure 8A shows representative fluorescent microscopy pictures capturing the expression of GFP in NIH3T3 cells transfected with tagged GSDMCN ter. Figure 8B shows quantification of GFP in NH43T3 cells transfected with tagged GSDMCN terat different doses. Figure 8C shows immunoblotting analysis of GFP in NIH3T3 cells transfected with tagged GSDMCN terat different doses. Figure 8D shows quantification of luciferase in NH43T3 cells transfected with tagged GSDMCN terat different doses. Figure 8E shows representative fluorescent microscopy pictures capturing the expression of GFP in LLC cells transfected with tagged GSDMCN ter. Figure 8F shows quantification of luciferase in LLC cells and MC38 cells transfected with tagged GSDMCN terat different doses.

[0063] Figures 9A-9M illustrate GSDMC promoting type-2 immunity in lECs, with cleavage triggered by helminth infection or commensal protist colonization. Figures 9A-9C show WT J AX mice infected with N.b. or PBS control. 7 days post-infection western blot for (A) GSDMC2cter, (B) full length GSDMC3 and GSDMC3N ter, (C) GSDMC4c-terin the jejunum and respective quantifications normalized to Actin are shown. Gsdmcl-4^ mice served as a control to show antibody specificity. Figures 9D-9G show Gsdmcl-4^ mice and littermate controls infected with N.b. or PBS control. 7 days post-infection (DE) mLNs were analyzed by flow cytometry for quantification of (D) IL-13+ILC2s (gated on KLRG1+Lin') and (E) IL-13+Th2 (gated on Gata3+Foxp3‘). (F) Tuft cells in the jejunum visualized by the expression of ACTIVE 131727896.1 |072396.1120

[0064] PATENT DCLK1 in red; epithelial cells (epithelial cell adhesion molecule; EPCAM) in green; and nuclei (DAPI) in blue. (G) Goblet cells in the jejunum visualized by PAS staining. (F-G) Representative images and quantification are shown; scale bars, 50 pm. Figures 9H and 91 show WT JAX mice colonized with T. arnold or PBS control. 12 days post-colonization western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin the jejunum and colon was performed (H). Quantification normalized to Actin is shown in (I). Representative experiment out of 2 independent experiments is shown. Figures 9J and 9K show WT JAX mice colonized with T. arnold or PBS control. 30 weeks post-colonization western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin the colon was performed (J). Quantification normalized to Actin is shown in (K). A representative experiment out of 2 independent experiments is shown. Figures 9L and 9M depict Western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin intestinal organoids of WT mice treated with 10 ng / ml IL-13 or PBS control for 72 hours (L). Quantification normalized to Actin is shown in (M). Representative experiment out of 2 independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = not significant.

[0065] Figures 10A-10H illustrate that CTSS cleaves human and murine GSDMC proteins. Figures 10A-10D show Western blot of (A) human GSDMC (hGSDMC), (B) mouse GSDMC2, (C) mouse GSDMC3, (D) mouse GSDMC4 (HA tagged at C-terminus) in the presence of various Cathepsins. Proteins were transiently expressed in HEK293T cells. Figures 10E-10G show cell lysis quantifications 9E-9F) and imaging (G) of HEK293T cells that were transiently transfected with the indicated plasmids are shown. C-terminus Ixflag tagged human GSDMC were co-transfected with CTSS or GFP for (E) 24 or (F) 48 hours. Plasmid ratio of CTSS: GSDMC = 3:2. Cell lysis (cytotoxicity) was measured by CytoTox-Glo. 100% of cell lysis was calculated based on HEK293T cells cultured under same conditions as shown in (E-F) treated with 1% Triton X-100. For (G), HEK293T cells were co-transfected with hGSDMC and CTSS (1 : 1) for 24 hours and then stained with To-Pro-3. Representative images are shown; scale bars, 100 pm. Figure 9H shows in vitro protease assay with mammalian cell purified recombinant human CTSS and GSDMC proteins (full length, His-SUMO tagged). Western blot recognizing amino acids 1-100 (left panel), amino acids 300-508 (middle panel) of human GSDMC and Coomassie blue staining (Gel -code Blue) (right panel) is shown. Neutral pH (7.5) conditions have been used for this assay. ***P < 0.001.

[0066] Figures 11A-11T illustrate that Cathepsin S is required for GSDMC cleavage during helminth infection and T. arnold colonization. Figures 11A and 11B depict Western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin intestinal colon organoids of ACTIVE 131727896.1 8072396.1120

[0067] PATENT

[0068] Ctssr / ~ mice and littermate controls treated with 10 ng / ml IL-13 or PBS control for 48 hours (A). Quantification of GSDMC3 (full length) and GSDMC3N ternormalized to Actin is shown (n = 4 / group) (B). Representative experiment out of 2 independent experiments is shown. Figures 11C and 11H show Ctss ^ mice and littermate controls colonized with T. arnold or PBS control. 12 days post-colonization western blot for (C) GSDMC2c ter, (E) full length GSDMC3 and GSDMC3N ter, (G) GSDMC4c terin the ileum and respective quantifications (D) GSDMC2c ter, (F) full length GSDMC3 and GSDMC3N ter, (H) GSDMC4C-ter normalized to Actin are shown. Gsdmcl-4^ mice served as a control to show antibody specificity, n.d. = not detected. Figures 1 II- 1 IN show Ctss^ mice and littermate controls infected with N.b. 7 days post-infection western blot for (I) GSDMC2c ter, (K) full length GSDMC3 and GSDMC3N ter, (M) GSDMC4c terin the jejunum and respective quantifications (J) GSDMC2c ter, (L) full length GSDMC3 and GSDMC3N ter, (N) GSDMC4c ternormalized to Actin are shown. Gsdmcl-4 ' mice served as a control to show antibody specificity. Figures 110-1 IS depict Ctss^ mice and littermate controls irradiated and reconstituted with CD45.1+WT bone marrow. 6-weeks post reconstitution, mice were infected with N.b. 7 days post-infection (O-P) mLNs were analyzed by flow cytometry for quantification of (O) IL-13+ILC2 (gated on KLRG1+Lin'), (P) IL-13+Th2 cells (gated on Gata3+Foxp3‘) and (Q) eosinophils. (R) Tuft cells in the jejunum visualized by the expression of DCLK1 in red; epithelial cells (EPCAM) in green; and nuclei (DAPI) in blue. (S) Goblet cells in the jejunum visualized by PAS staining. (R-S) Representative images and quantification are shown; scale bars, 50 pm. Figure 11T shows a model for CTSS-processed GSDMCN'terin promoting positive type-2 immune feedback for anti-helminth immunity. *P < 0.05, **P < 0.01, n.s. = not significant Figures 12A-12D illustrate that CTSS-processed GSDMCN'tercolocalizes to and penetrates RAB7+vesicles. Figure 12 A shows full length RFP-hGSDMC (upper panel), RFP-hGSDMC 284 aa (middle panel), RFP-hGSDMC 289 aa (lower panel) co-expressed with GFP-Rab7 in HeLa cells. White box shown as zoom in next to the cell images. Representative images are shown. Scale bar = 10 pm. Figure 12B shows RFP-mGSDMC2N'terco-expressed with GFP-Rab7 in HeLa cells. White box shown as zoom in next to the cell images. Representative images are shown. Scale bar = 10 pm. Figure 12C shows RFP-mGSDMC4N'terco-expressed with GFP-Rab7 in HeLa cells. White box shown as zoom in next to the cell images. Representative images are shown. Scale bar = 10 pm. Figure 12D shows C-ter (upper panel) or N-ter (middle panel) GFP-tagged hGSDMCN'terectopically co-expressed with mKate2-tagged Rab7 or mKate2-tagged Rab7 only (lower panel) in C. elegans. White arrows indicate Rab7 vesicles colocalized with hGSDMCN'ter. Blue arrows indicate Rab7 vesicles not colocalized with hGSDMCN'ter. ACTIVE 131727896.1 072396.1120

[0069] PATENT

[0070] Representative images are shown. Scale bar = 10 pm. Quantification of Rab7+vesicle (puncta) size in the presence or absence of hGSDMCN terco-expression is shown. Two-tailed unpaired / -test. ** < 0.01

[0071] Figures 13A-13L illustrate functional features of Cathepsin S-generated GSDMCN tertargeting Rab7+vesicles. Figures 13 A and 13B show HEK293T cells co-transfected with pBabe (empty vector), full length (FL) hGSDMC, the 1-284 aa GSDMCN teror 1- 289 aa GSDMCN terwith a plasmid expressing mCherry (20:1) for 48 hours. mCherry level were measured by western blot (A) or fluorescent microscopy (B). Representative images are shown. Scale bar = 1000 pm. Figure 13C shows HEK293T cells co-transfected with full length (FL) hGSDMC or 1-284 aa GSDMCN ter(both are Ampicillin resistance, Amp+) with a separate Kanamycin resistance plasmid (Kan+) at 1:1 ratio for 48 hours. The DNA extracted from the nucleic fraction of these transfected cells was transformed into competent A. coli. cells. Shown are the transformants counted after overnight culture. Figures 13D-13F show NIH3T3 cells stably transduced with BioID2-HA tagged vector control, full length human GSDMC (hGC FL), or GSDMCNter(hGC 289aa). Stably transduced cell lines were transfected with 2 ug GFP plasmid using lipofectamine 2000 for 48 hours to assess transfection efficiency in NH43T3 cells. (D) anti-HA western blot to verify stable transduction of hGC FL or hGC 289aa. GSDMCs were C-tagged with BioID2-HA tag. Actin served as a loading control. (E-F) GFP levels were measured by (E) fluorescent microscopy. Representative images are shown. Scale bar = 1000 pm; (F) flow cytometry. Representative pseudo color plots are shown. Figure 13G shows GSDMCN terco-expressed with GFP-ADRP in HeLa cells. 24 hours post transfection media was replaced without addition of FBS (starvation) or with FBS (control). Cells were imaged 48 hours post starvation. Representative images (left), Scale bar = 30 pm and quantification of the percentage of cells with GFP-ADRP forming dots of all GFP+cells (right) from 3 independent experiments is shown. Mean ± s.e.m. One-way ANOVA, Sidak’s post hoc test. Figure 13H shows intestinal organoids of Gsdmcl-4^ mice, Ctss^ mice, and littermate controls treated with 10 ng / ml IL-13 or PBS control for 72 hours. Lipid droplet (Nile red staining) mean fluorescence intensity (MFI) in live lECs was determined by flow cytometry. Shown is the fold change relative to WT PBS from 2 independent experiments. (n=6-7 / group). Figures 131 and 13 J show intestinal organoids of Gsdmcl-4^ mice and littermate controls treated with 10 ng / ml IL- 13 or PB S control for a total of 72 hours. (I) At 24- and 48-hours post-IL-13 treatment, organoids were treated with CID-1067700 (100 pM) or vehicle control. (J) At 24 hours post-IL-13 treatment, organoids were treated with Bafilomycin Al (100 nM) or vehicle control. Lipid droplet (Nile red staining) mean fluorescence intensity (MFI) in live ACTIVE 131727896.1 H)072396.1120

[0072] PATENT

[0073] lECs was determined by flow cytometry. Shown is the fold change relative to WT PBS from 2 independent experiments (n = 7-9 / group). Figures 13K and 13L show intestinal organoids of Gsdmcl-4~ ~ mice and littermate controls treated with 10 ng / ml IL-13 or PBS control for 72 hours. (L) 24 hours post-IL-13 treatment, organoids were treated with Bafilomycin Al (100 nm) or with vehicle control. (K-L) Intestinal organoids were stimulated with 500 ng / ml ionomycin in HBSS / calcium for 30 minutes. PGD2 in organoid supernatant was measured by ELISA (n=4-6 / group). For Figure 13C, center is mean, two-tailed unpaired / -test. For Figures 13G-13L, mean ± s.e.m. For Figures 13G-13J, and 13L, one-way ANOVA, Sidak’s post hoc test. For Figure 13K, Two-tailed unpaired / -test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = not significant

[0074] Figures 14A-14H illustrate that Rab7 inhibition restores impaired type-2 immunity to helminths in Gsdmcl-4~~ mice. Figures 14A-14D show Gsdmcl-4~~ mice and littermate controls infected with N.b. and treated daily with 16 mg / kg Rab7 GTPase inhibitor CID-1067700 or vehicle control. 7 days post-infection (A-B) mLNs were analyzed by flow cytometry for quantification of (A) IL-13+ILC2s (gated on KLRG1+Lin') and (B) IL-13+Th2 (gated on Gata3+Foxp3‘). (C) Goblet cells in the jejunum were visualized by PAS staining and (D) Tuft cells in the jejunum were visualized by the expression of DCLK1 in red and nuclei (DAPI) in blue. Representative images and quantification are shown; scale bars, 50 pm. Figures 14E-14G show Gsdmcl-4^ mice and littermate controls infected with N.b. and treated daily with 1 mg / kg CRTH2 inhibitor OC000459 or vehicle control. (E) 7 days post-infection goblet cells in the jejunum were visualized by PAS staining. Representative images and quantification are shown; scale bars, 50 pm. (F-G) mLNs were analyzed by flow cytometry for quantification of (F) IL-13+ILC2s (gated on KLRG1+Lin') and (G) IL-13+Th2 (gated on Gata3+Foxp3‘). Figure 14H shows model of CTSS-processed GSDMCN'tertargeting Rab7+vesicles to promote positive type-2 immune feedback in anti-helminth immunity. For Figures 14A-14C and 14E-14G, each dot represents an individual mouse. Center is mean, one-way ANOVA, Sidak’s post hoc test. For Figure 14D, center is median, one-way ANOVA, Sidak’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. = not significant.

[0075] Figures 15A-15K illustrate that completion of the lipid interaction motif in GSDMC enhances its pyroptotic activity. Figure 15A depicts alignment showing the lipid interaction motif of various gasdermins. GSDMC misses the first amino acid “L” or “W”. Figures 15B-15E show quantifications of cell lysis (B, D) and imaging (C, E) of HEK293T cells transiently transfected with the indicated plasmids are shown. Transfected plasmids include CTSS + WT hGSDMC, WT mGSDMC2, or versions containing the “L” or “W” insertion, for 24 hours. The ACTIVE 131727896.1 ii072396.1120

[0076] PATENT

[0077] plasmid ratio of CTSS: GSDMC = 3:2. Cell lysis (cytotoxicity) was measured using the CytoTox-Glo assay, with 100% cell lysis calculated based on HEK293T cells cultured under same conditions and treated with 1% Triton X-100. Topro-3 uptake (red, a Pl-like dye) was used to stain lysed cells in (C, E). Scale bar =100 pm. Figures 15F-15H show quantifications of cell lysis of HEK293T or HeLa cells transiently transfected with the indicated plasmids. Transfected plasmids included mGSDMC2 / 4N'terand their L-inserted versions, with a GFP tag on the C-ter (F-G) or an RFP tag on the N-ter of the mGSDMC2 / 4N'ter(H), for 24 hours. Cell lysis (cytotoxicity) was measured using the CytoTox-Glo assay as described in (B). Figure 151 shows RFP-mGSDMC2N ter(L inserted) co-expressed with GFP-Rab7 in HeLa cells. White box shown as zoom in next to the cell images. Representative images are shown. Scale bar = 10 pm. Figures 15J and 15K shows the oligomerization of (J) mGSDMC2N terand (K) mGSDMC4N'ter, along with their L-inserted versions, was detected by western blotting using an anti-GFP antibody. (GFP is tagged to the C-ter of the expressed protein). Cell lysates from HEK293T cells transfected with mGSDMC2N ter, mGSDMC4N'ter, or their respective L-inserted versions were separated using native PAGE. In Figure 15A, SEQ ID Nos: 31-41 are presented in order from top to bottom.

[0078] Figures 16A-16H illustrate GSDMC promoting type-2 immunity in lECs, with cleavage triggered by helminth infection or commensal protist colonization. Figure 16A shows WT JAX mice were infected with N.b. 7 days post-infection Gsdmc2-4 gene expression in the jejunum was assessed by RT-PCR. Figure 16B shows diagram depicting the location of the guide RNAs used to target the Gsdmc locus for disruption by CRISPR / Cas9. The Gsdmcl-4~~ line founder reported in this study is deleted between guide Gsdmc-5' and guide Gsdmc-3'. In Figure 16B, SEQ ID Nos: 42 and are presented in order from left to right. Figure 16C shows sequence alignment of Gsdmc, Gsdmc2, Gsdmc3, and Gsdmc4 from mouse. The alignment was generated using COBALT and presented using CLC Genomics Workbench 12. Non-identical residues are indicated by the background. In Figure 16C, SEQ ID Nos: 44-47 are presented in order from top to bottom. Figure 16D shows Gsdmc 1-4^ mice and littermate controls were infected with N.b. or PBS control. 7 days post-infection mLNs were analyzed by flow cytometry for quantification of eosinophils. Figure 16E shows Gsdmc 1-4 ^ mice and littermate controls were infected with N.b. 5 days post-infection worm burden in the small intestine was assessed. Figures 16F and 16G show WT JAX mice colonized with T. arnold. 12 days postcolonization. Gsdmc2-4 gene expression in the (F) jejunum and (G) colon was assessed by RT-PCR. Figure 16H shows WT JAX mice colonized with T. arnold. 12 days post-colonization western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin the duodenum, ACTIVE 131727896.1 il072396.1120

[0079] PATENT

[0080] jejunum, ileum and colon was performed. Quantification of GSDMC3 (full length) and GSDMC3N ternormalized to Actin is shown (n = 4 / group). Actin served as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001.

