Chimeric tumor necrosis factor receptor 1 compositions and methods of use thereof

Abstract

Disclosed are compositions comprising dominant negative chimeric tumor necrosis factor receptor 1 (TNFR1) fusion proteins and methods for use in cell therapies.

Description

CHIMERIC TUMOR NECROSIS FACTOR RECEPTOR 1 COMPOSITIONS AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No.63 / 634,636, filed on April 16, 2024. The contents of each of the aforementioned patent applications are incorporated herein by reference in their entireties. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The contents of the file named “POTH-095_001WO_SequenceListing_ST26”, which was created on April 8, 2025, and is 46,884 bytes in size, are hereby incorporated by reference in their entirety. FIELD

[0003] The disclosure is directed to molecular biology, and more specifically, to compositions comprising dominant negative chimeric tumor necrosis factor receptor 1 (TNFR1) fusion proteins and methods for use in cell therapies. BACKGROUND

[0004] The discovery of cell-based therapies capable of recognizing and binding to a specific target protein with high affinity and avidity has been a focus of the biopharmaceutical industry. There remains a need for more efficacious cell-based therapies that demonstrate reduced in vivo immunotoxicity compared to traditional cell-based therapies. SUMMARY

[0005] Provided herein are compositions comprising chimeric Tumor Necrosis Factor Receptor 1 (TNFR1) Fusion Proteins, in particular, TNFR1 Fusion Proteins comprising a TNFR1 extracellular domain, a TNFR1 transmembrane domain, and an intracellular signalingdomain, and methods of using such compositions to reduce TNF -mediated cytokine levelsand transaminase expression in a cell.

[0006] In certain aspects, provided are compositions comprising a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, comprising: (a) a TNFR1 extracellular domain; (b) a TNFR1 transmembrane domain; and (c) an insulin-like growth factor receptor 1 (IGFR1) intracellular signaling domain. In certain embodiments, the TNFR1 extracellular domain comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the TNFR1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the TNFR1 extracellular domain and TNFR1 transmembrane domain are comprised in amino acids 1-234 of the TNFR1 coding sequence set forth in SEQ ID NO: 7. In certain embodiments, IGFR1 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the chimeric TNFR1-IGFR1 fusion protein comprises the amino acid sequence of SEQ ID NO: 12.

[0007] In certain aspects, provided are compositions comprising a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, comprising: (a) a TNFR1 extracellular domain; (b) a TNFR1 transmembrane domain; (c) a c-MET intracellular signaling domain. In certain embodiments, the TNFR1 extracellular domain comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the TNFR1 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the TNFR1 extracellular domain and TNFR1 transmembrane domain are comprised in amino acids 1-234 of the TNFR1 coding sequence set forth in SEQ ID NO: 7. In certain embodiments, c-MET intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the chimeric TNFR1-c-MET fusion protein comprises the amino acid sequence of SEQ ID NO: 13.

[0008] In certain aspects, provided are polynucleotides encoding the chimeric TNFR1- IGFR1 or TGFR1-c-MET fusion proteins. In certain embodiments, the polynucleotide encoding the chimeric TNFR1-IGFR1 fusion protein comprises the nucleic acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the polynucleotide encoding the chimeric TNFR1-IGFR1 fusion protein comprises the nucleic acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the polynucleotide encoding the chimeric TNFR1-IGFR1 fusion protein comprises the nucleic acid sequence set forth in SEQ ID NO: 16. In certain embodiments, polynucleotides encoding the chimeric TNFR1-c-MET fusion protein include the nucleic acid sequence set forth in SEQ ID NO: 17. In certain embodiments, polynucleotides encoding the chimeric TNFR1-c-MET fusion protein include the nucleic acid sequence set forth in SEQ ID NO: 18. In certain embodiments, polynucleotides encoding the chimeric TNFR1-c-MET fusion protein include the nucleic acid sequence set forth in SEQ IDNO: 19. In certain embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is RNA. In certain embodiments, the RNA is mRNA. In certain embodiments, the mRNA comprises a 5’-CAP. In certain embodiments, 5’-CAP is a 5’ CleanCap®.

[0009] In certain aspects, provided are vectors comprising the polynucleotides encoding TNFR1-IGFR1 or TGFR1-c-MET fusion proteins.