[0081] Figures 17A-17H illustrate GSDMC promoting type-2 immunity in lECs, with cleavage triggered by helminth infection or commensal protist colonization. Figure 17A shows WT JAX mice colonized with T. arnold or PBS control. 12 days post-colonization of GSDMC2 / 3 and EPC AM protein expression in the jejunum was assessed by immunofluorescence. GSDMC2 / 3 is visualized in red, epithelial cells (epithelial cell adhesion molecule; EPCAM) in green, and nuclei (DAPI) in blue. White box shown as zoom in (lower left corner). Representative images are shown; scale bars, 50 pm. Gsdmcl-4^ mice served as a control to show antibody specificity. Figures 17B and 17C show Il4rcc / ' mice and littermate controls colonized with T. arnold. 12 days post-colonization (B-C) Gsdmc2-4 gene expression in the (B) jejunum and (C) colon was assessed by RT-PCR. Center is median, Mann-Whitney test. Figure 17D shows IMrcc^ mice and littermate controls colonized with T. arnold. 12 days post-colonization GSDMC3 showing full length GSDMC3 and GSDMC3N terwas assessed in the jejunum and colon. Actin served as a loading control. Quantification of GSDMC3 (full length) and GSDMC3N ternormalized to Actin is shown (n = 2 / tissue group). Figure 17E and 17F show T. arnold colonization was quantified by RT-PCR using DNA isolated from fecal contents. 28S was normalized to the host murine Ifnbl gene. Median is shown. Figures 17G and 17H show Gsdmcl-4~ ~ mice and littermate controls colonized with T. arnold or PBS control. 12 days post-colonization (G) Tuft cells in the jejunum visualized by the expression of DCLK1 in red; epithelial cells (EPCAM) in green; and nuclei (DAPI) in blue, (H) Goblet cells in the jejunum visualized by PAS staining. Representative images and quantification are shown; scale bars, 50 pm. Two independent experiments, n = 4-8 mice / group. Figure 171 shows intestinal organoids of WT mice treated with 10 ng / ml IL- 13 or PBS control for 24 hours to assess Gsdmc2-4 gene expression by RT-PCR. *P < 0.05, **P < 0.01, ***p < 0.001, ****P < 0.0001

[0082] Figures 18A-18J illustrate that CTSS cleaves human and murine GSDMC proteins. Figures 18A and 18B show Western blot of (A) GSDMD and (B) GSDME in intestinal organoids of WT mice treated with IL-13 or PBS control for 24 hours. Figure 18C shows WT JAX mice infected with N.b. or PBS control. 7 days post-infection western blot for caspase-8 (activated form not detected) in the jejunum was performed. Quantification normalized to Actin is shown. Gsdmcl-4^ mice served as a control. Figure 18D shows WT JAX mice colonized with T. arnold or PBS control. 12 days post-colonization western blot for caspase-8 (activated ACTIVE 131727896.1 ii072396.1120

[0083] PATENT

[0084] form not detected) in the jejunum was performed. Quantification normalized to Actin is shown. Gsdmcl-4^ mice served as a control. Figure 18E depicts Western blot for caspase-8 showing full length caspase-8 (activated form not detected) in Gsdmcl-4^ and WT intestinal organoids treated with 10 ng / ml IL- 13 or PBS control for 72 hours. Quantification normalized to Actin is shown. Figure 18F depicts Western blot for GSDMC3 showing full length GSDMC3 and GSDMC3N terin intestinal organoids of Casp 7 / 77 / - mice and Caspl / 1+ / +mice treated with 10 ng / ml IL-13 or PBS control for 72 hours. Quantification normalized to Actin is shown. Figure 18G shows in vitro protease assay with mammalian cell purified recombinant human CTSS and GSDMC proteins (full length, His and Myc-tagged) conducted under pH = 5.5 and pH = 7. 5 conditions. Western blot recognizing amino acids 300-508 of human GSDMC is shown. Figure 18H shows the non-tryptic peptides identified from the mass spectrum analysis of the enriched CTSS-cleaved GSDMCcter. Peptides directly captured by the mass spectrum are labeled in red. The amino acids before or after these peptides are labeled black. The putative cleavage sites were predicted from these captured peptides. Figure 181 shows part of the human GSDMC sequence (part of the linker region, 241-300). The putative cleavage sites from Figure 18H are marked in red dashed lines. Figure 18J shows Western blot of full length (FL) and truncated human GSDMC in the presence of CTSS or GFP (control). Proteins were transiently expressed in HEK293T cells. Each truncation sequence is shown in Figure 181. In Figure 18H, SEQ ID Nos: 21-25 are presented in order from top to bottom. In Figure 181, SEQ ID Nos: 26-29 are presented in order from top to bottom.

[0085] Figures 19A-19F illustrate that Cathepsin S is required for GSDMC cleavage during helminth infection and T. arnold colonization. Figure 19A shows WT JAX mice infected with N.b. 7 days post-infection CTSS and LAMPl protein expression in the jejunum was assessed by immunofluorescence. CTSS is visualized in red, LAMPl (lysosome marker) in green, and nuclei (DAPI) in blue. White box shown as zoom in (lower panel). White arrows indicate CTSS not colocalized with LAMPL Blue arrows indicate CTSS colocalized with LAMPL Representative images and quantification are shown; scale bars, 50 pm. Figure 19B shows WT JAX mice colonized with T. arnold. 12 days post-colonization CTSS and LAMPl protein expression in the colon was assessed by immunofluorescence. CTSS is visualized in red, LAMPl (lysosome marker) in green, and nuclei (DAPI) in blue. White box shown as zoom in (lower panel). White arrows indicate CTSS not colocalized with LAMPL Blue arrows indicate CTSS colocalized with LAMPL Representative images are shown; scale bars, 50 pm. Figure 19C shows CTSS and EPCAM protein expression in the small intestine of Ctss ^ mice assessed by immunofluorescence. CTSS is visualized in red, EPCAM in green, and nuclei (DAPI) in ACTIVE 131727896.1 ii072396.1120

[0086] PATENT

[0087] blue. Representative images are shown; scale bars, 100 pm. Figure 19D shows intestinal organoids treated with 10 ng / ml IL- 13 or PBS control for 24 hours to assess Ctss gene expression by RT-PCR (n = 4 / group); two-tailed paired / -test. Figure 19E shows WT JAX mice infected with N.b. or PBS control. 7 days post-infection CTSS protein expression in the jejunum was assessed by immunofluorescence. CTSS is visualized in red and nuclei (DAPI) in blue. White box shown as zoom in (right panel). Representative images and quantification (fluorescence intensity of CTSS in lECs and lamina propria separated by dotted line) are shown, (n = 3-4 mice / group); scale bars, 50 pm. Center is mean, two-tailed unpaired / -test. Figure 19F shows CD45.2+Ctss^ mice and littermate controls irradiated to deplete immune cells. Reconstitution with bone marrow derived from CD45.1+WT mice 7 weeks post irradiation was assessed in mLN by means of flow cytometry. *P < 0.05; n.s. = not significant Figures 20A-20J illustrate that CTSS-processed GSDMCN tercolocalizes to and penetrates RAB7+vesicles. Figure 20A shows intestinal organoids of Gsdmcl-4^ mice and littermate controls treated with 10 ng / ml IL-13 or PBS control for 48 hours to assess cell death. Cells were stained with propidium iodide (PI) and Hoechst and fluorescence was measured on a fluorescent plate reader. Quantification of percent cell death (PI / Hoechst) is shown. Figure 20B shows intestinal organoids of Gsdmcl-4^ mice, Ctss ^ mice, and littermate controls treated with 10 ng / ml IL- 13 for 72 hours to assess cell death. Cells were stained with PI and Hoechst and fluorescence was measured on a fluorescent plate reader. Quantification of percent cell death (PI / Hoechst) is shown. Figure 20C shows intestinal organoids of Gsdmcl-4^ mice, Cts / _mice, and littermate controls were treated with IL- 13 or PBS control for 72 hours. Images of twelve connecting sections were stitched together to one image. Representative images of whole domes of organoids are shown. 2% Triton treatment was used as a positive control for cell death. Scale bars, 2 mm. Figure 20D shows Gsdmcl-4^ mice and littermate controls infected with N.b. or PBS control. 7 days post-infection apoptotic cells in the jejunum were visualized by TUNEL staining. TUNEL + region of interest (ROI) was enumerated in villous epithelial cells. Quantification is shown. Center is mean, one-way ANOVA, Sidak’s post hoc test. Figure 20E shows Gsdmcl-4~ ~ mice and littermate controls colonized with T. arnold. 12 days post-colonization apoptotic cells in the colon were visualized by TUNEL staining. TUNEL + ROI was enumerated in colonic mucosa. Quantification is shown. Center is mean, two-tailed unpaired / -test. Figures 20F and 20G show WT JAX mice colonized with T. arnold or PBS control. 30 weeks post-colonization colon histology H&E and colon length was assessed. Representative images are shown. Scale bar 100pm. Figures 20H and 201 show Gsdmcl-4 ' mice and littermate controls were infected with N.b. 7 days post-infection jejunum ACTIVE 131727896.1 ii072396.1120

[0088] PATENT

[0089] explants were cultured in RPMI for 3 hours. (H) IL- 1 J3 and (I) IL- 18 production were measured in the supernatant, and cytokine levels were normalized to tissue weight. Figure 20J shows intestinal organoids of WT mice were treated with 10 ng / ml IL- 13 or PBS control for 24 hours (circles) or 48 hours (triangles) to assess IL-33 cytokine levels. IL-33 was measured in organoid culture supernatants and in lysed organoids treated with 1% NP-40 (positive control) using electrochemiluminescence, n.s. = not significant.

[0090] Figures 21 A and 21B show quantifications of cell lysis of HEK293T cells transiently transfected with the mGSDMC2N terplasmids with a GFP tag on the C-ter. Cells were treated with CID-1067700 (100 pM) or vehicle control ~12 hr post transfection. Cell lysis (cytotoxicity) was measured using the CytoTox-Glo assay, with 100% cell lysis calculated based on HEK293T cells cultured under same conditions and treated with 1% Triton X-100. Intestinal organoids of Gsdmcl-4^ mice and littermate controls were treated with 10 ng / ml IL-13 or PBS control for a total of 72 hours. At 24- and 48-hours post-IL-13 treatment, organoids were treated with CID-1067700 (100 pM) or vehicle control. % of live ZombieNeg lECs are shown. Two-tailed unpaired Ltest

[0091] Figures 22A and 22B show Western blot verification of the expression of mGSDMC2 / 4N'terand their L-inserted versions, with (Figure 22A) an RFP tag on the N-ter and (Figure 22B) a GFP tag on the C-ter HEK293T cells.

[0092] Figures 23 A-23D depict GSDMCN tertargeted endosomes are leakier. Figures 23 A and 23B show HEK293T cells co-transfected with pBabe (empty vector), full length (FL) hGSDMC, the 1-284 aa GSDMCNter or 1- 289 aa GSDMCN terwith a plasmid expressing mCherry (20: 1) for 48 hours. mCherry levels were measured by western blot (Figure 23 A) or fluorescent microscopy (Figure 23B). Representative images are shown. Figure 23C shows HEK293T cells co-transfected with full length (FL) hGSDMC or 1-284 aa GSDMCN ter(both are Ampicillin resistance, Amp+) with a separate Kanamycin resistance plasmid (Kan+) at 1:1 ratio for 48 hours. The DNA extracted from the nucleic fraction of these transfected cells was transformed into competent A. coli. cells. Shown are the transformants counted after overnight culture. Center is mean. Figure 23D shows HEK293T cells co-transfected with lOng GFP mRNA with GSDMCN terfor 48 hr. GFP was measured by fluorescent microscopy. Scale bar = 1000 pm in (B) and (D).

[0093] Figure 24 shows GSDMCN terincreases transfection efficiency. NIH3T3 cells were stably transduced with BioID2-HA tagged vector control, full length human GSDMC (hGC FL) or GSDMCNter (hGC 289aa). Stably transduced cell lines were transfected with 2 ug GFP plasmid using lipofectamine 2000 for 48 hours to assess transfection efficiency in NH43T3 ACTIVE 131727896.1072396.1120

[0094] PATENT

[0095] cells. GFP levels were measured by fluorescent microscopy. Representative images are shown. Scale bar = 1000 pm.

[0096] Figure 25 shows GSDMCN terdisrupts RAB7+vesicles. TEM of HeLa cells expressing GSDMCN ter. E is endosome. Red arrows indicated the damaged sites of endosomes. Scale bar, 500 nm.

[0097] Figure 26 shows GSDMCN terfacilitates mRNA-nanoparticles delivery in vivo. Mice were injected with nanoparticles containing mRNA encoding luciferase, with or without GSDMCN ter. These nanoparticles specifically delivered mRNA to the spleen. 1 day after i.v. injection, luciferase activity was imaged. Representative images are shown.

[0098] DETAILED DESCRIPTION

[0099] The present disclosure is based, in part, on the observation that the inclusion of a polynucleotide encoding a fusion polypeptide comprising a tag and a cleaved Gasdermin C increases the payload release from lipid nanoparticles (LNPs).

[0100] The subject matter of the present disclosure is described with reference to the Figures. It should be understood that numerous specific details, relationships, and methods are set forth in this Detailed Description, Examples, and accompanying Figures to provide a more complete understanding of the subject matter disclosed herein.

[0101] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0102] 1. Definitions;

[0103] 2. Fusion Polypeptides;

[0104] 3. Lipid-Based Carriers and Lipid Nanoparticles;

[0105] 4. Pharmaceutical Compositions;

[0106] 5. Kits; and

[0107] 6. Methods.

[0108] 1. Definitions

[0109] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them.

[0110] ACTIVE 131727896.1072396.1120

[0111] PATENT

[0112] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Still further, the terms “having,” “including,” “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open-ended terms.

[0113] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0114] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0115] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0116] As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0117] An “effective amount” or “therapeutically effective amount” is an amount effective, at dosages and for periods of time necessary, that produces a desired effect, e.g., the desired therapeutic or prophylactic result. In certain embodiments, an effective amount can be formulated and / or administered in a single dose. In certain embodiments, an effective amount can be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0118] As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed ACTIVE 131727896.1 ii072396.1120

[0119] PATENT

[0120] either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing aneurysms, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0121] By “preventing” progression of a disease or disorder, a treatment can prevent deterioration due to a disorder (e.g., a cancer) in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment can prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0122] By “increase” is meant to alter positively by at least about 5%. A positive alteration can be an increase of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0123] By “reduce” or “decrease” is meant to alter negatively by at least about 5%. A negative alteration can be a decrease of about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or more, even by about 100%.

[0124] The terms “nucleic acid sequence” and “polynucleotide,” as used herein, refer to a single or double-stranded covalently-linked sequence of nucleotides in which the 3’ and 5’ ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide can include deoxyribonucleotide bases or ribonucleotide bases, and can be manufactured synthetically in vitro or isolated from natural sources.

[0125] The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0126] ACTIVE 131727896.1 ii072396.1120

[0127] PATENT

[0128] As used herein, “a functional fragment” of a molecule or polypeptide includes a fragment of the molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0129] The term “gene” refers to a region of a genomic sequence associated with regulatory regions, transcribed regions, and / or other functional sequence regions. A gene typically includes a coding sequence encoding a gene product, such as an RNA molecule or a polypeptide.

[0130] As used herein, the term “mutation” refers to a mutation in an amino acid sequence or in a nucleic acid sequence. In certain embodiments, a mutation in an amino acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least one amino acid in the amino acid sequence. In certain embodiments, a mutation in a nucleic acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least nucleotide of the nucleic acid sequence.

[0131] As used herein, the term “substantially identical” or “substantially homologous” refers to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

[0132] As used herein, “conservative” amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, no more than one, no more than two, no more than three, no

[0133] ACTIVE 131727896.1072396.1120

[0134] PATENT

[0135] more than four, no more than five residues within a specified sequence are altered. Exemplary conservative amino acid substitutions are shown in Table 1 below.

[0136] Table 1

[0137]

[0138] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( / .< ., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0139] ACTIVE 131727896.1 it072396.1120

[0140] PATENT

[0141] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.

[0142] 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0143] As used herein, the terms “antibody” and “antigen-binding fragment” refer to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and specifically binds an epitope of an antigen (e.g., amphiregulin) or a fragment thereof. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Antibodies include intact immunoglobulins and variants thereof. Functional fragments (antigen-binding fragments) of antibodies, that specifically bind an antigen (e.g., amphiregulin) are well known in the art, such as Fab fragments, Fab’ fragments, F(ab)’2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”) that specifically bind the target antigen. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker. In dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. In certain embodiments, the term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). A naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (1) and kappa (K). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region. Light and heavy chain variable regions contain four (4) regions (e.g., FR1, FR2, FR3, and FR4) interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDR.” The extent of the framework region and CDRs have been defined by designation systems known in the art such as Kabat, Clothia, IMGT, etc. The CDRs are primarily responsible for binding to an epitope of an antigen.

[0144] ACTIVE 131727896.1 22072396.1120

[0145] PATENT

[0146] The term “dosage” is intended to encompass a formulation expressed in terms of total amounts for a given timeframe, for example, as pg / kg / hr, pg / kg / day, mg / kg / day, or mg / kg / hr. The dosage is the amount of an ingredient administered in accordance with a particular dosage regimen. A “dose” is an amount of an agent administered to a mammal in a unit volume or mass, e.g., an absolute unit dose expressed in mg of the agent. The dose depends on the concentration of the agent in the formulation, e.g., in moles per liter (M), mass per volume (m / v), or mass per mass (m / m). The two terms are closely related, as a particular dosage results from the regimen of administration of a dose or doses of the formulation. The particular meaning, in any case, will be apparent from the context.

[0147] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Ranges disclosed herein, for example, “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y. With respect to sub-ranges, “nested sub-ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0148] As used herein, the term “pharmaceutical formulation” refers to a composition or preparation that allows an effective the biological activity of the active ingredient to be effective. Pharmaceutical formulations can include excipients (e.g., pharmacologically inactive substances used as a carrier for the active ingredient. In certain embodiments, the pharmaceutical formulations are sterile.

[0149] As used herein, the term “sterile” refers to a formulation free or essentially free from all living microorganisms or other life forms.

[0150] The term “endogenous,” as used herein, refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0151] The term “exogenous,” as used herein, refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides. By “exogenous” ACTIVE 131727896.1 13072396.1120

[0152] PATENT

[0153] nucleic acid is meant a nucleic acid not present in a native wild-type cell; for example, an exogenous nucleic acid can vary from an endogenous counterpart by sequence, by position / location, or both. For clarity, an exogenous nucleic acid can have the same or different sequence relative to its native endogenous counterpart; it can be introduced by genetic engineering into the cell itself or a progenitor thereof, and can optionally be linked to alternative control sequences, such as a non-native promoter or secretory sequence.

[0154] 2. _ Fusion Polypeptides

[0155] In certain embodiments, the present disclosure provides compositions (e.g., lipid nanoparticles) comprising a polynucleotide encoding a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. Additionally or alternatively, the present disclosure provides compositions (e.g., lipid nanoparticles) comprising a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide.

[0156] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is provided below.

[0157] MPSMLERISKNLVKEIGSKDLTPVKYLLXATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSL NDILEPXSSVLETWTGPFHFXDIMIQKHKADMGVNVGIEVXVSGEASVDHGCSLEFQIVTI PSPNLEDFQKRKLLDPEPSFLKECRRRGDNLYWTEAVELINNTVLYDSXXVNILGKIALWI TYGKGQGQGEXLRVKKKALTLQKGMVMAYKRKQLVIKEKAILISDDDEQRTFQDEYEISEMV GYCAARXEGLLPSFHTISPTLFNASSNDMKLKPELFLTQQF [SEQ ID NO: 1 ]

[0158] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is provided below.

[0159] ACTIVE 131727896.1072396.1120

[0160] PATENT MPSMLERISKNLVKEIGSKDLTPVKYLLXATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSL NDILEPXSSVLETWTGPFHFXDIMIQKHKADMGVNVGIEVXVSGEASVDHGCSLEFQIVTI PSPNLEDFQKRKLLDPEPSFLKECRRRGDNLYWTEAVELINNTVLYDSXXVNILGKIALWI TYGKGQGQGEXLRVKKKALTLQKGMVMAYKRKQLVIKEKAILISDDDEQRTFQDEYEISEMV GYCAARXEGLLPSFHTISPTLFNASSNDMKLKPELF [SEQ ID NO: 2 ]

[0161] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 3 is provided below.

[0162] MPSMLERISKNLVKEIGSKDLTPVKYLLSATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSL NDILEPSSSVLETWTGPFHFSDIMIQKHKADMGVNVGIEVSVSGEASVDHGCSLEFQIVTI PSPNLEDFQKRKLLDPEPSFLKECRRRGDNLYWTEAVELINNTVLYDSSSVNILGKIALWI TYGKGQGQGESLRVKKKALTLQKGMVMAYKRKQLVIKEKAILISDDDEQRTFQDEYEISEMV GYCAARSEGLLPSFHTISPTLFNASSNDMKLKPELFLTQQF [SEQ ID NO: 3]

[0163] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is provided below.

[0164] MPSMLERISKNLVKEIGSKDLTPVKYLLSATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSL NDILEPSSSVLETWTGPFHFSDIMIQKHKADMGVNVGIEVSVSGEASVDHGCSLEFQIVTI PSPNLEDFQKRKLLDPEPSFLKECRRRGDNLYWTEAVELINNTVLYDSSSVNILGKIALWI TYGKGQGQGESLRVKKKALTLQKGMVMAYKRKQLVIKEKAILISDDDEQRTFQDEYEISEMV GYCAARSEGLLPSFHTISPTLFNASSNDMKLKPELF [SEQ ID NO: 4 ]

[0165] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid

[0166] ACTIVE 131727896.1 it072396.1120

[0167] PATENT

[0168] sequence set forth in SEQ ID NO: 9. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NO: 9 is provided below.

[0169] MPSMLERISKNLVKEIGSKDLTPVKYLLSATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSL NDILEPSSSVLETWTGPFHFSDIMIQKHKADMGVNVGIEVSVSGEASVDHGCSLEFQIVTI PSPNLEDFQKRKLLDPEPSFLKECRRRGDNLYWTEAVELINNTVLYDSSSVNILGKIALWI TYGKGQGQGESLRVKKKALTLQKGMVMAYKRKQLVIKEKAILISDDDEQRTFQD [SEQ ID NO: 9]

[0170] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NO: 16 is provided below.

[0171] MSYTFDWLSKDWKKLQGRDLRPVKCLSDATKFCLFNILQETSSRLALKTEYIPVGFTLLHL LEPNIPVPEPEVSAPIPLKHTISQKLKADLDVETIAGGEAGFVKSCGYDIEVQSKSIPNPKL ESLQNRKLLDQLPTFMKTCWKDGKNLYWTEAYEVTKDTVLEGTSNSKFAIKGI INQLVKVG GSGQWQTEKTDSIPIQKGSVLAYKKQQLVIEDNTCVILTSANTKKKMTFPMRFVGMSGHLRY QDLVIETGSWINDIDPIGTIKEPTHLDFMC [SEQ ID NO: 16]

[0172] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 17. SEQ ID NO: 17 is provided below.

[0173] MGYSFDRASKDWKKLQGRDLRPVECLSDATKFRLFHILQETPRSGWETEDIPVGFTLLDLL EPNFPVPEPEVSAPKPFIHVQSTDLEANLNVADIARGGVGYVGYGGYNIEVQSTSIPNPKLE ILQNRKLLDNLPTFMKFCRMERKNLYWTEAYEVSKDTMLTGLSSVNLSVKGFFKQLFKVRG KAGRSEKYSIPIPKGSVLAYKKQQLVIENNTCVILPSATKKKMTFPGTPKYASASEPTEIYR TELQGLWINDIVPIGRIQEPAHLDFMC [SEQ ID NO: 17 ]

[0174] ACTIVE 131727896.1 B072396.1120

[0175] PATENT

[0176] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below.

[0177] MGYSFDRASKDWKKLQGRDLRPVECLSDATKFRLFHILQETPRSGWETEDIPVGFTLLDLL EPNFPVPEPEVSAPKPFIHVQSTDLEANLNVADIARGGVGYVGYGGYNIEVQSTSIPNPKLE ILQNRKLLDKLPTFMKFCRMERKNLYWTEAYEVSKDTMLTGLSSVNLLVKGFFKQLFKVRG KAGRSEKYSIPIPKGSVLAYKKQQLVIENNTCVILPSATKKKMTFPGTPKYASASEPTEIYR TELQGLWINDIEPIGRIQEPAHLDFKC [SEQ ID NO: 18 ]

[0178] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 19. SEQ ID NO: 19 is provided below.

[0179] MGYSFDRASKDWKKLQGRDLRPVECLSDATKFRLFHILQETPRSGWETEDIPVGFTLLDLL EPNFPVPEPEVSAPKPFIHVQSTDLEANLNVADIARGGVGYVGYGGYNIEVQSTSIPNPKLE ILQNRKLLDKLPTFMKFCRMERKNLYWTEAYEVSKDTMLTGLSSVNLLVKGFFKQLFKVRG KAGRSEKYSIPIPKGSVLAYKKQQLVIENNTCVILPSATKKKMTFPDRPLKLYDLPVTLRYQ EEVIETGSWIDDIDPIGTIEEPANLNFMC [SEQ ID NO: 19]

[0180] In certain embodiments, the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the GSDMC polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NO: 20 is provided below.

[0181] MLYTFDQVSKDWKKLQGKDLRPVRCLSDATKFRQFDILQKTPQSLFFKSEDTPVGYSLLQI LEPNFPVPETEVSAPMPLKHITSQKWKADVDVKATIADGGASAEFVQSCGYDIEVQSRSIPD ACTIVE 131727896.1072396.1120

[0182] PATENT SKLESLQNRKLLDKKLSFVTDCQMGRNNLYWTEVFEVTKDTWQGSSSIDLSGKALVSQLV KGEAQGQWQRETTDLVPIPKGAVLAYKKKQLVIENNTCAILLSANAKKKTFPGIFNFGMSSR SQTMEIVPYSANGLGSWIDYIPPIGRIEEPVH [SEQ ID NO: 20]

[0183] In certain embodiments, the tag polypeptide comprises a FLAG polypeptide, a polyhistidine (His) tag polypeptide, a hemagglutinin (HA) tag polypeptide, a Myc tag polypeptide, a Strep-tag polypeptide, a glutathione S-transferase (GST) tag polypeptide, a calmodulin-binding protein (CBM) polypeptide, a maltose-binding protein (MBP) polypeptide, a T7 tag polypeptide, a V5 tag polypeptide, a green fluorescent protein (GFP) polypeptide, a blue fluorescent protein (BFP) polypeptide, a cyan fluorescent protein (CFP) polypeptide, a yellow fluorescent protein (YFP) polypeptide, a FMN-binding fluorescent protein (FbFP) polypeptide, or a red fluorescent protein (RFP) polypeptide. In certain embodiments, the tag polypeptide comprises a FLAG polypeptide. In certain embodiments, the FLAG polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the FLAG polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the FLAG polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 5. SEQ ID NO: 5 is provided below.

[0184] MDYKDHDGDYKDHDIDYKDDDDKLAAANSGRP [SEQ ID NO: 5]

[0185] In certain embodiments, the tag polypeptide comprises an RFP polypeptide. In certain embodiments, the RFP polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the RFP polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the RFP polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6. SEQ ID NO: 6 is provided below.

[0186] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILS PQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGWTVTQDSSLQDGEFIYKVKL RGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKP VQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGALYKGRP [SEQ ID NO: 6]

[0187] In certain embodiments, the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are connected by a linker. The linker is usually rich in glycine for flexibility, as

[0188] ACTIVE 131727896.1072396.1120

[0189] PATENT

[0190] well as serine or threonine for solubility. In certain embodiments, the linker can link the C-end of the GSDMC polypeptide and the N-end of the tag polypeptide. In certain embodiments, the linker can link the C-end of the tag polypeptide and the N-end of the GSDMC polypeptide. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910- 1917 (2008) and International Patent Publication No. WO 2021 / 178971, the contents of which are incorporated by reference in their entirety.

[0191] In certain embodiments, the linker is a G4S linker. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 10. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 11. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 12. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 13. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 14. In certain embodiments, the linker comprises the amino sequence set forth in SEQ ID NO: 15. SEQ ID Nos: 10-15 are provided below.

[0192] GSGG [ SEQ ID NO : 10 ]

[0193] GSGGSG [ SEQ ID NO : 11 ]

[0194] GSGGGG [ SEQ ID NO : 12 ]

[0195] GSG [ SEQ ID NO : 13 ]

[0196] GSGGSGGSGGSGGG [ SEQ ID NO : 14 ]

[0197] GGGSGGGSGG [ SEQ ID NO : 15 ]

[0198] In certain embodiments, the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are covalently linked without a linker. In certain embodiments, the C-end of the GSDMC polypeptide is covalently linked to the N-end of the tag polypeptide. In certain embodiments, the C-end of the tag polypeptide is covalently linked to the N-end of the GSDMC polypeptide.

[0199] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the fusion polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO: 7 is provided below.

[0200] ACTIVE 131727896.1072396.1120

[0201] PATENT MDYKDHDGDYKDHDIDYKDDDDKLAAANSGRPMPSMLERISKNLVKEIGSKDLTPVKYLLXA TKLRQFVILRKKKDSRSSFWEQSDYVPVEFSLNDILEPXSSVLETWTGPFHFXDIMIQKHK ADMGVNVGIEVXVSGEASVDHGCSLEFQIVTIPSPNLEDFQKRKLLDPEPSFLKECRRRGDN LYWTEAVELINNTVLYDSXXVNILGKIALWITYGKGQGQGEXLRVKKKALTLQKGMVMAYK RKQLVIKEKAILISDDDEQRTFQDEYEISEMVGYCAARXEGLLPSFHTISPTLFNASSNDMK LKPELFLTQQF [SEQ ID NO: 7 ]

[0202] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the fusion polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 8 is provided below.

[0203] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILS PQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGWTVTQDSSLQDGEFIYKVKL RGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKP VQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGALYKGRPMPSMLERISKNLVKEIG SKDLTPVKYLLXATKLRQFVILRKKKDSRSSFWEQSDYVPVEFSLNDILEPXSSVLETWTG PFHFXDIMIQKHKADMGVNVGIEVXVSGEASVDHGCSLEFQIVTIPSPNLEDFQKRKLLDPE PSFLKECRRRGDNLYWTEAVELINNTVLYDSXXVNILGKIALWITYGKGQGQGEXLRVKKK ALTLQKGMVMAYKRKQLVIKEKAI L I SDDDEQRT FQDE YE I SEMVGYCAARXEGLLPS FHT I SPTLFNASSNDMKLKPELFLTQQF [SEQ ID NO: 8 ]

[0204] In certain embodiments, the polynucleotide encoding a fusion polypeptide comprises a codon-optimized sequence. As used herein, the term “codon-optimized” refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system or species. For example, a presently disclosed polynucleotide could be codon optimized for expression in humans. Codon optimization does not alter the amino acid sequence of the encoded protein.

[0205] In certain embodiments, the polynucleotide encoding a fusion polypeptide comprises a codon-degenerated sequence. As used herein, the term “codon-degenerated” refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as ACTIVE 131727896.1072396.1120

[0206] PATENT

[0207] long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0208] 3. Lipid-Based Carriers and Lipid Nanoparticles

[0209] In certain embodiments, the polynucleotide encoding the fusion polypeptide disclosed herein is encapsulated or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids) or with a lipid-based carrier (e.g., lipid nanoparticles, liposomes, lipoplexes, or nanoliposomes). In certain embodiments, the polynucleotide encoding a fusion polypeptide disclosed herein is encapsulated in lipid nanoparticles disclosed herein. In certain embodiments, the polynucleotide encoding a fusion polypeptide disclosed herein can be located in the interior space of the lipid nanoparticle, liposome, lipoplex, or nanoliposome. In certain embodiments, the polynucleotide encoding a fusion polypeptide disclosed herein can be located within the lipid layer / membrane of the lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0210] Additionally or alternatively, the fusion polypeptide disclosed herein is encapsulated or associated with one or more lipids (e.g., cationic lipids and / or neutral lipids), thereby forming lipid-based carriers (e.g., lipid nanoparticles, liposomes, lipoplexes, or nanoliposomes). In certain embodiments, the fusion polypeptide disclosed herein is encapsulated in lipid nanoparticles disclosed herein. In certain embodiments, the fusion polypeptide disclosed herein can be located in the interior space of the lipid nanoparticle, liposome, lipoplex, or nanoliposome. In certain embodiments, the fusion polypeptide disclosed herein can be located within the lipid layer / membrane of the lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0211] In certain embodiments, the lipid-based carrier is a lipid nanoparticle. In certain embodiments, the lipid-based carrier comprises a cationic lipid (e.g., an ionizable lipid), a noncationic lipid (e.g., phospholipid), a structural lipid (e.g., cholesterol), a PEG-modified lipid, or a combination thereof. In certain embodiments, one or more lipids of the lipid-based carrier is a naturally occurring lipid. In certain embodiments, one or more lipids of the lipid-based carrier is a synthetic lipid.

[0212] In certain embodiments, the lipid-based carrier comprises positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.

[0213] In certain embodiments, the lipid-based carrier comprises positively charged (cationic) lipids. Non-limiting examples of cationic lipids include N,N'-dimethyl-N,N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3 -[N-(N',N'- dimethylaminoethyl)carbamoyl)

[0214] ACTIVE 131727896.1 33072396.1120

[0215] PATENT

[0216] cholesterol (DC-chol), l,2-dioleoyloxy-3- [trimethylammonio]-propane (DOTAP), 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and l,2-dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), l,2-Dioleoyl-3-Dimethylammonium-propane (DODAP), l,2-Dioleoylcarbamyl-3 -Dimethylammoniumpropane (DOCDAP), l,2-Dilineoyl-3- Dimethylammonium-propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta- oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en- 3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis, cis-9',12'-octadecadienoxy)propane (CpLin DMA), and N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA).

[0217] In certain embodiments, the lipid-based carrier comprises non-cationic lipids. In certain embodiments, the non-cationic lipid is a phospholipid. In certain embodiments, the noncationic lipid is negatively charged (anionic) lipid. Non-limiting examples of non-cationic lipids includedistearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylchohne (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), di stearoyl -phosphatidyl -ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-0-monom ethyl PE), dimethylphosphatidylethanolamine, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamme (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimynstoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl- sn-glycero-3 -phosphocholine (DLPC), Sodium 1,2-ditetradecanoyl-sn-glycero-3 -phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine. Additional examples of non-cationic lipids include distearoylphosphatidylchohne / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, dimyrystoylphosphatidylchohne / cholesterol, INACTIVE 131727896.1 ii072396.1120

[0218] PATENT

[0219] Dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol, egg sphingomyelin / cholesterol, stearyl amine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.

[0220] In certain embodiments, the lipid-based carrier can further include one or more phospholipids.

[0221] In certain embodiments, the lipid-based carrier includes one or more structural lipids. As used herein, the term “structural lipid” refers to sterols (e.g., cholesterol) and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle can mitigate the aggregation of other lipids in the particle. Non-limiting examples of structural lipids include cholesterol, cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol.

[0222] In certain embodiments, the lipid-based carrier includes one or more polymers or copolymers, e.g., poly(lactic-co-glycolic acid) (PF AG) nanoparticles.

[0223] In certain embodiments, the lipid-based carrier includes one or more polyethylene glycol (PEG) lipid. Non-limiting examples of PEG-lipids include l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Poly ethylene glycol)-350] (mPEG 350 PE); 1 ,2-Diacyl-sn- Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl- sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000] (mPEG 1000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy (Polyethylene glycol)-2000] (mPEG 2000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Poly ethylene glycol)-3000] (mPEG 3000 PE); 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (mPEG 5000 PE); N-Acyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol) 750] (mPEG 750 Ceramide); N- Acyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG 2000 Ceramide); and N- Acyl-Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol) 5000] (mPEG 5000 Ceramide).

[0224] In certain embodiments, the lipid-based carrier includes one or more conjugated lipids. Non-limiting examples of conjugated lipids include PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (Cl 4), PEG-dipalmitoylglycerol (Cl 6), PEG-disterylglycerol (Cl 8), PEG- ACTIVE 131727896.1 33072396.1120

[0225] PATENT

[0226] dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (Cl 6), PEG-disterylglycamide (Cl 8), PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG), and PEG dialkoxypropylcarbam.

[0227] 3.1. Payloads

[0228] In certain embodiments, the presently disclosed lipid-based carriers (e.g., lipid nanoparticles) comprise a payload. In certain embodiments, the payload is a polynucleotide that regulates the expression of a protein or a gene. For example, but without any limitation, the payload can be a siRNA, a microRNA, an mRNA, a DNA sequence encoding a gene, and the like.

[0229] In certain embodiments, the payload is a polynucleotide that encodes an antigenic polypeptide. An antigenic polypeptide is a polypeptide that is able to trigger a humoral (innate) or cell-mediated immune response. In certain embodiments, the antigenic polypeptide is derived from a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell (e.g., a cancer cell). In certain embodiments, the antigenic polypeptide is associated with one or more diseases or conditions including, for example and without any limitation, infectious diseases, autoimmune diseases, and cancer.

[0230] In certain embodiments, the antigenic polypeptide is isolated from any portion of a virus. For example, but without any limitation, the antigenic polypeptide can be a natural viral capsid structure or a portion thereof, or a composite of a structure of multiple strains of the virus.

[0231] Non-limiting examples of viruses from which an antigenic polypeptide is obtained or derived include dsDNA viruses (e.g., adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus), ssDNA viruses (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6), dsRNA viruses (e.g., reovirus), (+)ssRNA viruses (e.g., picornavirus, coxsackievirus, hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus), (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses (including Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala, virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ACTIVE 131727896.1072396.1120

[0232] PATENT

[0233] ebola), ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus (HIV)), and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other viruses not listed above.

[0234] In certain embodiments, the antigenic polypeptide is a viral antigenic polypeptide isolated from a Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus).

[0235] In certain embodiments, the antigenic polypeptide is isolated from any portion of a bacteria. For example, but without any limitation, the antigenic polypeptide can be a polypeptide of the cell membrane of the bacteria. Non-limiting examples of bacteria from which an antigenic polypeptide is obtained or derived include Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisellatularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g.; Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis). As one of skill in the art would appreciate, antigenic polypeptides can be derived from other bacteria not listed above.

[0236] In certain embodiments, the antigenic polypeptide is isolated from any portion of a parasite. Non-limiting examples of parasites from which an antigenic polypeptide is obtained or derived include Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium ACTIVE 131727896.1 it072396.1120

[0237] PATENT

[0238] dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium), Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other parasites not listed above.

[0239] In certain embodiments, the antigenic polypeptide is isolated from any portion of a tumor also known as tumor antigen (TA). Non-limiting examples of tumor antigens from which an antigenic polypeptide is obtained or derived include gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE- 12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N-acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA); carcinoma- associated mutated mucins (i.e., MUC-1 gene products); EBNA gene products of EBV (i.e., EBNA-I); E7, E6 proteins of human papillomavirus; prostate specific antigen (PSA); prostate specific membrane antigen (PSMA); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes; KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, Kras, Histone, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96. As one of skill in the art would appreciate, antigenic polypeptides can be derived from other tumor antigens not listed above.