[0010] In certain aspects, provided are compositions comprising the polynucleotides encoding TNFR1-IGFR1 or TGFR1-c-MET fusion proteins.

[0011] In certain aspects, a composition comprising a polynucleotide is encapsulated in at least one lipid nanoparticle composition (LNP). In some embodiments, the LNP comprises at least one RNA molecule and / or at least one DNA molecule. In some embodiments, the LNP comprises about 40% of HMA-404 by moles, about 52.5% of cholesterol by moles, about 5% of DOPC by moles, about 2% of DMG-PEG2000 by moles, and about 0.5 mol% of a targeting ligand comprising GalNac, wherein the at least one RNA molecule comprises a polynucleotide encoding a chimeric TNFR1 fusion protein, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w), and wherein HMA-404 comprises the following structure:In certain embodiments, the RNA molecule is an mRNA molecule, preferably wherein the mRNA molecule further comprises a 5’-CAP. In certain embodiments, the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, preferably wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a helitron transposase, a Tol2 transposase, a TcBuster transposase or a mutant TcBuster transposase. In certain embodiments, the DNA molecule is a circular DNA molecule, DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule. In certain embodiments, the at least one DNA molecule comprises a nucleic acid sequence encoding at least onetransposon. In certain embodiments, the LNP comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding at least one therapeutic protein. In certain embodiments, the LNP comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding at least one transposon, wherein the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.

[0012] In certain aspects, provided are pharmaceutical compositions, comprising a composition of the present disclosure and at least one pharmaceutically-acceptable excipient or diluent.

[0013] In certain aspects, provided are methods of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure. In some embodiments, the at least one cell is a liver cell. In some embodiments, the liver cell is a hepatocyte, a hepatic stellate cell, Kupffer cell or liver sinusoidal endothelial cell.

[0014] In some aspects, provided are methods of genetically modifying at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure. In some embodiments, the at least one cell is a liver cell. In some embodiments, the liver cell is a hepatocyte, a hepatic stellate cell, Kupffer cell or liver sinusoidal endothelial cell.

[0015] In some aspects, provided are methods of treating at least one disease or disorder in a subject in need thereof comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure. In some embodiments, the at least one disease or disorder is a liver disease or disorder.

[0016] In certain aspects, provided are methods for reducing TNF -mediated cytokinelevels in a cell, comprising contacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein,wherein the TNF -mediated cytokine levels in the cell are reduced at least 2-fold comparedto TNF -mediated cytokine levels in a cell that is contacted with a composition that does notcomprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein. In certain embodiments, the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a fusion protein of the present disclosure. In certain embodiments, the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a polynucleotide of the present disclosure. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte, a hepatic stellate cell, Kupffer cell or liver sinusoidal endothelial cell. In some embodiments, a composition comprising apolynucleotide is encapsulated in at least one lipid nanoparticle composition (LNP), for example in an LNP of the present disclosure.

[0017] In certain aspects, provided are methods for reducing transaminase expression in a cell, comprising contacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, wherein the transaminase expression in the cell is reduced at least 2-fold compared to transaminase expression in a cell that is contacted with a composition that does not comprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein. In certain embodiments, the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a fusion protein of the present disclosure. In certain embodiments, the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a polynucleotide of the present disclosure. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte, a hepatic stellate cell, Kupffer cell or liver sinusoidal endothelial cell. In some embodiments, a composition comprising a polynucleotide is encapsulated in at least one lipid nanoparticle composition (LNP), for example in an LNP of the present disclosure.

[0018] Any of the aspects and / or embodiments described herein can be combined with any other aspect and / or embodiment described herein.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0020] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited hereinare not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGs.1A-1B are images of Western Blots of total mRNA isolated from huh7 hepatocytes transfected with increasing concentrations of an mRNA encoding a chimeric TNFR1-IGFR receptor (FIG.1A) fusion protein or an mRNA encoding a chimeric TNFR1- cMET receptor (FIG.1B) fusion protein detected using an anti-IGFR, an anti-c-MET or an anti-p65 primary antibody. Dose-dependent increases in TNFR1-IGFR or TNFR1-c-MET expression were observed with increasing mRNA concentrations. In addition, in the presenceof tumor necrosis factor alpha (TNF ), a concomitant dominant negative reduction in pro-apoptotic signaling was observed as shown by decreased levels of phosphorylated p65 (indicated by arrows).