[0240] In certain embodiments, the payload is a polynucleotide that encodes a therapeutic protein (e.g., an antibody, a transmembrane protein, a growth factor, an enzyme, an antibody, a chimeric antigen receptor, or a structural protein). Non-limiting examples of therapeutic protein include transforming growth factor-beta (TGF-beta), interferon-alpha, interferon-beta, ACTIVE 131727896.1072396.1120

[0241] PATENT

[0242] interferon-gamma, granulocyte colony stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), interleukin- 1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin- 10, interleukin- 12, interleukin- 13, interleukin- 15, interleukin- 17, interleukin- 18, interleukin-22, interleukin-23, interleukin-35, amylin, anti-Mullerian hormone, calcitonin, cholecystokinin, corticotropin, endothelin, enkephalin, erythropoietin (EPO), follicle-stimulating hormone, gallanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human growth hormone (hGH), inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, luteinizing hormone releasing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, GLP-1, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, vasoactive intestinal peptide, vasopressin, trastuzumab emtansine, brentuximab vedotin, T-DM1, polyvalent IgG2a Fc (M045), SMN1, coagulation factors (e.g., F8 or F9), hemoglobin, ARSA, ABCD1, DDC, ADA, RPE65, ASP A, ARG1, DMD, COL7A1, BCL11A, cas9, WAS, HTT, CTNS, CPS1, OTOF, IDS, TTR, LAMP2, UGT1A1, PAH, KLKB1, ATP7B, GAN, agalsidase beta, imiglucerase, velaglucerase alfa, taliglucerase, alglucosidase alfa, laronidase, idursulfase, galsulfase, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, altumomab pentetate, amatuximab, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, cantuzumab ravtansine, capromab pendetide, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, dacetuzumab, demcizumab, detumomab, drozitumab, ecromeximab, eculizumab, elotuzumab, ensituximab, epratuzumab, etaracizumab, farletuzumab, figitumumab, flanvotumab, galiximab, gemtuzumab ozogamicin, girentuximab, ibritumomab tiuxetan, imgatuzumab, ipilimumab, labetuzumab, lexatumumab, lorvotuzumab mertansine, nimotuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, tacatuzumab tetraxetan, tositumomab, trastuzumab, totumumab, and zalutumumab.

[0243] In certain embodiments, the payload is a codon-degenerated polynucleotide. In certain embodiments, the payload is a codon-optimized polynucleotide.

[0244] 3.2. Modified Polynucleotides

[0245] In certain embodiments, any of the presently disclosed polynucleotides (e.g., the polynucleotide encoding a fusion polypeptide disclosed herein, the payload polynucleotide) can include one or more modified nucleosides (“modified polynucleotides”), which have useful

[0246] ACTIVE 131727896.1 37072396.1120

[0247] PATENT

[0248] properties including the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.

[0249] Modified polynucleotides can have enhanced efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, reduced immunogenicity, or a combination thereof.

[0250] In certain embodiments, the modified polynucleotide includes one or more different nucleoside modifications. In certain embodiments, the modified polynucleotide exhibits reduced degradation in a cell into which the polynucleotide is introduced, relative to a corresponding unmodified polynucleotide. For example, but without any limitation, the degradation rate of the modified polynucleotide acid is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater than about 90%, compared to the degradation rate of the corresponding unmodified polynucleotide.

[0251] In certain embodiments, the modified polynucleotide includes one or more different modified nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiomidine, 4-thio-pseudomidine, 2-thio-pseudowidine, 5-hydroxyuridine, 3-methylmidine, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudoutidine, 5-propynyl-uridine, 1-propynyl-pseudomidine, 5-taurinomethyluridine, 1-taurinom ethyl -pseudouridine, 5-taurinomethyl-2-thio-utidine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudomidine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydromidine, 2-thio-dihydropseudoulidine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudomidine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4- thiopseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-m ethyl- 1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- ACTIVE 131727896.1072396.1120

[0252] PATENT

[0253] hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, 5'-O-(l-Thiophosphate)-Adenosine, 5'-O-(l-Thiophosphate)-Cytidine, 5'-O-(l-thiophosphate)-Guanosine, 5'-O-(l-Thiophophate)-Uridine, and 5'-O-(l - Thiophosphate)-Pseudouridine.

[0254] The a-thio substituted phosphate moiety confers stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked nucleic acids also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0255] In certain embodiments, the modified polynucleotide includes one or more different modified nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8- aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, J-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0256] In certain embodiments, the modified polynucleotide includes a modified uridine or 1-methyl-pseudouridine. Polynucleotides (e.g., mRNAs) including 1 -methylpseudouridine in place of uridine can be translated at a higher level or for a longer duration than the mRNA that contained uridine. In certain embodiments, the modified polynucleotide includes 1-methylpseudouridine, pseudouridine, 5 -methylcytosine (m5C), 5-methyluridine (m5U), 2'-O-methyluridine, 2-thiouridine, N6-methyladenosine (m6A), or a combination thereof.

[0257] Additionally or alternatively, the modified polynucleotide can include certain modified nucleosides or modifications (e.g., different nucleic acid bases, different sugar moieties, different internucleotide linkages) based on desired activity.

[0258] In certain embodiments, the modified polynucleotide can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing polynucleotides are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: apractical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005).

[0259] ACTIVE 131727896.1072396.1120

[0260] PATENT

[0261] Modified polynucleotides of the present disclosure do not need to be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions in the polynucleotides. For example, but without any limitation, nucleotide analogs or other modification(s) can be located at any position(s) of the polynucleotide.

[0262] In certain embodiments, the modified polynucleotide includes a 5' terminal modification or a 3' terminal modification.

[0263] 3.3. Exemplary Lipid Nanoparticles

[0264] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprises a payload.

[0265] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload.

[0266] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload.

[0267] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising e first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload.

[0268] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the ACTIVE 131727896.1 ii072396.1120

[0269] PATENT

[0270] second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0271] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0272] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising e first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0273] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising e first polynucleotide and a second polynucleotide. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0274] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the polynucleotide comprises a payload.

[0275] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the

[0276] ACTIVE 131727896.1 H072396.1120

[0277] PATENT

[0278] tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the polynucleotide comprises a payload.

[0279] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the polynucleotide comprises a payload.

[0280] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the polynucleotide comprises a payload.

[0281] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the polynucleotide comprises a payload.

[0282] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the polynucleotide comprises a payload.

[0283] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the polynucleotide comprises a payload.

[0284] ACTIVE 131727896.1072396.1120

[0285] PATENT

[0286] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and (b) a polynucleotide. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the polynucleotide comprises a payload.

[0287] 4. Pharmaceutical Compositions

[0288] The present disclosure further provides pharmaceutical compositions that include a lipid-based carrier comprising a polynucleotide encoding the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide.

[0289] In certain embodiments, the pharmaceutical composition includes a lipid-based carrier comprising a first polynucleotide encoding the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and a second polynucleotide (e.g., a payload described above).

[0290] In certain embodiments, the pharmaceutical composition includes a lipid-based carrier comprising the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide and a polynucleotide (e.g., a payload described above).

[0291] In certain embodiments, the pharmaceutical composition includes a first lipid-based carrier comprising a polynucleotide encoding the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and a second lipid-based carrier comprising a second polynucleotide (e.g., a payload described above).

[0292] In certain embodiments, the pharmaceutical composition includes a first lipid-based carrier comprising the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and a second lipid-based carrier comprising a second polynucleotide (e.g., a payload described above).

[0293] In certain embodiments, the pharmaceutical composition can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combinations thereof in oils, in solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combinations thereof.

[0294] In certain embodiments, the pharmaceutical composition described herein further includes a pharmaceutically acceptable carrier, e.g., an excipient. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier which does not interfere with the

[0295] ACTIVE 131727896.1072396.1120

[0296] PATENT

[0297] effectiveness of the biological activity of the active ingredients and / or that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. In certain embodiments, the pharmaceutically acceptable carrier can be a buffering agent. Non-limiting examples of suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As a buffering agent, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide other calcium salts, and combinations thereof.

[0298] In certain embodiments, the pharmaceutical compositions can include one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients to stabilize the polynucleotides and / or the lipid-based carriers, to prolong the shelf-life, to facilitate administration of the pharmaceutical composition, and / or to enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients include parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride, chelators (e.g., EDTA), and the like. In certain embodiments, the pharmaceutical compositions can be provided as a frozen liquid form or a lyophilized form. A variety of cryoprotectants can be used including, but without limitations, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. Once formulated with the cryoprotectant, the pharmaceutical compositions can be frozen (or lyophilized and cryopreserved) at -20° C. to -80° C.

[0299] Additionally or alternatively, in certain embodiments, the pharmaceutical compositions can be provided to a patient in an aqueous buffered solution — thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution. In certain embodiments, the buffered solution is isotonic and suitable for intramuscular or intradermal injection. In certain embodiments, the buffered solution is a phosphate-buffered saline (PBS). ACTIVE 131727896.1 Il072396.1120

[0300] PATENT

[0301] Furthermore, the present disclosure provides an article of manufacture that provides the pharmaceutical composition in a single container, or provides the pharmaceutical composition in one container and a physiological buffer for reconstitution in another container. The container can include a single-use dosage or multi-use dosage. The containers can be pretreated glass vials or ampules. The article of manufacture can include instructions for use as well.

[0302] In certain embodiments, the pharmaceutical composition is provided for use in intramuscular (IM) injection. The pharmaceutical composition can be injected into a subject at, e.g., his / her deltoid muscle in the upper arm. In certain embodiments, the pharmaceutical composition is provided in a pre-filled syringe or injector (e.g., single-chambered or multichambered). In certain embodiments, the pharmaceutical composition is provided for use in inhalation and is provided in a pre-filled pump, aerosolizer, or inhaler.

[0303] 4.1. Exemplary Pharmaceutical Compositions

[0304] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprises a payload.

[0305] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload.

[0306] In certain embodiments, the present disclosure provides a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload.

[0307] ACTIVE 131727896.1072396.1120

[0308] PATENT

[0309] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload.

[0310] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0311] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising a first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0312] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising e first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0313] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising e first polynucleotide and a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked. ACTIVE 131727896.1 ii072396.1120

[0314] PATENT

[0315] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the polynucleotide comprises a payload.

[0316] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the polynucleotide comprises a payload.

[0317] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the polynucleotide comprises a payload.

[0318] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide. In certain embodiments, the fusion polypeptide is encapsulated in lipid nanoparticle, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the polynucleotide comprises a payload.

[0319] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the GSDMC polypeptide ACTIVE 131727896.1 ii072396.1120

[0320] PATENT

[0321] comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the polynucleotide comprises a payload.

[0322] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the polynucleotide comprises a payload.

[0323] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the polynucleotide comprises a payload.

[0324] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle comprising (a) a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, (b) a polynucleotide, and (c) a pharmaceutically acceptable carrier. In certain embodiments, the fusion polypeptide is located in the interior space of the lipid nanoparticle. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the polynucleotide comprises a payload.

[0325] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprises a payload.

[0326] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. ACTIVE 131727896.1 ii072396.1120

[0327] PATENT

[0328] In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload.

[0329] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload.

[0330] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload.

[0331] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0332] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. In certain embodiments, the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked. ACTIVE 131727896.1 II072396.1120

[0333] PATENT

[0334] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0335] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a first lipid nanoparticle comprising a first polynucleotide, a second lipid nanoparticle comprising a second polynucleotide, and a pharmaceutically acceptable carrier. In certain embodiments, the first polynucleotide encodes a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second polynucleotide comprises a payload. In certain embodiments, the first polynucleotide and the second polynucleotide are operably linked.

[0336] 5. Kits

[0337] The present disclosure also provides kits for the culture and transfection of cells comprising at least one container comprising a polynucleotide encoding the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide. Such kits can also comprise at least one component (or a combination thereof) selected from the group consisting of a polynucleotide encoding the fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, a cell medium, at least one cell, a polynucleotide encoding a payload, a lipid-based carrier, a buffer or buffering salt, and instructions for using the kit to introduce a payload into a cell. Preferably, the medium is a serum-free medium and / or a chemically defined medium and / or a protein-free or low protein medium and / or a medium lacking animal derived components.

[0338] 6. Methods

[0339] In certain embodiments, the present disclosure provides methods for preventing an infectious disease in a subject in need thereof. In certain embodiments, the present disclosure provides methods for treating an infectious disease in a subject in need thereof. In certain embodiments, the present disclosure provides methods for inducing an immune response against an infectious disease in a subject in need thereof. In certain embodiments, the methods

[0340] ACTIVE 131727896.1072396.1120

[0341] PATENT

[0342] comprise administering to the subject a therapeutically effective amount of a lipid-based carrier disclosed herein or a composition thereof. In certain embodiments, the subject is human.

[0343] In certain embodiments, the infectious disease is a viral disease. Non-limiting examples of viruses from which the infectious disease is derived include dsDNA viruses (e.g., adenovirus, herpesvirus, Epstein Barr virus, herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type 8, human cytomegalovirus, varicella-zoster virus, poxvirus), ssDNA viruses (e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6), dsRNA viruses (e.g., reovirus), (+)ssRNA viruses (e.g., picornavirus, coxsackie virus, coronavirus (e.g., SARS, CoVid-19, etc.), hepatitis A virus, poliovirus, togavirus, rubella virus, flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile virus, coronavirus), (-)ssRNA viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, metapneumovirus, arenaviruses (including Lassa virus, LuJo virus, Junin virus, Machupo virus, Bear Canyon virus, Wenzhou virus, Guanarito virus, Latino virus, Sabia virus, Chapare virus, Whitewater Arroyo virus, Pichinde virus, LCMV virus, Mopeia virus, Mobala, virus, Ippy virus, Mobala virus, and Morogoro virus, rhabdovirus, rabies virus, ebola), ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus (HIV)), and dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B).

[0344] In certain embodiments, the infectious disease is a bacterial disease. Non-limiting examples of bacteria from which the infectious disease is derived include Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisellatularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g.; Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonella typhinurium), ACTIVE 131727896.1 if072396.1120

[0345] PATENT

[0346] Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (Yersinia pestis).

[0347] In certain embodiments, the infectious disease is a parasitic disease. Non-limiting examples of parasites from which the infectious disease is derived include Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g., botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei, Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium), Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and Wuchereria bancrofti.

[0348] The pharmaceutical compositions of the present disclosure can be administered to subjects in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against a target pathogen for a sufficient amount of time (e.g., one year, two years, five years, ten years, or lifetime). Sufficient immune protection can be, for example, prevention or alleviation of symptoms associated with infections by the pathogen. In certain embodiments, multiple doses (e.g., two doses) of the pharmaceutical composition can be injected into subjects in need thereof to achieve the desired prophylactic effects. The doses (e.g., prime and booster doses) can be separated by an interval of e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, or ten years.

[0349] In certain embodiments, the present disclosure provides methods for preventing cancer in a subject in need thereof. In certain embodiments, the present disclosure provides methods for treating cancer in a subject in need thereof. In certain embodiments, the methods comprise ACTIVE 131727896.1072396.1120

[0350] PATENT

[0351] administering to the subject a therapeutically effective amount of a lipid-based carrier disclosed herein or a composition thereof. For example, but without any limitation, the cancer can be an adrenal cancer, a breast cancer, a colon cancer, a leukemia, a bile duct cancer, a bone cancer, a lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor), a bladder cancer, a brain cancer, a bronchial cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, an ependymoma, a retinoblastoma, a gallbladder cancer, a gastric cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, a heart cancer, a liver cancer, a pancreatic cancer, a melanoma, a kidney cancer, a laryngeal cancer, a lip or oral cancer, a lymphoma, a mesothelioma, a mouth cancer, a myeloma, a nasopharyngeal cancer, a neuroblastoma, an oropharyngeal cancer, an ovarian cancer, a thyroid cancer, a penile cancer, a pituitary cancer, a prostate cancer, a rectal cancer, a renal cancer, a salivary gland cancer, a sarcoma, a skin cancer, a stomach cancer, a testicular cancer, a throat cancer, a uterine cancer, a vaginal cancer, and a vulvar cancer. In certain embodiments, the subject is human.

[0352] In certain embodiments, the present disclosure provides methods for preventing cancer in a subject in need thereof. In certain embodiments, the present disclosure provides methods for treating cancer in a subject in need thereof. In certain embodiments, the methods comprise administering to the subject a therapeutically effective amount of a lipid-based carrier disclosed herein or a composition thereof comprising a payload. In certain embodiments, the payload comprises or encodes for an antibody. For example, but without any limitation, the payload can comprise or encode for abagovomab, adecatumumab, afutuzumab, alacizumab pegol, altumomab pentetate, amatuximab, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, cantuzumab ravtansine, capromab pendetide, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, dacetuzumab, demcizumab, detumomab, drozitumab, ecromeximab, eculizumab, elotuzumab, ensituximab, epratuzumab, etaracizumab, farletuzumab, figitumumab, flanvotumab, galiximab, gemtuzumab ozogamicin, girentuximab, ibritumomab tiuxetan, imgatuzumab, ipilimumab, labetuzumab, lexatumumab, lorvotuzumab mertansine, nimotuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, tacatuzumab tetraxetan, tositumomab, trastuzumab, totumumab, and zalutumumab. In certain embodiments, the subject is human.

[0353] In certain embodiments, the present disclosure provides methods for inducing immune response against a tumor antigen in a subject in need thereof. In certain embodiments, the methods comprise administering to the subject a therapeutically effective amount of a lipid- ACTIVE 131727896.1 ii072396.1120

[0354] PATENT

[0355] based carrier disclosed herein or a composition thereof. Non-limiting examples of tumor antigens include gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE- 12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA); carcinoma- associated mutated mucins (i.e., MUC-1 gene products); EBNA gene products of EBV (i.e., EBNA-I); E7, E6 proteins of human papillomavirus; prostate specific antigen (PSA); prostate specific membrane antigen (PSMA); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes; KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, Kras, Histone, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96. In certain embodiments, the subject is human.

[0356] In certain embodiments, the present disclosure provides methods for preventing an inflammatory disease in a subject in need thereof. In certain embodiments, the present disclosure provides methods for treating an inflammatory disease in a subject in need thereof. In certain embodiments, the methods comprise administering to the subject a therapeutically effective amount of a lipid-based carrier disclosed herein or a composition thereof comprising a payload. In certain embodiments, the payload comprises or encodes for an antibody. For example, but without any limitation, the payload can comprise or encode for adalimumab, alemtuzumab, atlizumab, canakinumab, certolizumab, certolizumab pegol, daclizumab, efalizumab, fontolizumab, golimumab, infliximab, mepolizumab, natalizumab, omalizumab, ruplizumab, ustekinumab, visilizumab, zanolimumab, vedolizumab, belimumab, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, eculizumab, briakinumab, atacicept, abatacept, alefacept, etanercept, and rilonacept.

[0357] In certain embodiments, the payload comprises or encodes for a cytokine. For example, but without any limitation, the payload can comprise or encode for transforming growth factorbeta (TGF-beta), interferon-alpha, interferon-beta, interferon-gamma, granulocyte colony stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), interleukin- 1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin- 10, interleukin- 12, interleukin- 13, interleukin- 15, interleukin- 17, interleukin- 18, interleukin-22, interleukin-23, and interleukin-35.

[0358] In certain embodiments, the present disclosure provides methods of delivering a payload to a cell (e.g., one described in Section 3.1). In certain embodiments, the methods ACTIVE 131727896.1072396.1120

[0359] PATENT

[0360] comprise contacting the cell with an effective amount of a lipid-based carrier or composition described herein. In certain embodiments, the methods are in vitro. In certain embodiments, the methods are ex vivo. In certain embodiments, the methods are in vivo. In certain embodiments, the cell can be any primary cell. For example, but without any limitations, the cell can be a blood cell, a cancer cell, an immune cell (e.g., a T cell), an epithelial cell (e.g., a skin cell), an endocrine cell, a muscle cell, a liver cell, or a neuron.

[0361] In certain embodiments, the present disclosure provides methods for increasing the transfection efficiency of a payload into a cell. In certain embodiments, the methods comprise contacting the cell with an effective amount of a lipid-based carrier or composition described herein. In certain embodiments, the methods are in vitro. In certain embodiments, the methods are ex vivo. In certain embodiments, the methods are in vivo. In certain embodiments, the cell can be any primary cell. For example, but without any limitations, the cell can be a blood cell, a cancer cell, an immune cell (e.g., a T cell), an epithelial cell (e.g., a skin cell), an endocrine cell, a muscle cell, a liver cell, or a neuron.

[0362] EXAMPLES

[0363] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.

[0364] Example 1

[0365] Nanoparticle liposome-mRNA delivery can be used to express proteins for use in vitro (e.g., cell culture transfection) and in vivo (e.g., mRNA vaccines, mRNA-based therapies). Increasing the mRNA-mediated protein expression is always one key to achieving a robust phenotype and / or therapeutic efficacy.

[0366] The present disclosure found that the cleaved (active) form of human Gasdermin C (GSDMC) N-terminus (GSDMCNter) can target and penetrate Rab7+late endosomes, facilitating cargo release. Physiologically, after nanoparticle liposome-mRNA transfection, the RNA must be released from the endosomes (e.g., mRNA escape) and transferred to the cytoplasm. The cleaved (active) form of human GSDMC, when co-delivered with the mRNA of interest, can help with more mRNA release and translation, further helping protein expression.

[0367] Although the cleaved (active) form of human GSDMCNtermRNA can lyse the endosome, it can also make pores / damage the plasma membrane, causing cell death, usually

[0368] ACTIVE 131727896.1072396.1120

[0369] PATENT

[0370] defined as pyroptosis. Thus, the natural form of the cleaved (active) form of human GSDMCNtercannot be used for nanoparticle liposome-mRNA transfection, as it kills cells. However, the present disclosure discovered that the tagged GSDMCNtercannot induce pyroptosis but still can facilitate mRNA-mediated protein expression. Thus, the presently disclosed tagged GSDMCNterfacilitated nanoparticle liposome-mRNA delivery mediated protein expression.

[0371] Results

[0372] Given the potential role of Gasdermin C (GSDMC) in the regulation of the immune system, it was determined which peptidase would be responsible for its cleavage. As shown in Figures 1A-1D, the peptidase Cathepsin S (CTSS) was capable of cleaving human and murine GSDMC polypeptides. A mass spectrometry approach was carried out to determine which amino residues would be the cleavage site. As outlined in Figures 2A-2C, multiple putative sites were identified including A252, H263, T264, F284, L285, and F289. Notably, mutants including deletion of the identified sites showed reduced cleavage of GSDMC by CTSS (Figure 2C).

[0373] In order to confirm the cellular function of the cleaved and active form of GSDMC (GSDMCNter), an RFP-tagged GSDMCNterwas expressed in HeLa cells and C. elegans (Figures 3 A and 3B, respectively). Notably, GSDMCNterlocalized and even penetrated Rab7+vesicles in both systems.

[0374] Next, HEK293T and bacterial cells were transfected with plasmids encoding the mCherry protein with a full length GSDMC (hGC FL) and two variants of the cleaved GSDMCNter. Cells transfected with GSDMCNterincreased the release of payloads from nanoparticles (e.g., lipid nanoparticles) (Figures 4 and 5). To further investigate the observed improved payload release, NIH3T3 cells, which are traditionally difficult to be transfected, were with plasmids expressing GPF and including either the hGC FL or the hGC variant (hGC 289aa). The presence of GSDMCNtersignificantly improved the transfection of these cells.

[0375] It is known that cleaved and active GSDMC can induce cell death by pyroptosis. Thus, transfected cells were stained for TO-PRO-3, a dead cell indicator. Surprisingly, cells expressing a tagged GSDMCNter, including a FLAG polypeptide and GSDMCNter, did not show increased cell death (Figure 7). Indeed, further experiments (see Figures 8A-8G) showed that tagged GSDMCNterwas able to boost mRNA transfection efficiency in multiple cell systems without inducing cell death.

[0376] As seen in Figures 23 A-23D, when GFP mRNA was co-expressed with the GSDMCN' ter plasmid, the initially released plasmid encoding the presently disclosed fusion protein (i.e., ACTIVE 131727896.1072396.1120

[0377] PATENT

[0378] the CTSS-generated GSDMCN ter) was releasing more GFP mRNAs from the “leaky” endosomes once transcribed and translated. Since GSDMCN tercompromises endosomes and facilitates the release of plasmids and mRNAs and the following protein expressions, it was hypothesized that the expression of the GSDMCN tercould increase transfection efficiency and, in turn, increase the expression of the transfected proteins of interest. Therefore, it was determined whether GSDMCN-ter could render cell lines that are traditionally difficult to be transfected more amenable to transfection. NIH3T3 cell lines were prepared to stably espress either hGSDMCFLor hGSDMCN terand it was found that hGSDMCN'ter-expression strongly increased the transfection efficiency (more GFP+cells) (Figure 24). Importantly, these cells tolerated well the stably expressed hGSDMCN terwithout observed cell death.

[0379] Overall, the present disclosure provides compositions including a tagged GSDMCNterthat can significantly improve the transfection efficiency without undesired side effects (e.g., pyroptosis).

[0380] Example 2

[0381] Gasdermin C (GSDMC), predominantly expressed in the gut, belongs to the Gasdermin (Gsdm) protein family, which becomes functional after the protease cleavage-mediated release of the N-terminus from their self-inhibited C-terminus. However, the direct protease that cleaves intestinal GSDMC is unknown. The present example shows that the protease Cathepsin S (CTSS) is the direct protease for GSDMC. Further, type 2 immune-promoting microbes, including the commensal gut protist Tritrichomonas arnold and helminth parasite Nippostrongylus brasiliensis, induced GSDMC cleavage, processed by CTSS, specifically in intestinal epithelial cells. CTSS-processed GSDMCN terlocalized to and penetrated RAB7+ vesicles. Consequently, targeting of RAB7+ vesicles resulted in lipid droplet accumulation and impacted lipid release, thus modulating anti-helminth immunity. This newly described biological function in RAB7+ vesicle regulation expands the role of Gsdm beyond pyroptosis and cytokine release.

[0382] Introduction

[0383] The Gasdermin (Gsdm) family includes six paralogous genes encoding GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59, with GSDMB being absent in mice. Gsdm proteins play a crucial role in inflammation, particularly in initiating pyroptosis, a form of programmed necrotic cell death. For example, GSDMD in macrophages and other myeloid cells, as well as GSDME in certain types of cancer, can form pores on the plasma membrane (PM), leading to cell lysis and pyroptosis. Gsdm proteins are increasingly recognized as

[0384] ACTIVE 131727896.1 37072396.1120

[0385] PATENT

[0386] important players in gastrointestinal health and disease. Several Gsdms are prominently expressed in the mammalian gut where they have a diverse functional repertoire influenced by stimuli and cell type-specific expression. Whereas in the intestine Gsdms are expressed in several cell types including lymphocytes, at least in the mouse intestine, Gasdermin C (GSDMC) expression is only found at low levels in intestinal epithelial cells (lECs) and its expression is dramatically increased during infection with the type-2 promoting helminth Heligmosomoides polygyrus, suggesting potential functions in intestinal inflammation and during homeostasis, which are poorly characterized.

[0387] Unlike many other immune modulators, transcriptional regulation of Gsdm family proteins is not sufficient to execute their biological functions. Gsdms A-E share highly conserved N-terminal (N-ter) and C-terminal (C-ter) domains separated by a variable linker. The C-term exhibits self-inhibition by completely masking the hydrophobic pocket of the N-ter that binds lipids. Thus, even if Gsdm expression is transcriptionally upregulated, in most cases, Gsdm is typically non-functional until cleaved by a protease to release the N-ter from the self-inhibited C-ter. In addition to protease-mediated cleavage, recent studies have identified other post-translational modifications that may activate Gsdms. For instance, palmitoylation of full-length GSDMD has been shown to induce liposome leakage and form pores similar to those formed by GSDMDN ter. One key reason the functions of intestinal GSDMC remain unclear is that its activating protease has not yet been identified. Examples of proteases that directly activate Gsdm proteins include, SpeB for GSDMA, Granzyme A for GSDMB, Caspase-8 for GSDMC (in certain cancer cells), Caspase- 1, 4, 5, 8, 11 for GSDMD, and Granzyme B or Caspase-3, 6 for GSDME5. Thus, the proteases that activate Gsdm play a key role in Gsdm biology, and in order to fully understand the function and regulation of intestinal GSDMC, identification of the activating protease is critical.

[0388] The present disclosure found that the cysteine protease Cathepsin S (CTSS) can cleave human and murine GSDMC. Colonization with the commensal protist Tritrichomonas (T) arnold, or infection with the helminth Nippostrongylus brasiliensis (N.b.), was sufficient to mediate CTSS-induced GSDMC. Deficiency in either GSDMC or CTSS resulted in impaired type 2 responses to helminth infections. CTSS-generated GSDMCN tertranslocated to and penetrated Rab7+vesicles. Inhibition of Rab7 restored impaired type-2 immunity to helminths in Gsdmcl-4^ mice. These findings establish a new paradigm for CTSS-generated GSDMCN'terin protective mucosal type 2 immunity and highlight a novel role for Gsdm proteins in modulating Rab7+ vesicles, such as late endosome / lipid droplet functions.

[0389] ACTIVE 131727896.1072396.1120

[0390] PATENT GSDMC is cleaved upon helminth infection or commensal protist colonization and promotes type-2 immunity.

[0391] Intestinal helminths, such as the hookworm N.b., trigger protective type-2 immune mediated inflammation characterized by IL- 13 -producing innate lymphoid cells (ILC2), eosinophilia, and goblet and tuft cell hyperplasia. N.b. infection of JAX WT mice also coincided with robust induction of GSDMC2, GSDMC3, and GSDMC4 expression and cleavage in the jejunum compared to controls (Figures 9A-9C and 16A), suggesting that GSDMC can regulate type-2 immunity to helminths. Of note, while GSDMC3 was detected using an antibody capable of recognizing both the full-length protein and the cleaved N-ter fragment, the antibodies used for GSDMC2 and GSDMC4 detection only recognized the cleaved C-ter fragment and not the full-length protein (Figures 9A-9C). Nevertheless, as Gsdmcl-4^ cells were used as a negative control, the signals observed in these blots (Figures 9A-9C) was attributed to GSDMC proteins, rather than non-specific or unrelated proteins that are also induced with N.b. infection. Further, although mice have four GSDMC genes, it was only detected gene expression for GSDMC2-4 (Figure 16A), as previously reported.

[0392] To study the role of the GSDMC in N.b. infection, a mouse model was developed including deletion of the entire 160kb Gsdmc locus including Gsdmc, Gsdmc2, Gsdmc3, and Gsdmc4 in the C57BL / 6 background (Figure 16B). Based on the highly homologous GSDMC protein sequences (Figure 16C) and potentially redundant functions, it was determined to delete the entire Gsdmc locus using a CRISPR / Cas9 approach.

[0393] N.b. infection of WT mice induced a significant increase in IL- 13 -producing ILC2, Th2 cells, and eosinophils in jejunum-draining mesenteric lymph nodes (mLN) compared to uninfected mice (Figures 9D-9E and 16D). In contrast, N.b. infection of Gsdmcl-4- / - mice failed to induce a significant increase in IL- 13 -producing ILC2 and eosinophils compared to uninfected controls and showed significantly decreased IL- 13 -producing ILC2, Th2 cells, and eosinophils compared to infected littermate controls (Figures 9D-9E and 16D). Furthermore, N.b. infection of Gsdmcl-4- / - mice resulted in significantly decreased tuft and goblet cell expansion compared to infected littermate controls (Figures 9F and 9G). The impaired type-2 immune response in N.b. infected Gsdmcl-4- / -mice resulted in an increased worm burden compared to littermate controls, indicating that GSDMC is required for robust worm clearance (Figure 16E).

[0394] It is well-accepted that the microbiota plays an important role in intestinal inflammation and homeostasis. Therefore, it was tested whether colonization with murine commensal protists Tritrichomonas spp., including Tritrichomonas (T.) arnold. could induce GSDMC cleavage, ACTIVE 131727896.1 it072396.1120

[0395] PATENT

[0396] As previously reported, T. arnold promotes mucosal innate type-2 immunity, initiated by the intestinal tuft cell-derived alarmin interleukin-25 (IL-25) in response to sensing protists or helminths. IL-25 drives a feed-forward tuft cell-ILC2 signaling circuit, in which tuft cell-derived IL-25 acts on local intestinal ILC2s to produce IL-13. This cytokine, in turn, acts on lECs expressing the IL-13 / IL4 receptor complex, promoting their differentiation into tuft and goblet cells (Tuft-ILC2 circuit). Experimental colonization of Tritrichomonas-free WT mice with T. arnold was sufficient to potently induce GSDMC expression and cleavage in lECs of the jejunum and GSDMC cleavage in the colon (Figures 9H-9I and 16F-16G). Further examination showed that T. arnold colonization results in GSDMC cleavage across the intestine (duodenum to colon) (Figure 16H) and that GSDMC is specifically expressed in epithelial but no other cells in the gut (Figure 17A).

[0397] In contrast to N.b. infection, which is cleared at around 10 days, commensal T. arnold colonization and the associated moderate but heightened type-2 immunity persisted throughout the mouse’s lifetime without causing noticeable gut pathology under homeostatic conditions. GSDMC activation remained detectable up to 30-weeks post-T. arnold colonization (Figures 9J and9K). Using IL4ra- / - mice, it was demonstrated that T. arnold-vaQ a GSDMC expression and cleavage depend on IL-4 / IL-13 signaling (Figures 17B-17D), indicating that the T. arnold-vaQ a tuft-ILC2 circuit can be important for GSDMC expression and cleavage. However, T. arnold colonization was not impacted by the presence or absence of GSDMC (Figure 17E): this is in line with previous findings that, in contrast to N.b. infection, type-2 immunity is not controlling commensal Tritrichomonas colonization (Figure 17F). Colonization of mice with T. arnold resulted in a significant increase in tuft and goblet cell numbers compared to non-protist colonized mice; however, tuft cell number increase was less robust in T. arnold colonized Gsdmcl-4- / - mice compared to T. arnold colonized littermate controls while no differences were observed in goblet cell numbers between genotypes (Figures 17G and 17H). These findings show that GSDMC’s role in anti-helminth immunity differs from its role during commensal protist colonization.

[0398] In support of these findings, treatment of intestinal organoid cultures with recombinant IL- 13 was sufficient to promote GSDMC expression and cleavage in lECs (Figures 9L-9M and 171). Given that intestinal organoid cultures are characterized by epithelial cells and therefore lack immune cells and other cell types, it was postulated that IL-13-induced GSDMC cleavage was lEC-intrinsic, occurring independently of support from other cell types.

[0399] ACTIVE 131727896.1072396.1120

[0400] PATENT

[0401] In summary, the present disclosure established two independent in vivo models showing GSDMC cleavage in the presence of a commensal protist and during helminth infection, generating GSDMC2-4 cleavage products around 30 kDa, processed by an unknown protease.

[0402] Cathepsin S cleaves human and murine GSDMC proteins.

[0403] GSDMC is likely not functional until it is processed by a protease to release the N-terminus from the self-inhibited C-terminus. Thus, the proteases that activate GSDMC play a central role in GSDMC biology. Caspase-8 has been reported as a protease that can cleave human GSDMC (in certain cancer cells), GSDMD (in macrophages), and GSDME (in certain cancer cells) via direct and indirect mechanisms that lead to cell lysis and pyroptosis. However, in contrast to GSDMC, IL- 13 was insufficient to mediate cleavage of GSDMD or GSDME in lECs (Figures 9L, 9M, 18A, and 18B). Importantly, caspase-8 was not activated by IL-13 in intestinal organoids or under type-2 stimulating conditions in vivo (Figures 18C-18E). Using caspase 1 / 11~ ~ mice, it was also found that the GSDMD-activating proteases, caspase-1 / 11, are not required for IL- 13 -mediated GSDMC cleavage in intestinal organoid cultures (Figure 18F). Altogether, caspase- 1, -8, and -11 were excluded as potential proteases for intestinal GSDMC cleavage.

[0404] Hence, a different protease was processing GSDMC in lECs. GSDMC expression in IEC is regulated in a STAT6-dependent manner in response to type 2 immunity. It was hypothesized that the protease that cleaves GSDMC is also downstream of STAT6. In macrophages, STAT6-activating cytokines IL-4 and IL- 13 alone or in combination with other related cytokines (IL-6 and -10) induce six cathepsin (CTS) proteases. Cathepsins that have strict exopeptidase functions (e.g., CTSC and CTSZ) or only function in an acidic environment (e.g., CTSH and CTSL) such as lysosomes were excluded since GSDMC cleavage is most likely occurring in the cytosol. The two remaining cathepsins, CTSB and CTSS, which are capable of functioning at a pH greater than 7, were further evaluated. It was found that CTSS, but not CTSB, cleaved both human and mouse GSDMC proteins when co-expressed in HEK293T cells, generating a 25-30 kDa C-terminal fragment (Figures 10A-10D). Coexpression of CTSS with GSDMC also induced moderate cell lysis (-20%) in HEK293T cells, further suggesting that CTSS can functionally activate GSDMC (Figures 10E-10G).

[0405] To further prove that CTSS is the direct protease for GSDMC, in vitro protease assays were performed using purified recombinant proteins. The purified recombinant CTSS produced robust GSDMC cleavage products (Figures 10H and 18G). In the tested conditions, GSDMC had a 6-His- and SUMO tag at its N-terminus, thus the full length was -70kDa. Importantly, CTSS cleaved GSDMC effectively in both pH=5.5 and pH=7.5 buffers in vitro ACTIVE 131727896.1 ii072396.1120

[0406] PATENT

[0407] (Figures 10H and 18G), in agreement with previous findings showing that CTSS can function in both acid and neutral pH.

[0408] Further, potential CTSS cleavage sites on GSDMC were investigated and whether they align with the ~30kDa C-terminal fragments identified in Figure 10 A. Unlike caspase-8, which exclusively cuts after an aspartic acid (D), CTSS cleavage is not restricted to a specific amino acid or amino acid sequence. Multiple cleaved fragments of CTSS-processed GSDMC C-ter were observed around 25-30 kDa (Figure 10A), suggesting multiple cleavage sites for CTSS. To overcome the challenge of N-ter sequencing (Edman sequencing), which requires a uniform clearcut C-ter protein fragment, mass spectrometry was performed to identify cleavage sites of CTSS on GSDMC. Taking advantage of the fact that trypsin cuts at lysine (K) and arginine (R), and that the Pl residue preference of CTSS is not K or R25(Pl residue is the amino acid right before the cleavage site), the CTSS-cleaved C-ter GSDMC fragments were enriched by HA-IP (HA is C-tagged to the human GSDMC) from HEK293T cells. These fragments were then subjected to mass spectrometry to identify non-tryp sin-generated peptides. Thus, the peptides not generated with a K- or R- cleavage were considered to be generated via CTSS-cleavage. Five (5) non-trypsin-generated peptides were identified, matching the molecular weight of the cleaved C-ter of GSDMC (approx. 30 kDa) (Figures 10A and 18H). Consequently, it was hypothesized that the possible cleavage sites include alanine (A)252, histidine (H)263, threonine (T)264, phenylalanine (F)284, leucine (L)285, and (F)289. Notably, the F284 site and F289 site share a similar Pl’ L residue, indicating cleavage between F-L (with Pl’ residue being the amino acid immediately after the cleavage site) (Figure 18H). To verify whether these sites were indeed cleaved by CTSS, GSDMC variants including deletion were generated (A250-294aa; A284-295aa; and A7) (Figure 181) followed by coexpression with CTSS in HEK293T cells. The presently disclosed data shows two of the small deletions (A7 and A250-294aa) successfully diminished CTSS-mediated GSDMC cleavage, while A284-295aa deletion largely, but not completely, prevented CTSS-mediated cleavage (Figure 18J), indicating that CTSS has multiple cleavage sites on GSDMC.