[0022] FIGs.2A-2C show the experimental design for inducing pro-survival signaling of chimeric TNFR1 fusion proteins expressed in Hepa1-6 cells (FIG.2A). FIGs.2B and 2C are images of Western Blots of total mRNA isolated from Hepa1-6 cells (control) or Hepa1-6 cells expressing either a chimeric TNFR1-IGFR1 fusion protein (FIG.2B) or a chimeric TNFR1-c-MET fusion protein (FIG.2C) using an anti-IGFR, an anti-c-MET, an anti-p56 or an anti-AKT primary antibody. Increased phosphorylation of the pro-survival markers AKTand p56 in the presence of TNF was observed in the presence, but not the absence of, thechimeric TNFR1 fusion protein.

[0023] All documents cited herein, including any cross referenced or related patent or application are hereby incorporated herein by reference in its entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.DETAILED DESCRIPTION

[0024] The present disclosure relates to chimeric tumor necrosis factor receptor 1 (TNFR1) fusion proteins, cells and / or compositions comprising the same. In certain embodiments, the chimeric TNFR1 fusion proteins of the present disclosure compete with the endogenousTNFR1 cellular receptors for TNF binding, thereby reducing undesired TNF -mediated pro-apoptotic cell signaling, TNF -mediated cytokine induction, and TNF -mediatedtransaminase expression. In certain embodiments, binding of TNF to the chimeric TNFR1fusion proteins of the present disclosure promotes pro-survival cell signaling through the Insulin Growth Factor Receptor 1 (IGFR1) or c-MET intracellular signaling domains resulting in a desired dominant negative effect on cells expressing said receptors (e.g., liver cells). In some embodiments, the chimeric TNFR1 fusion proteins of the present disclosures can be used for cell therapies. Chimeric Tumor Necrosis Factor Receptor 1 (TNFR1) Fusion Proteins A. Chimeric TNFR1-Insulin Growth Factor Receptor 1 (IGFR1)

[0025] In certain embodiments, compositions of the disclosure comprise chimeric TNFR1- IGFR1 fusion proteins. In certain embodiments, the chimeric TNFR1-IGFR1 fusion protein comprises: (a) a TNFR1 extracellular domain; (b) a TNFR1 transmembrane domain; and (c) an IGFR1 intracellular signaling domain.

[0026] In certain embodiments, the amino acid sequence of the TNFR1 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 8: MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICC TKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISS CTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAG FFseqLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTT (SEQ ID NO: 8).

[0027] In certain embodiments, the amino acid sequence of the TNFR1 extracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8.

[0028] In certain aspects, the amino acid sequence of the TNFR1 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 9: VLLPLVIFFGLCLLSLLFIGL (SEQ ID NO: 9).

[0029] In certain embodiments, the amino acid sequence of the TNFR1 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9.

[0030] In certain embodiments, the coding sequence for the IGFR1 intracellular signaling domain comprises amino acids 930-1337 of the IGFR1 coding sequence. In some embodiments, the IGFR1 intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 10: RKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYE GVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQ GQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNA NKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESL KDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDM LFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPEN MESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNE RALPLPQSSTC (SEQ ID NO: 10).

[0031] In certain embodiments, the amino acid sequence encoding the IGFR1 signaling domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10.

[0032] In certain embodiments, the coding sequence for the TNFR1 extracellular domain and the TNFR1 transmembrane domain comprise amino acids 1-234 of the TNFR1 coding sequence. In some embodiments, the TNFR1 extracellular domain and transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 7: MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICC TKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISS CTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAG FFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIG LMY (SEQ ID NO: 7).

[0033] In certain embodiments, the amino acid sequence encoding the TNFR1 extracellular domain and TNFR1 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7.

[0034] In certain aspects, the amino acid sequence of the TNFR1-IGFR1 fusion protein comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 12:MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICC TKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISS CTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAG FFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIG LMYRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGM VYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGV VSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAY LNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMS PESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDN CPDMLFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELD LEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNG GRKNERALPLPQSSTC* (SEQ ID NO: 12).