[0409] Cathepsin S is required for GSDMC cleavage.

[0410] Ctss' / ~ animals were studied to further confirm the requirement of CTSS in GSDMC cleavage. First, a significantly reduced GSDMC cleavage was observed in IL- 13 -stimulated Ctss' / ~ intestinal organoid cultures compared to CTSS-sufficient organoid cultures (Figure 11 A), demonstrating that CTSS cleaves GSDMC in an lEC-intrinsic manner, bypassing the requirement for immune cell co-cultures.

[0411] ACTIVE 131727896.1 62072396.1120

[0412] PATENT

[0413] Next, it was assessed whether GSDMC cleavage was mediated by CTSS in lECs using conditions that led to GSDMC cleavage in the presence of T. arnold colonization or N.b. infection (Figures 11A-11C and 11H). It was found that GSDMC2 and GSDMC4 cleavage was not detectable in Ctss^ mice colonized with T. arnold or infected with N.b. compared to littermate controls (Figures 11C-11D, 11G-11J, and 11M-11N). In addition, GSDMC3 cleavage was significantly reduced in Ctss / - mice colonized with T. arnold or infected with N.b. compared to littermate controls (Figures 1 IE-1 IF and 1 IK-1 IL).

[0414] Having identified CTSS as an important protease for lEC-specific GSDMC, it was next assessed CTSS localization and expression in lECs in the small intestine and colon under conditions that led to CTSS-mediated GSDMC cleavage during N.b. infection or T. arnold colonization (Figures 11C-1 IN). In lECs, CTSS is constantly active after self-processing and can retain catalytic activity in both acidic and neutral pH (e.g., pH = 7.5 and Figures 11H and 18G), implying that CTSS can function inside and outside of lysosomes. Around 80% of CTSS was localized inside lysosomes (colocalized with Lampl+, blue arrows) and 20% of CTSS localized outside of lysosomes (a diffusion pattern, white arrows) in lECs of the small intestine and colon (Figures 19A and 19B), indicating that CTSS and GSDMC, which are both present in the cytosol, could potentially interact in the cytosol. Of note, specificity for CTSS staining was validated in Ctss^ mice (Figure 19C). Moreover, IL- 13 stimulation of intestinal organoid cultures led to a significant increase in CTSS gene expression in lECs and N.b. infection of mice resulted in a significant upregulation of CTSS protein expression, specifically in lECs but not in the lamina propria (Figures 19D and 19E).

[0415] Different from GSDMC, which is exclusively expressed in lECs (Figure 17A), CTSS is expressed in both lECs and immune cells (Figure 19E). Thus, to test the importance of CTSS expression in lECs in protecting from N.b. helminth infection, CD45.2+Ctss^ mice, littermate controls, and reconstituted mice with bone marrow (BM) derived from CD45.1+WT mice were irradiated (Figure 19F). N.b. infection 6-weeks after successful reconstitution revealed a significant decrease in IL- 13 producing ILC2 and Th2 cells and a substantial reduction in eosinophils in BM chimeric Ctss ^ mice compared to BM chimeric littermate controls (Figures 110-1 IQ). Furthermore, N.b. infection of BM chimeric Ctss^ mice resulted in significantly decreased tuft and goblet cell expansion compared to infected littermate controls (Figures 11R and 11 S). Based on the significant phenotypic differences between Ctss^ and WT mice, CTSS was identified as the primary protease responsible for murine GSDMCs cleavage under type-2 stimulating conditions. However, since Ctss^ mice did not completely abolish GSDMC3 cleavage (Figures 11 A, 1 IB, 1 IE, 1 IF, 1 IK, and 1 IL), it is possible that another unidentified ACTIVE 131727896.1 ii072396.1120

[0416] PATENT

[0417] protease could act alongside CTSS to contribute to GSDMC3 cleavage. In summary, CTSS-processed GSDMCN terin lECs promoted positive type-2 immune feedback for anti-helminth immunity (Figure 11T).

[0418] Cathepsin S-generated murine GSDMCN~teris not effective in inducing cell death in lECs under type-2 stimulating conditions.

[0419] Next, it was aimed to mechanistically understand how CTSS-processed GSDMCN termodulates type 2 immunity. Although caspase- or Granzyme-processed members of the Gsdm family members usually typically can use their N-ter fragments to form pores on the PM, leading to pyroptosis, including caspase-8-processed human GSDMC in certain cancer cell lines, it has also been reported that cell death is not an obligatory outcome of murine intestinal GSDMC activation. It was confirmed that the activation of mGSDMCs in lECs does not result in pyroptosis under type-2 stimulating conditions.

[0420] IL- 13 treatment of intestinal organoid cultures failed to induce cell death, indicated by propidium iodide (PI) staining, despite the observation of the CTSS-mediated cleavage of GSDMC (Figures 11 A and 20A). Moreover, no differences were observed in cell death (PI+) and no morphological differences of IL- 13 treated GSDMC-deficient, CTSS-deficient and WT intestinal organoid cultures were observed for as long as 72 hours (Figures 20B and 20C).

[0421] In addition, TUNEL staining did not reveal differences in the apoptotic index of lECs between Gsdmcl-4~~ mice and littermate controls under N. b. infection or T. arnold colonization conditions (Figures 20D and 20E). Notably, intestinal inflammation or any noticeable histopathology was not observed in T. arnold-colonized mice compared to non-colonized controls, despite detectable GSDMC activation in T. arnold-colonized mice up to 30 weeks post-colonization (Figures 9J, 9K, 20F, and 20G). This is a sharp contrast to GSDME, which can induce massive pyroptosis and inflammation in intestines when activated by chemotherapy drugs such as cisplatin and 5-fluorouracil, marked by severely disrupted crypts and villi, which is attenuated in Gsdme ^ mice.

[0422] Further, Gsdm-induced pores have also been implicated in the release of intracellular IL-1 cytokine family members such as IL-1, IL- 18, and IL-33, mediating inflammation. GSDMC has been implicated in IL-33 release from goblet cells in the H. polygyrus infection model. No differences were observed in IL-13 or IL-18 release in intestinal explants of Gsdmcl-4~ ~ mice and littermate controls of N.b. infected mice (Figures 20H and 201). Of note, IL-33 release under the same experimental conditions was below the detection range. Similarly, despite the observed cleavage of GSDMC in intestinal organoids, IL- 13 or GSDMC-dependent IL-33 release was not observed, showing that at least in organoid cultures CTSS- ACTIVE 131727896.1 61072396.1120

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[0424] generated GSDMCN terdoes not promote pore formation that can result in pyroptosis, or IL-33 release from lECs (Figure 20J), at least under IL- 13 stimulating conditions. This aligns with previous studies that did not observe or report that GSDMC activation in intestinal organoids alone is sufficient to promote either IL-33 release or pyroptosis. However, one study showed that co-culture of mast cells with the CMT-93 cell line can indeed promote GSDMC-dependent IL-33 release. Altogether, these findings indicate that unlike intestinal GSDMs (e.g., GSDME), the active GSDMCN terin lECs probably does not effectively promote pyroptosis but leads to adaptation of lECs to heightened baseline type-2 immunity.

[0425] Cathepsin S-generated GSDMCN~tercolocalizes to and penetrates Rab7 vesicles. Next, to investigate the functional consequences of CTSS-generated GSDMCN ter, subcellular localizations of the CTSS-generated GSDMCN terwere assessed. Red fluorescent protein (RFP)-tagged CTSS-generated GSDMCN tercolocalized with, and even penetrated into Rab7+vesicles, when expressed in HeLa cells, compared to the full-length (FL) GSDMC protein (Figure 12A). Similarly, the murine GSDMC2N terand GSDMC4N'teralso colocalized with and penetrated into Rab7+vesicles (Figures 12B and 12C). To confirm these findings, colocalization studies were performed using the Caenorhabditis (C.) elegans model system, where Rab7+vesicles are more readily visualized. Indeed, when ectopically expressed in C. elegans, both N-terminal tagged, or C-terminal tagged GSDMCN tercolocalized with Rab7+vesicles. Additionally, Rab7+vesicles that colocalized with GSDMCN ter(white arrows) were significantly larger in size compared to Rab7+vesicles that did not colocalize with GSDMCN'ter(blue arrows) (Figure 12D), indicating that GSDMCN tercan compromise the integrity of Rab7+vesicles.

[0426] Functional consequences of Cathepsin S-generated GSDMCN~tertargeting Rab7+vesicles.

[0427] Next, functional assays were performed to further validate that CTSS-generated GSDMCN terindeed compromised Rab7+vesicles. Rab7+vesicles are largely late endosomes, which play critical roles in endocytosis / phagocytosis. Plasmids, when transfected into cells, undergo endocytosis / phagocytosis and are initially trapped in endosomes. Only upon release from endosomes into the cytosol do plasmids translocate to the nucleus, where they are transcribed. Thus, the plasmid “leak out” can serve as an indicator of Rab7+vesicle integrity. Co-transfection experiments in HEK293T cells were performed with plasmids expressing CTSS-generated GSDMCN teror controls with a separate plasmid expressing the red fluorescent protein mCherry. It was found that when co-transfected with CTSS-generated GSDMCN ter, the mCherry expression level was strongly increased compared to when co-transfected with ACTIVE 131727896.1 ii072396.1120

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[0429] empty vector (pBabe) control or full length GSDMC as shown by western blot and microscopy (Figures 13A and 13B). Further, co-transfection experiments were performed in HEK293T cells with plasmids expressing CTSS-generated GSDMCN teror full-length GSDMC which carried an ampicillin resistance gene, with a separate plasmid carrying a kanamycin resistance gene. Then the nuclei were isolated, the DNA was extracted , and these DNAs were transformed into E. coli competent cells. The transformants on both ampicillin and kanamycin resistance agar plates were counted. In both cases, it was observed significantly more transformants when CTSS-generated GSDMCN terwas present compared to full length GSDMC (Figure 13C). These data suggest that more plasmids were released from the “leakier” endosomes and entered the nuclei in the presence of CTSS-generated GSDMCN ter. Further, it was determined whether the expression of the GSDMCN tercould render cell lines that are traditionally difficult to be transfected more amenable to transfection. As a proof of principle, NIH3T3 cell lines stably expressing either GSDMCFLor GSDMCN terwere analyzed and it was found that using the GSDMCN'ter-expressing cell line strongly increased the numbers of GFP+cells, boosting the transfection efficiency (Figures 13D-13F).

[0430] Rab7+vesicles have been implicated in the late endocytic pathway and are known to associate with lipid droplet (LD) breakdown and regulate lysosome recruitment for lipid turnover. Thus, it was tested whether LDs might also be regulated by GSDMCN tervia targeting Rab7+vesicles. FBS starvation typically leads to LD breakdown and turnover, as shown by GFP-ADRP, a marker for LDs (Figure 13G). It was found that overexpressed GSDMCN-ter significantly inhibited FBS starvation-induced LD breakdown and turnover in HeLa cells, indicated by less dotted GFP-ADRP+cells (Figure 13G). Next, it was assessed whether CTSS-processed GSDMCN terin intestinal organoids regulates LDs under IL- 13 stimulation conditions. Using Nile red staining to assess LDs, no differences were found in the abundance of lipid droplets between untreated GSDMC-deficient, CTSS-deficient, and WT intestinal organoid cultures (Figure 13H). In contrast, IL-13 treatment led to a significant accumulation of LDs in WT intestinal organoids compared to untreated WT organoids. However, no increase was observed in IL-13-treated GSDMC-deficient and CTSS-deficient organoids compared to corresponding untreated controls (Figure 13H). These findings indicate that the active forms (N-ter) of GSDMCs in IL-13-treated WT organoids suppress LD breakdown and turnover, leading to increased LD accumulation.

[0431] In GSDMC-deficient and CTSS-deficient organoids, even with IL-13 treatment, the absence of active GSDMCs allowed LD breakdown and lipid turnover to proceed. Importantly, treatment with either a Rab7 GTPase inhibitor (CID-1067700) or a lysosomal inhibitor ACTIVE 131727896.1 ii072396.1120

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[0433] (bafilomycin Al) elevated LDs in Gsdmcl-4^ organoids to levels comparable to those observed WT organoids (Figures 131 and 13J). This occurred because CID-1067700 and bafilomycin Al mimicked the effects of GSDMC activation by inhibiting LD breakdown and lysosome recruitment for lipid turnover. In summary, GSDMCN terplays an inhibitory role in LD breakdown and lipid turnover via targeting Rab7+vesicles.

[0434] Since the CTSS-processed GSDMCN tercan regulate LD breakdown or turnover, it was further tested whether it might modulate lipid-based immune modulator(s), instead of cytokines. LDs serve as lipid reservoirs that can be used for synthesizing immunomodulatory eicosanoids such as Prostaglandin D2 (PGD2), which can dampen intestinal anti-helminth type 2 immunity. IL-13-treated Gsdmcl-4^ intestinal organoid cultures released significantly more PGD2 compared to WT controls when stimulated with ionomycin for 30 minutes (Figure 13K). This increase is possibly due to the efficient turnover of LDs in Gsdmcl-4^ organoids, providing lipid resources for lipid synthesis, whereas in WT organoids, LDs cannot be efficiently turned over and utilized for lipid synthesis. Supporting this possibility, treatment with bafilomycin Al, which inhibits LD turnover, lowered PGD2 in Gsdmcl-4~ ~ organoids to levels mimicking those observed in WT organoids (Figure 13L).

[0435] Rab7 inhibition restores impaired type-2 immunity to helminths in Gsdmcl-4~ ~ mice.

[0436] Based on the in vitro data, it was tested whether the role of GSDMCs in type-2 immunity to the helminth N.b. is also mediated by Rab7 inhibition in vivo. It was found that blockade of Rab7 using a Rab7 GTPase inhibitor CID-1067700 restored impaired type-2 immunity in N.b. infected Gsdmcl-4^ mice as evident by full restoration of goblet cells, tuft cells, and Th2 and ILC2 immune responses comparable to those observed in N.b. infected WT animals (Figures 14A-14D). Of note, CID-1067700 treatment did not affect cell death in organoid cultures or in HEK293T-overexpression mGSDMC2N terconditions, further supporting the idea that GSDMCs contribute to type 2 immunity by targeting Rab7+vesicles rather than modulating cell death (Figures 21 A and 21B).

[0437] Given the findings that CTSS-processed GSDMCN tersuppresses PGD2 release via Rab7-LDs in in vitro intestinal organoid cultures, along with the observations that Gsdmcl-4~ / _mice and BM chimeric Ctss ^ mice exhibit impaired goblet cell hyperplasia when infected with N.b. (Figures 9G and Figure US), and the established role of PGD2 in suppressing intestinal anti-helminth immunity by engaging the PGD2 receptor CRTH2 on lECs resulting in diminished goblet cell hyperplasia upon N.b. infection, it was tested whether blockade of CRTH2 could restore at least part of the impaired type-2 immunity, particularly the goblet cell hyperplasia defect, in Gsdmcl-4^ mice. Indeed, CRTH2 blockade significantly restored goblet ACTIVE 131727896.1 6072396.1120

[0438] PATENT

[0439] cell hyperplasia in N.b. infected Gsdmcl-4^ mice to levels comparable to those observed in N.b. infected WT animals (Figure 14E). However, the blockade was insufficient to restore impaired Th2 and ILC2 immune responses (Figures 14F and 14G).

[0440] In summary, CTSS processes GSDMC in response to type 2 immune activation triggers. Once activated by CTSS, the GSDMCN'tertargets Rab7+vesicles to modulate LDs and lipid synthesis, at least partly by limiting the production of PGD2, a suppressor of intestinal type 2 immunity. Thus, CTSS-processed GSDMCN terin lECs promotes a positive type-2 immune feedback loop critical for anti-helminth immunity.

[0441] Since blockade of CRTH2 only rescued the goblet cell hyperplasia phenotype and not others, it was concluded that GSDMCN tercan modulate additional unknown lipids or even lipid-independent but Rab7-dependent functions, that are important for type-2 immunity against helminths (Figure 14H).

[0442] Completion of the lipid interaction motif in the bl-b2 loop of GSDMC enhances its pyroptotic activity.

[0443] Upon comparing protein sequences, it was found that the conserved motif for lipid interaction, featuring a hydrophobic tip flanked by basic residues (“LFW” or “WFW”) at the 131-132 loop, which is present in most pyroptosis-inducing GSDMs (such as GSDMD, GSDMA, and GSDME), is not intact in GSDMC. Specifically, GSDMC lacks the first amino acid “L” or “W” (Figure 15 A). Mutating the “L” in GSDMA3 or the “W” in GSDMD to “E” partially impairs their pore-forming activity. Here, by introducing either an “L” or a “W” into GSDMC, the present disclosure was able to enhance its function in robust pyroptosis induction (Figures 15B and 15C). CTTS co-expression doubled the cell death induction of hGSDMC “L” compared to WT hGSDMC. Even more strikingly, full-length hGSDMC “W” induced strong cell death even without CTSS (Figure 7B-C). Similarly, CTSS co-expression doubled the cell death induction of murine GSDMC2 “L” compared to WT GSDMC2 (Figures 15D and 15E). Similarly, CTSS co-expression doubled the cell death induction of murine GSDMC2 “L” compared to WT GSDMC2 (Figures 15D and 15E). Expression of GSDMC2N terand GSDMC4N terwith “L” insertion resulted in exacerbated pyroptosis compared to WT GSDMC2N terand GSDMC4N ter, with general higher pyroptotic activity in HEK293T cells compared to HeLa cells and general higher pyroptotic activity when GFP / RFP-tag was put at C-ter of GSDMC2N terand GSDMC4N ter(Figures 15F-15H, 21 A, and 2 IB). Of note, despite the increased pyroptotic activity of GSDMC2N ter“L” insertion compared to WT GSDMC2N'ter(Figure 15H), GSDMC2N ter“L” retained the ability to target Rab7 vesicles (Figure 151), indicating that the lipid binding motif at the 131-132 loop is required for enhancing membrane ACTIVE 131727896.1 ii072396.1120

[0444] PATENT

[0445] pore-forming activity but not a determinant factor of subcellular targeting, such as Rab7+vesicles. Functionally, oligomerization of GSDMCN teris important for membrane pore formation. Importantly, GSDMC2N terand GSDMC4N terwith “L” insertion resulted in increased oligomerization compared to WT GSDMC2N ter, using native PAGE (without sodium dodecyl sulfate (SDS)) (Figures 15J and 15K), providing an explanation for the L-insertion mediated enhancement of GSDMC’s pyroptotic activity.

[0446] Together, these findings demonstrate that, unlike other Gsdms that form pores at the PM, CTSS-generated GSDMCN terlocalizes to and targets Rab7+endosomes, promoting lipid droplet accumulation and modulating anti-helminth immunity, partially dependent on PGD2. This newly described biological function of a Gsdm family member expands the role of Gsdm pore-forming activities beyond pyroptosis and IL-1 family cytokine release (Figure 14H).

[0447] Discussion

[0448] In this example, it was shown that the protease CTSS directly cleaves GSDMC in the linker region to generate GSDMCN ter. In the intestine, type-2 - IL- 13 promoting commensal protist T. arnold or the parasite helminth N.b. was sufficient to mediate CTSS-dependent cleavage of GSDMC. N.b. infection revealed an important role for GSDMCN terin mounting potent immunity to parasites. Intriguingly, Cathepsin S-generated GSDMCN tercolocalized to and penetrated Rab7+ vesicles. Rab7 targeting of GSDMCN-ter resulted in LD accumulation and modulating anti-helminth immunity, which was in part dependent on PGD2. CTSS-cleaved intestinal GSDMCN terexhibited weak pore-forming activity. This weak pyroptotic activity was augmented by completing an incomplete lipid interaction motif in GSDMC. Thus, the present disclosure reveals a novel mechanism for GSDMC biology under type-2 stimulating conditions that are independent of pyroptosis but reliant on Rab7.

[0449] Different from other Gsdms, the CTSS-processed GSDMCN terhas a unique localization with Rab7+vesicles. Although many Gsdms primarily target the plasma membrane, recent studies have shown that both GSDMD and GSDME also target mitochondria before causing damage to the plasma membrane. For example, activated GSDMD can cause cardiolipindependent mitochondrial destruction, in both inner and outer membrane. This process is key for pyroptosis as it leads to ROS production, loss of transmembrane potential, and attenuated oxidative phosphorylation. Facilitated by GSDMD, mitochondria also release PNPT1 to cause global mRNA decay during pyroptosis. Similarly, neuronal GSDME also drives local mitochondrial damage and axon loss prior to pyroptosis and is involved in neurodegenerative diseases. The question remains as to why GSDMCN terpreferentially targets Rab7+vesicles. For GSDMDN'ter-mediated mitochondrial targeting, the lipid component cardiolipin is required. ACTIVE 131727896.1 ii072396.1120

[0450] PATENT

[0451] Genetic ablation of cardiolipin synthase Crlsl or the scramblase Plscr3 suppressed GSDMDN'ter-mitochondria co-localization. It is worth investigating whether GSDMCN terhas a unique lipid-binding pattern distinct from other Gsdms or, alternatively, whether other protein components on Rab7+vesicles facilitate the preference of GSDMCN terfor lipid membranes of Rab7+vesicles.

[0452] On a functional level, it is shown that GSDMCN tercan penetrate Rab7+vesicles to facilitate endocytic cargo release from late endosomes resulting in enhanced protein production during plasmid transfection and improved transfectability of cell lines that were previously difficult to transfect, which could serve as a tool to enhance the efficiency of delivering exogenous nucleic acids for gene therapies. Furthermore, Rab7 associates with LDs and regulates lysosome recruitment to LDs. CTSS-generated GSDMCN terresulted in impaired LD degradation under starvation conditions and increased lipid droplets in IL- 13 -stimulated organoids. Although mechanisms and biological consequences for the increase in LD formation in lECs under IL- 13 simulating conditions are unclear, LDs harbor lipids and enzymes that can readily be made available to synthesize immunomodulatory mediators like PGD2, which has been shown to modulate anti-helminth immunity. While the presently disclosed data indicate a role of CTSS-generated GSDMCN terin PGD2 release and the restoration of goblet cell hyperplasia in response to helminth infection, the partial restoration of type-2 immunity upon PGD2 signaling blockade — compared to the full restoration achieved with Rab7 inhibition — indicates that GSDMC can modulate other lipids or exert unknown lipid-independent functions that are Rab7-dependent and critical for type-2 immunity to helminths. In addition, GSDMCN tercan also modulate endocytosis / phagocytosis activity in lECs. For instance, lECs can engulf dead / dying cell corpses, which undergo phagosomelysosome flux and thus, involve Rab7. Therefore, GSDMCN tercan affect lECs to clear dead / dying cells by phagocytosis. Alternatively, GSDMCN termay affect Rab7+vesicles to indirectly modulate the secretion pathway, especially for certain cytokines, chemokines, and lipids.

[0453] A previous study found that GSDMC plays a protective role in Heligmosomoides (H.) polygyrus helminth infection and that this protection is mediated through GSDMC-dependent IL-33 release specific to goblet cells. Further, that study showed that the increased susceptibility of G mc-deficient mice could be rescued by giving mice recombinant IL-33. Although the presently disclosed data do not support that CTSS-processed GSDMCN terdirectly promotes IL-33 release at the plasma membrane, it is possible that the GSDMC-dependent IL- 33 release is specific to goblet cells (GSDMC is expressed in all lECs upon helminth infection), ACTIVE 131727896.1 ii072396.1120

[0454] PATENT

[0455] the infection model used, or specifically mediated by one of the four murine GSDMC proteins that are cleaved by another unknown protease. In that regard, a recent study showed that mast cell-derived proteases may contribute to GSDMC cleavage in the intestine using the H. polygyrus infection model. Hence, it is possible that mast cell-derived proteases indirectly facilitate the lEC-intrinsic, CTSS-mediated, direct GSDMC cleavage. Alternatively, mast cell proteases may promote GSDMC cleavage independent of CTSS; however, different from the present example, a direct proteolytic function for mast cell proteases on GSDMC using purified recombinant proteins has not been demonstrated. In line with these findings, a previous study found increased expression of GSDMC genes in helminth-infected mice; however, in stark contrast to a proposed correlation between GSDMC and pyroptosis in said study, the present disclosure has not found evidence for that claim. The fact that GSDMC cleavage can be promoted and sustained in mice colonized with the commensal protist T. arnold also argues against a role of GSDMC in pyroptosis.

[0456] While expression of GSDMC genes in small intestinal lECs is barely detectable in the absence of type-2 inducing microbes, GSDMC gene expression increases up to 100-fold during T. arnold colonization or helminth infections, resulting in simultaneous GSDMC cleavage. In contrast, in the colon GSDMC is potently expressed in the absence of IL-13 signaling and type-2 inducing microbes but requires T. arnold colonization to be cleaved. This observation highlights that lEC-specific genes are regulated in a tissue-specific context and warrant careful consideration when studying IEC biology in the gut. These findings suggest that GSDMC is tightly regulated and plays an important role in type-2 mediated IEC responses.

[0457] Overall, the present example demonstrates the role of GSDMC in regulating the gut immune system and its use in improving payload and cargo release in transfection systems.

[0458] Experimental Model and Subject Detads

[0459] Mice. All mice used in this example are on a C57BL / 6 background. .Gsdmcl-E^ mice were generated by pronuclear injection of Cas9 mRNA and two sgRNA (guide: Gsdmc- 5' GTTTCATTAGATAGTTGTCCTGG; guide: Gsdmc-3' TCCATTCAACGAACTGTTCCTGG) into fertilized C57BL / 6J zygotes at the Innovative Technologies Development Core at the Department of Immunology at the University of Pittsburgh School of Medicine. BALB / c-I14ratmlSz / J (IL4ra-deficient) mice were purchased from Jackson Laboratories and backcrossed to the C57BL / 6 background for 10 generations. B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) mice were purchased from Jackson Laboratories. Ctss^ mice were previously described. Both female and male mice were used for experiments; no notable sex-dependent differences were found for the reported experiments. Mice were housed ACTIVE 131727896.1 ii072396.1120

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[0461] under specific pathogen-free (SPF) conditions, where cages were changed on a weekly basis; ventilated cages, bedding, food, and water (non-acidified) were autoclaved before use, ambient temperature maintained at 23 C, and 5% Clidox-S was used as a disinfectant. Absence of Tritrichomonas in breeding and experimental mice was confirmed by qPCR. Experimental cages were randomly housed on two different racks in the vivarium and all cages were kept on automatic 12-h light / dark cycles. Germ-free C57BL / 6 WT mice were maintained in flexible film isolators.

[0462] Mouse helminth infection and protist colonization. Mice were infected subcutaneously with 500 N. brasiliensis stage 3 larvae (L3) and were euthanized at day 7 post infection to collect tissues. In some experiments, Gsdmcl-4^ mice received daily intraperitoneal injections of OC000459 (Cayman Chemical, 1 mg / kg body weight), CID 1067700 (Medchem Express, 16 mg / kg body weight), or a vehicle control for the duration of the experiment. Worm burden was enumerated across the entire small intestine at day 5 post infection. Wild-type mice naturally colonized with T. arnold were used as a source of protist. T. arnold were purified from cecal contents of colonized mice using low speed centrifugation, Percoll and antibiotics. T. arnold were orally gavaged into experimental mice. T. arnold colonization status was confirmed by qPCR .

[0463] Bone marrow chimeras. 6- to 8-week-old Ctss- / - mice and littermate controls were fullbody irradiated with 8 Gray (Gy) using an X-ray Precision SmART+ (225 kV) and injected intravenously with 107 WT CD45.1 bone marrow cells. Chimeric mice were infected with A. brasiliensis 6 weeks after irradiation.

[0464] Cell lines. HEK293T and HeLa cells were maintained at 37°C, 5% v / v CO2 in a humidified incubator in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 200 U / ml penicillin-streptomycin, and 50 pg / mL Plasmocin (Invivogen). For transient transfections, GSDMCs, Cathepsins, Rab7 and GFP / mCherry control encoding vectors or empty vectors were co-transfected into HEK293T or HeLa cells by Lipofectamine 2000 or TransIT-VirusGen.

[0465] Intestinal organoid cultures. Organoids were cultured in 50% L-WRN conditioned medial3 in the presence of lx B27. 10 pM ROCK inhibitor Y-27632 and 50 ng / ml mEGF were added at the start of intestinal organoid cultures and omitted upon the first medium change. Organoids were treated with 10 ng / ml IL-13 at the indicated times.

[0466] C. elegans transgenic strains and plasmid injections. C. elegans with transgenes Pcol-19-mKate2::RAB-7(zjuSil88) were grown in NGM plates at a temperature of 20°C. For injection, C. elegans strains were grown to the young adult stage and placed onto 4% agarose ACTIVE 131727896.1072396.1120

[0467] PATENT

[0468] pads inside halogen oil to minimize mobility. The plasmid injections were accomplished under an injection microscope (Nikon Eclipse Ti) and injected with a plasmid mix composed of 10 ng / pL gene of interest codon plasmid, 50 ng / pL co-injection marker, 50 ng / pL empty vector (pBluescript plasmid) for the extrachromosomal arrays.

[0469] RNA processing and qPCR. Small and large intestines were removed and transferred into cold PBS. A piece (~5 mm) of whole intestinal tissue (jejunum and colon) was soaked in RNAlater (Qiagen, 76106) at 4 °C for 48 h and then stored at -80 °C until further analysis. For RNA extraction a Tissue-Tearor Homogenizer (Biospec) was used. RNA was prepared using the RNeasy Mini Kit (Qiagen, 74136). cDNA synthesis was performed using iScriptTM (BioRad, 1708891BUN) according to the manufacturer’s instructions. Expression analysis was performed in duplicate via qPCR on a Bio-Rad CFX384 using iTaqTM SYBR (Bio-Rad, 1725125). Expression levels were quantified and normalized to Gapdh.

[0470] Antibodies and flow cytometry. Single-cell suspensions were pelleted and resuspended in FACS buffer (PBS, 2% FBS) for immunostaining and subsequent flow cytometry analysis. Cell suspensions were incubated with Fc Block prior to staining with fluorophore-conjugated monoclonal antibodies. All fluorophore-conjugated antibodies used are listed in Key Resources Table. Zombie NIR Fixable Viability Kit and Fixable Aqua Dead Cell Stain Kit were used to determine living cells. For analysis of transcription factors and cytokine expression cells were incubated in RPMI media in the presence of 50 ng / ml phorbol 12-myristate 13-acetate (PMA), 500 ng / ml ionomycin, 1.3 pl / ml Golgi Stop and 1 pl / ml Golgi Plug for 3 hours at 37 °C, 5% CO2. For intracellular staining, cells were permeabilized with the Foxp3 fixation / permeabilization kit. Flow cytometry analysis was performed on a Cytek Aurora (Cytek). Cell sorting was performed on a BD Aria IIU (BD Biosciences). Data was analyzed with FlowJo (Treestar).

[0471] Cell dissociation and isolation. Mesenteric lymph nodes (mLN) were dissected followed by digestion with 1 mg / ml collagenase VIII (Sigma) in a shaking incubator at 37 °C, 220rpm for 30 minutes. Cells were mashed through a 100 pm cell strainer to obtain a single cell suspension.

[0472] Western blotting of intestinal tissues and organoids. Intestinal organoids were lysed in Lysis buffer (137mM NaCl, 20mM Tris, ImM EDTA, ImM EGTA, 200pM Sodium orthovanadate, 10% Glycerol, 1% NP-40, 500pM DTT, and protease / phosphatase inhibitor), vortexed for 30 seconds, and tumbled for 30 minutes at 4 °C. Intestinal tissue pieces were flash frozen in liquid nitrogen, lysis buffer was added, and pieces were homogenized using a Tissue-Tearor and tumbled for 2 hours at 4 °C.. All samples were spun down at 17,000 x g for 10 ACTIVE 131727896.1 l072396.1120

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[0474] minutes to separate the lysate from the insoluble pellet. For western blotting, lysate samples were denatured by boiling for 5 minutes in lx Laemmli sample buffer. Cell lysates were subjected to reducing SDS-PAGE, dry transfer to nitrocellulose, and western blotted. Enhance chemiluminescence was used for antigen detection, and images analyzed using the Bio-Rad Image Lab system. Membranes were re-blotted with Actin as a loading control.

[0475] Histology for PAS (Periodic acid Schiff) and Hematoxylin / eosin staining. A 5mm piece of jejunum and colon were fixed in 10% formalin for 24 hours at RT, transferred to 70% ethanol, and processed for embedding in paraffin. PAS (jejunum) and hematoxylin and eosin (H&E, colon) staining was performed on 4 pm paraffin-embedded intestinal sections. Staining was performed by the Clinical Biospecimen Repository and Processing Core. PAS slides were digitized for representative sections on a Keyence BZX-810 widefield microscope.

[0476] Immunofluorescence staining of intestinal tissues. The jejunum and colon were removed, cut longitudinally, and pinned down onto wax. Swiss roles of the jejunum or colon were fixed in 4% PFA for 3 hours at 4 °C and dehydrated in 30% w / v sucrose overnight before being embedded in OCT and sectioned on a cryostat (Leica CM 1950). 10-12 pm thick sections were used for immunofluorescence staining. Slides were washed in blocking buffer (1% w / v bovine serum albumin, 0.1% Tween-20, lx PBS) for 10 minutes at RT. Sections were surrounded using a hydrophobic barrier (PAP pen, Thermo-Fi scher) and permeabilized by incubating tissue in permeabilization buffer (0.4% Triton-XlOO, 1% fetal bovine serum, lx PBS) for 10 minutes at RT twice. Sections were then incubated with primary antibodies in blocking buffer for 1 hour at RT or overnight at 4 °C, washed three times in PBS, and incubated with fluorophore-labelled secondary antibodies for 30 minutes at RT, and washed 3 times in PBS and stained with 5 pg / ml 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) for 5 minutes at RT. Slides were washed an additional three times in PBS and were then mounted in Fluoromount-G. Slides were stored light protected at 4 °C until digitized using a Nikon Ti inverted microscope equipped with a Nikon Al point scanning confocal scan head and a 1.40 N.A. 20x Objective or Keyence BZX-810 Widefield microscope equipped with an 0.75 N.A. lOx, 20x, and 40x objective.

[0477] Nile red staining and PGD2 measurements in intestinal organoids. Intestinal organoids derived from the colon were treated with 10 ng / ml IL-13 or PBS control for 72 hours. For Nile red staining organoid cultures were dissociated with TrypLE Express and incubated in a shaking incubator for 30 min at 37°C to obtain single cell suspension. Organoid cultures were washed in PBS and incubated with Zombie NIR for 10 min at RT, followed by Nile Red and EPCAM staining in Nile Red staining buffer for 30 min at 37°C. Lipid droplet (Nile red ACTIVE 131727896.1 ii072396.1120

[0478] PATENT

[0479] staining) mean fluorescence intensity (MFI) in live lECs was determined by flow cytometry. For PGD2 measurements organoid domes were washed 3 times for 5 minutes at 50 rpm. Organoids were stimulated with 400 pl of 500 ng / ml ionomycin in HBSS containing 1.26 mM CaCl for 30 minutes, 37 °C, at 50 rpm. Supernatants were harvested and assayed with the Prostaglandin D2 ELISA kit.

[0480] LD measurements using GFP-ADRP. HeLa cells were first transfected with LD marker pEFIRES-P-EGFP-ADRP (Addgene #87157, 200 ng) along with either GSDMCN-ter (1-289aa, IxN-flag-tagged, 1800 ng) or a control plasmid (pBabe, 1800 ng) for 24 hours. Subsequently, cells were cultured with or without 10% FBS for an additional 48 hours. Cells were then observed using the Lionheart microscope. In all groups, the ratio of cells displaying EGFP-ADRP puncta (dotted GFP-ADRP) to the total number of GFP+ cells were quantified. A total of 3 independent experiments were conducted, with approximately 800 cells counted per experiment.

[0481] mGSDMC2 Oligomerization assays. HEK293T cells were transfected with pEGFP-N2-Murine Gsdmc2 (lx C-GFP tagged, N-ter,l-286aa), pEGFP-N2-Murine Gsdmc4 (lx C-GFP tagged, N-ter,l-288aa), and their L-inserted plasmid versions for 24 hours. Cell proteins were extracted using RIP A buffer, and the proteins were subjected to native-PAGE (Thermo Fisher XP04200BOX, without SDS) followed by Western blotting using an anti-GFP antibody.

[0482] CTSS-mediated GSDMC cleavage. To test the protease activity on GSDMC processing, multiple Cathepsins were co-transfected into HEK293T cells via lipofectamine 2000. 36-48 hours post transfection, cells were collected and lysed with PBS + 0.5% Triton X-100. After 8,000 rpm centrifugation for 2 min and quantification (BCA Protein Assay), cell lysates were subjected to SDS-PAGE and dry western blot (iBlot2 system, Invitrogen). Various antibodies, including anti-HA, anti-mCherry, and anti-actin were used as primary antibody. For in vitro cleavage, 1 pg of CTSS and GSDMC were incubated in reaction buffer containing 25 mM Tris (pH=7.5) and 50 ug / ml dextran sulfate sodium at 37 °C for 30 min to 3 hours. Reactions were stopped by adding loading buffer for SDS-PAGE analysis and Gelcode Blue staining.