[0035] In certain embodiments, the amino acid sequence of the chimeric TNFR1-IGFR1 fusion protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 12.

[0036] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1-IGFR1 fusion protein include the nucleic acid sequence (the mRNA start codon is highlighted in bold and italicized font, and the polyA sequence is shown in lower case letters): AGGAGACAAGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGAC ACCGCCACCATGGGCCTGAGCACAGTTCCTGATCTGCTGCTGCCTCTGGTGCTGCTGGAA CTGCTCGTGGGCATCTATCCTAGCGGCGTGATCGGACTGGTGCCTCACCTGGGCGATAGAG AAAAGCGGGATAGCGTGTGCCCTCAGGGCAAGTACATTCACCCTCAGAACAACAGCATCT GCTGCACCAAGTGCCACAAGGGCACCTACCTGTACAACGACTGTCCTGGACCAGGCCAG GATACCGACTGTAGAGAGTGTGAAAGCGGCAGCTTCACCGCCAGCGAGAACCACCTGAG ACACTGCCTGAGCTGCAGCAAGTGTCGGAAAGAGATGGGCCAAGTCGAGATCAGCAGCT GCACCGTGGACAGAGATACCGTGTGCGGCTGCCGGAAGAACCAGTACAGACACTATTGGA GCGAGAATCTGTTCCAGTGCTTCAACTGCAGCCTGTGCCTGAACGGCACAGTGCACCTGT CCTGCCAAGAGAAGCAGAACACCGTGTGTACCTGCCACGCCGGATTCTTTCTGCGCGAGA ATGAGTGCGTGTCCTGCAGCAACTGCAAGAAAAGCCTGGAATGCACCAAGCTGTGCCTGC CTCAGATCGAGAACGTGAAGGGCACAGAGGATAGCGGCACCACAGTGCTGCTCCCTCTCG TGATCTTCTTCGGCCTGTGTCTGCTGAGCCTGCTGTTCATCGGCCTGATGTACCGGAAGAG AAACAACAGCCGGCTCGGCAACGGCGTGCTGTACGCTTCTGTGAACCCCGAGTACTTCTC TGCCGCCGATGTGTACGTGCCCGACGAATGGGAAGTCGCCCGGGAAAAGATCACCATGAG CAGAGAACTCGGCCAGGGCAGCTTCGGCATGGTGTATGAAGGCGTGGCCAAGGGCGTCG TGAAGGACGAGCCTGAAACAAGAGTGGCCATCAAGACCGTGAACGAGGCCGCCAGCATG AGAGAGAGAATCGAGTTCCTGAATGAGGCCAGCGTGATGAAGGAATTCAACTGCCACCA CGTCGTCCGGCTGCTGGGAGTTGTGTCTCAAGGACAGCCCACACTGGTCATCATGGAACT GATGACACGGGGCGACCTGAAGTCCTACCTGAGATCTCTGCGGCCCGAGATGGAAAACAA CCCTGTGCTGGCTCCTCCTAGCCTGAGCAAGATGATTCAGATGGCCGGCGAGATCGCCGA CGGCATGGCTTATCTGAACGCCAACAAGTTCGTGCACCGCGACCTGGCCGCCAGAAATTG CATGGTGGCCGAGGACTTCACCGTGAAGATCGGCGATTTCGGCATGACCCGGGACATCTA CGAGACAGACTACTACCGGAAAGGCGGCAAGGGACTGCTGCCCGTCAGATGGATGTCTCC CGAGTCTCTGAAGGACGGCGTGTTCACCACCTACAGCGACGTGTGGTCTTTCGGCGTGGT GCTGTGGGAGATTGCCACACTGGCCGAGCAGCCTTATCAGGGCCTGTCCAACGAACAGGT GCTGAGATTCGTGATGGAAGGCGGCCTGCTGGACAAGCCCGACAACTGTCCTGACATGCT GTTCGAGCTGATGCGGATGTGCTGGCAGTACAACCCCAAGATGAGGCCCAGCTTCCTGGA AATCATCAGCAGCATCAAAGAAGAGATGGAACCCGGCTTCAGAGAGGTGTCCTTCTACTA CAGCGAGGAAAACAAGCTGCCCGAGCCTGAGGAACTGGACCTGGAACCTGAGAACATGGAAAGCGTGCCACTGGACCCTAGCGCCAGCTCTAGTTCTCTCCCACTTCCTGATCGGCACTC CGGACACAAGGCCGAGAATGGACCAGGACCTGGCGTGCTGGTTCTGAGAGCCAGCTTCG ATGAGAGACAGCCCTACGCTCACATGAATGGCGGACGGAAGAACGAGAGAGCCCTGCCT CTGCCTCAGTCCAGCACATGCTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCC TTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGA TTCTGCCTAATAAAAAACATTTATTTTCATTGCGCGGCCGCGGATCCCCGGGTACCGAATTC GATAtctctatagtgtcacctaaatttaattaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaa (SEQ ID NO: 14).