[0483] CTSS-mediated GSDMCN tertargeting of Rab7+ vesicles. To determine the subcellular localization of GSDMCN-ter in HeLa cells, RFP-tagged (N-terminus) GSDMCN-ter, full length GSDMC (control), and GFP-Rab7 were co-transfected into HeLa cells using lipofectamine 2000. 36-48 hours post transfection, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10~20 min. After mounting on the slides, images were taken with a Nikon AX-R scanning confocal microscope. To determine the subcellular ACTIVE 131727896.1 i072396.1120

[0484] PATENT

[0485] localization of GSDMCN terin C. elegans, worms were injected with either GFP-tagged (N-terminus) GSDMCN ter, or GFP-tagged (C -terminus) GSDMCN terplasmids. Young adult stage worms were used for live imaging experiments. Prior to imaging, worms were picked and immobilized on 4% agarose pads using a small drop of 12 mM levamisole. Worms were mounted to Nikon Eclipse Ni and Z-stack images were acquired with a 100* magnification using a Nikon Plan Apo 100 X / 1.40 oil microscope. A 488 nm laser and a 525 / 50 filter were used to detect the GFP signal, and a 561 nm laser and a 620 / 60 filter were used to detect the mKate2 signal. All cluster analyses were performed in ImageJ. Background intensity was used as a threshold. Cluster size was measured using Analyze Particles (a function in ImageJ software) with a minimum size set at 0.075 square micrometers. To measure the integrity of GSDMCN tertargeted Rab7+ vesicles, GSDMC or GSDMCN terplasmids were co-transfected into HEK293T cells with mCherry plasmid with a 20:1 ratio. At 48 hours post-transfection, cells were imaged with a BioTek Lionheart microscope for mCherry and phase contrast. Cells were then lysed as described above and blotted with anti-mCherry and anti-Actin antibodies. In a separate experiment, GSDMC or GSDMCN-ter plasmids (with Amp+ resistance) were cotransfected into HEK293T cells with Kan+ resistance plasmid at a 1:1 ratio. At 48 hours posttransfection, cells were lysed, and the nucleic fractions were enriched by 8,000 rpm centrifugation for 2 min. The DNA from the nucleic fraction was extracted by Zymo Research Corporation Quick-DNATM Miniprep Plus Kit, which contained both the genomic DNA from HEK293T cells (no antibiotics resistance) and the transfected plasmids released from the endosome and relocated into the nuclei (with either Amp+ or Kan+ resistance). Isolated DNAs were transformed into E. coli competent cells (NEB-lObeta), and the transformants were counted after 18 hours.

[0486] Cell lysis measurements. To measure cell lysis, CytoTox-Glo (Promega) was added into the extracellular media with HEK293T cells (with or without various transfections) in the wells from a tissue culture multi -well plate and measured according to the manufacturer’s protocol. 100% of cell lysis was calculated from HEK293T cells under the same culture conditions and then added with up to 1% Triton X-100. To measure cell lysis in intestinal organoids, culture media was removed from intestinal organoids and washed in PBS. A staining solution of 10 pg / ml propidium iodide and 10 pg / ml Hoechst 33342 in PBS was added to the organoids and incubated for 30 min at 37°C, 5% v / v CO2 in a humidified incubator. The staining solution was removed, and the organoids were washed with PBS three times. The plate was read on a Spectramax i3 (Molecular Devices) set at the following parameters: Fluorescence read from the top, propidium iodide excitation at 535 nm and emission at 617 ACTIVE 131727896.1072396.1120

[0487] PATENT

[0488] nm, Hoechst excitation at 361 nm and emission at 486 nm. Whole domes were imaged with the EVOS M7000 microscope set at brightfield 4x magnification. Images of 12 connected sections were taken and stitched together.

[0489] Mass spectrometry analysis. CTSS-cleaved GSDMCCter was enriched and sent for in gel protein ID. To enrich the GSDMCcter, HA C-tagged GSDMC (human) was co-transfected with CTSS followed by HA immunoprecipitation. HA beads were eluted with HA peptides and subject to SDS-PAGE. The PAGE gel was stained with Gelcode Blue to reveal the cleaved bands matching the size of the GSDMCcterfragments. These bands were excised from the gel and subjected to downstream mass spectrometry analysis. After the stained region was excised, it was washed with HPLC water, and destained with 50% acetonitrile (ACN) / 25mM ammonium bicarbonate until no visible staining remained. Gel pieces were dehydrated with 100% ACN, reduced with lOmM dithiothreitol (DTT) at 56°C for 1 hour, followed by alkylation with 55mM iodoacetamide (IAA) at room temperature for 45min in the dark. Gel pieces were then dehydrated with 100% ACN to remove excess DTT and IAA and then incubated with 20 ng / pl trypsin in 25 mM ammonium bicarbonate overnight at 37°C. The resultant tryptic peptides were extracted with 70% ACN / 5% formic acid (FA), vacuum dried, and re-constituted in 20 pl 3%ACN / 0.1% FA.

[0490] Mass spectrometry analysis was conducted on a Bruker timsTOF Pro2 coupled to a NanoElute. Approximately 200 pg of peptide was loaded onto an Aurora Ultimate C 18 column (1.7 pm, 25 cm x 75 pm, lonOpticks) and eluted at 300 nl / min over a 60-minute gradient (mobile phase A (MPA): 0.1% FA; mobile phase B (MPB): 0.1% FA in acetonitrile). A linear gradient of 2-35% MPB was applied for 60 min, followed by a 5 min wash at 95% MFB before equilibrating the column at 2% MFB for 6 min. The timsTOF Pro2 was set to PASEF scan mode and DDA with a scan range of 100 - 1700 m / z with 10 PASEF ramps per 1.1 sec cycle. The TIMS was set to a 100 ms ramp and accumulation time (100% duty cycle) with a ramp rate of 9.43 Hz. Linear precursor repetitions were set to a target intensity of 20,000 and a target threshold of 2500 and active exclusion was set to 0.40 min. Collision energy was set to a base of 1.60 1 / K0 [V-s / cm2] at 59 eV and 0.60 1 / K0 [V-s / cm2] at 20 eV. An isolation width was set to 2 m / z for <700 m / z and 3 m / z for >800 m / z. The collected MS data were analyzed using MSFragger V3.845 against the Gasdermin-C human protein sequence from SwissProt. The search parameters were set as follows: strict trypsin digestion, nonspecific cleavage, missing cleavage up to 2, carbamidomethylation of cysteine as static modification, oxidization of methionine and protein N-terminal acetylation as variable modification, a maximal mass

[0491] ACTIVE 131727896.1072396.1120

[0492] PATENT

[0493] tolerance of 15 ppm for the precursor ions and 20ppm for the fragment ions, and false detection rate (FDR) was set to be 1%.

[0494] Statistical Analysis. Mice were allocated to experimental groups based on their genotype and randomized within the given sex- and age-matched groups. Similar variance between the different experimental groups was assumed. All experimental and control animals were littermates, and none were excluded from the analysis at the time of collection. The number of mice per group is described in the corresponding figure legends as n and all quantitative data are presented as mean + / - standard error of the mean (s.e.m.), unless otherwise indicated. Data were analyzed using a paired or unpaired two-tailed Student’s t-test or Mann-Whitney test for single comparisons, and one-way or two-way ANOVA for multiple comparisons unless otherwise indicated. ANOVA analysis was followed by a Sidak’s post hoc test. Figures and statistical analysis were generated using GraphPad Prism 8 (GraphPad Software). The statistical test used, and P values are indicated in each figure legend. P values of <0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***p < 0.001 and 0.0001.

[0495] References

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[0536] Yang, L., He, H., Guo, X.K., Wang, J., Wang, W., Li, D., Liang, S., Shao, F., Liu, W., and Hu, X. (2024). Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity. Immunity . 10.1016 / j .immuni .2024.03.017.

[0537] Example 3

[0538] The above examples demonstrate that the cleaved (active) form of human Gasdermin C (GSDMC) N-terminus (GSDMCNter) can target and penetrate Rab7+late endosomes, facilitating cargo release. The inventors of the present disclosure further studied the ability of the presently disclosed tagged GSDMCNterto facilitated nanoparticle liposome-mRNA delivery mediated protein expression in vivo. Initially, the presently disclosed tagged GSDMCNterwas confirmed to disrupt RAB7-vesicles (see Figure 15). Next, in vivo experiments were performed. Briefly, mice were injected with nanoparticles containing mRNA encoding luciferase and the presently disclosed tagged GSDMCNter. As illustrated in Figure 26, the presently disclosed tagged GSDMCNterdelivered within 1 day (after i.v. injection) the payload mRNA to the spleen. These data confirm that the presently disclosed tagged GSDMCNtercan increase the delivery efficiency in vivo any desired payload (e.g., antigen) without undesired side effects (e.g., pyroptosis).

[0539] * * *

[0540] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily

[0541] ACTIVE 131727896.1 81072396.1120

[0542] PATENT

[0543] appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0544] For any patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of all of which are incorporated herein by reference in their entireties for all purposes.

[0545] ACTIVE 131727896.1

Claims

072396.1120PATENT CLAIMS1. A lipid-based carrier comprising a polynucleotide encoding a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide.

2. The lipid-based carrier of claim 1, wherein the carrier is a lipid nanoparticle, a liposome, a lipoplex, or a nanoliposome.

3. The lipid-based carrier of claim 1 or 2, wherein the carrier is a lipid nanoparticle.

4. The lipid-based carrier of any one of claims 1-3, wherein the carrier comprises a cation lipid, a non-cationic lipid, a structural lipid, a PEG-modified lipid, or a combination thereof.

5. The lipid-based carrier of any one of claims 1-4, wherein the GSDMC polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 9.

6. The lipid-based carrier of any one of claims 1-5, wherein the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 9.

7. The lipid-based carrier of any one of claims 1-6, wherein the GSDMC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

8. The lipid-based carrier of any one of claims 1-7, wherein the tag polypeptide comprises a FLAG polypeptide, a polyhistidine (His) tag polypeptide, a hemagglutinin (HA) tag polypeptide, a Myc tag polypeptide, a Strep-tag polypeptide, a glutathione S-transferase (GST) tag polypeptide, a calmodulin-binding protein (CBM) polypeptide, a maltose-binding protein (MBP) polypeptide, a T7 tag polypeptide, a V5 tag polypeptide, a green fluorescent protein (GFP) polypeptide, a blue fluorescent protein (BFP) polypeptide, a cyan fluorescentACTIVE 131727896.1 83072396.1120PATENTprotein (CFP) polypeptide, a yellow fluorescent protein (YFP) polypeptide, a FMN-binding fluorescent protein (FbFP) polypeptide, or a red fluorescent protein (RFP) polypeptide.

9. The lipid-based carrier of any one of claims 1-8, wherein the tag polypeptide comprises a FLAG polypeptide.

10. The lipid-based carrier of claim 9, wherein the tag polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5.

11. The lipid-based carrier of claim 9 or 10, wherein the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

12. The lipid-based carrier of any one of claims 1-8, wherein the tag polypeptide comprises an RFP polypeptide.

13. The lipid-based carrier of claim 12, wherein the tag polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 6.

14. The lipid-based carrier of claim 12 or 13, wherein the tag polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.

15. The lipid-based carrier of any one of claims 1-14, wherein the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are connected by a linker.

16. The lipid-based carrier of claim 15, wherein the linker comprises the amino sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

17. The lipid-based carrier of any one of claims 1-14, wherein the Gasdermin C (GSDMC) polypeptide and the tag polypeptide are connected without a linker.ACTIVE 131727896.1072396.1120PATENT18. The lipid-based carrier of any one of claims 1-17, wherein the fusion protein comprises, from N-end to C-end, the tag polypeptide and Gasdermin C (GSDMC) polypeptide.

19. The lipid-based carrier of any one of claims 1-17, wherein the fusion protein comprises, from N-end to C-end, Gasdermin C (GSDMC) polypeptide and the tag polypeptide.

20. The lipid-based carrier of any one of claims 1-14, 17, and 18, wherein the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7.

21. The lipid-based carrier of claim 20, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.

22. The lipid-based carrier of any one of claims 1-14, 17, and 18, wherein the fusion polypeptide comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 8.

23. The lipid-based carrier of claim 22, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8.

24. The lipid-based carrier of any one of claims 1-23, further comprising a second polynucleotide.

25. The lipid-based carrier of claim 24, wherein the second polynucleotide encodes a payload.

26. The lipid-based carrier of claim 24, wherein the second polynucleotide encodes an antigenic polypeptide derived from a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell.ACTIVE 131727896.1 s072396.1120PATENT27. The lipid-based carrier of claim 24, wherein the second polynucleotide encodes a tumor antigen.

28. The lipid-based carrier of claim 27, wherein the tumor antigen is selected from gplOO, MART-l / Melan A, gp75 (TRP-I), tyrosinase, NY-ESO-I, melanoma proteoglycan, MAGE family antigens (i.e., MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, and MAGE-12), BAGE family antigens, GAGE family antigens (i.e., GAGE-1, GAGE-2), RAGE family antigens, N- acetylglucosaminyltransferase-V, pl 5, P-catenin, MUM-I, cyclin dependent kinase-4 (CDK4), p21-ras, BCR- abl, p53, pl85 HER2 / neu, epidermal growth factor receptor (EGFR), carcinoembryonic antigens (CEA); carcinoma- associated mutated mucins (i.e., MUC-1 gene products); EBNA gene products of EBV (i.e., EBNA-I); E7, E6 proteins of human papillomavirus; prostate specific antigen (PSA); prostate specific membrane antigen (PSMA); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes; KSA, kinesin 2, HIP-55, TGFP-1 anti- apoptotic factor, tumor protein D52, HIFT, Kras, Histone, NY-BR-I, NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96.

29. The lipid-based carrier of claim 24, wherein the second polynucleotide encodes a therapeutic protein.

30. The lipid-based carrier of claim 29, wherein the therapeutic protein is selected from transforming growth factor-beta (TGF-beta), interferon-alpha, interferon-beta, interferongamma, granulocyte colony stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), interleukin- 1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin- 10, interleukin- 12, interleukin- 13, interleukin- 15, interleukin- 17, interleukin- 18, interleukin-22, interleukin-23, interleukin-35, amylin, anti-Miillerian hormone, calcitonin, cholecystokinin, corticotropin, endothelin, enkephalin, erythropoietin (EPO), follicle-stimulating hormone, gallanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human growth hormone (hGH), inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, luteinizing hormone releasing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, GLP-1, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, vasoactive intestinal peptide, vasopressin, trastuzumab emtansine, brentuximab vedotin, T-ACTIVE 131727896.1072396.1120PATENT DM1, polyvalent IgG2a Fc (M045), SMN1, coagulation factors (e.g., F8 or F9), hemoglobin, ARSA, ABCD1, DDC, ADA, RPE65, ASP A, ARG1, DMD, C0L7A1, BCL11A, cas9, WAS, HTT, CTNS, CPS1, OTOF, IDS, TTR, LAMP2, UGT1A1, PAH, KLKB1, ATP7B, GAN, agalsidase beta, imiglucerase, velaglucerase alfa, taliglucerase, alglucosidase alfa, laronidase, idursulfase, galsulfase, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, altumomab pentetate, amatuximab, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, cantuzumab ravtansine, capromab pendetide, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, dacetuzumab, demcizumab, detumomab, drozitumab, ecromeximab, eculizumab, elotuzumab, ensituximab, epratuzumab, etaracizumab, farletuzumab, figitumumab, flanvotumab, galiximab, gemtuzumab ozogamicin, girentuximab, ibritumomab tiuxetan, imgatuzumab, ipilimumab, labetuzumab, lexatumumab, lorvotuzumab mertansine, nimotuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, tacatuzumab tetraxetan, tositumomab, trastuzumab, totumumab, and zalutumumab.

31. A composition comprising the lipid-based carrier of any one of claims 1-30.

32. A composition comprising a first lipid-based carrier comprising a polynucleotide encoding a fusion polypeptide comprising a Gasdermin C (GSDMC) polypeptide and a tag polypeptide, and a second lipid-based carrier comprising a second polynucleotide.

33. The composition of claim 31 or 32, which is a pharmaceutical composition comprising a pharmaceutical acceptable excipient.

34. A method of treating a disease in a subject in need thereof, the method comprising administering an effective amount of the lipid-based carrier of any one of claims 1-30 or the composition of any one of claims 31-33.

35. A method of treating a cancer in a subject in need thereof, the method comprising administering an effective amount of the lipid-based carrier of any one of claims 1-30 or the composition of any one of claims 31-33.ACTIVE 131727896.1 87072396.1120PATENT36. The method of claim 35, wherein the cancer is selected from an adrenal cancer, a breast cancer, a colon cancer, a leukemia, a bile duct cancer, a bone cancer, a lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor), a bladder cancer, a brain cancer, a bronchial cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, an ependymoma, a retinoblastoma, a gallbladder cancer, a gastric cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, a heart cancer, a liver cancer, a pancreatic cancer, a melanoma, a kidney cancer, a laryngeal cancer, a lip or oral cancer, a lymphoma, a mesothelioma, a mouth cancer, a myeloma, a nasopharyngeal cancer, a neuroblastoma, an oropharyngeal cancer, an ovarian cancer, a thyroid cancer, a penile cancer, a pituitary cancer, a prostate cancer, a rectal cancer, a renal cancer, a salivary gland cancer, a sarcoma, a skin cancer, a stomach cancer, a testicular cancer, a throat cancer, a uterine cancer, a vaginal cancer, and a vulvar cancer.

37. The method of any one of claims 34-36, wherein the subject is human.

38. The lipid-based carrier of any one of claims 1-30 or the composition of any one of claims 31-33 for use in treating a disease.

39. The lipid-based carrier of any one of claims 1-30 or the composition of any one of claims 31-33 for use in treating a cancer.

40. The lipid-based carrier or the composition for use of claim 39, wherein the cancer is selected from an adrenal cancer, a breast cancer, a colon cancer, a leukemia, a bile duct cancer, a bone cancer, a lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and lung carcinoid tumor), a bladder cancer, a brain cancer, a bronchial cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, an ependymoma, a retinoblastoma, a gallbladder cancer, a gastric cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, a heart cancer, a liver cancer, a pancreatic cancer, a melanoma, a kidney cancer, a laryngeal cancer, a lip or oral cancer, a lymphoma, a mesothelioma, a mouth cancer, a myeloma, a nasopharyngeal cancer, a neuroblastoma, an oropharyngeal cancer, an ovarian cancer, a thyroid cancer, a penile cancer, a pituitary cancer, a prostate cancer, a rectal cancer, a renal cancer, a salivary gland cancer, a sarcoma, a skin cancer, a stomach cancer, a testicular cancer, a throat cancer, a uterine cancer, a vaginal cancer, and a vulvar cancer.ACTIVE 131727896.1 88072396.1120PATENT41. A kit comprising the lipid-based carrier of any one of claims 1-30 or the composition of any one of claims 31-33.

42. The kit of claim 41 for use in culturing and transfecting a cell.

43. A method of delivering a payload to a cell, comprising contacting the cell with an effective amount of the lipid-based carrier of any one of claims 1-30, the composition of any one of claims 31-33, or the kit of claim 41 or 42.

44. A method of increasing the transfection efficiency of a payload into a cell, comprising contacting the cell with an effective amount of the lipid-based carrier of any one of claims 1-30, the composition of any one of claims 31-33, or the kit of claim 41 or 42.ACTIVE 131727896.1 81

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