[0037] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1-IGFR1 fusion protein include the nucleic acid sequence comprising, consisting essentially of, or consisting of the DNA sequence of SEQ ID NO: 15: ATGGGCCTGAGCACAGTTCCTGATCTGCTGCTGCCTCTGGTGCTGCTGGAACTGCTCGTGG GCATCTATCCTAGCGGCGTGATCGGACTGGTGCCTCACCTGGGCGATAGAGAAAAGCGGG ATAGCGTGTGCCCTCAGGGCAAGTACATTCACCCTCAGAACAACAGCATCTGCTGCACCA AGTGCCACAAGGGCACCTACCTGTACAACGACTGTCCTGGACCAGGCCAGGATACCGACT GTAGAGAGTGTGAAAGCGGCAGCTTCACCGCCAGCGAGAACCACCTGAGACACTGCCTG AGCTGCAGCAAGTGTCGGAAAGAGATGGGCCAAGTCGAGATCAGCAGCTGCACCGTGGA CAGAGATACCGTGTGCGGCTGCCGGAAGAACCAGTACAGACACTATTGGAGCGAGAATCT GTTCCAGTGCTTCAACTGCAGCCTGTGCCTGAACGGCACAGTGCACCTGTCCTGCCAAGA GAAGCAGAACACCGTGTGTACCTGCCACGCCGGATTCTTTCTGCGCGAGAATGAGTGCGT GTCCTGCAGCAACTGCAAGAAAAGCCTGGAATGCACCAAGCTGTGCCTGCCTCAGATCGA GAACGTGAAGGGCACAGAGGATAGCGGCACCACAGTGCTGCTCCCTCTCGTGATCTTCTT CGGCCTGTGTCTGCTGAGCCTGCTGTTCATCGGCCTGATGTACCGGAAGAGAAACAACAG CCGGCTCGGCAACGGCGTGCTGTACGCTTCTGTGAACCCCGAGTACTTCTCTGCCGCCGAT GTGTACGTGCCCGACGAATGGGAAGTCGCCCGGGAAAAGATCACCATGAGCAGAGAACT CGGCCAGGGCAGCTTCGGCATGGTGTATGAAGGCGTGGCCAAGGGCGTCGTGAAGGACG AGCCTGAAACAAGAGTGGCCATCAAGACCGTGAACGAGGCCGCCAGCATGAGAGAGAG AATCGAGTTCCTGAATGAGGCCAGCGTGATGAAGGAATTCAACTGCCACCACGTCGTCCG GCTGCTGGGAGTTGTGTCTCAAGGACAGCCCACACTGGTCATCATGGAACTGATGACACG GGGCGACCTGAAGTCCTACCTGAGATCTCTGCGGCCCGAGATGGAAAACAACCCTGTGCT GGCTCCTCCTAGCCTGAGCAAGATGATTCAGATGGCCGGCGAGATCGCCGACGGCATGGC TTATCTGAACGCCAACAAGTTCGTGCACCGCGACCTGGCCGCCAGAAATTGCATGGTGGC CGAGGACTTCACCGTGAAGATCGGCGATTTCGGCATGACCCGGGACATCTACGAGACAGA CTACTACCGGAAAGGCGGCAAGGGACTGCTGCCCGTCAGATGGATGTCTCCCGAGTCTCT GAAGGACGGCGTGTTCACCACCTACAGCGACGTGTGGTCTTTCGGCGTGGTGCTGTGGGA GATTGCCACACTGGCCGAGCAGCCTTATCAGGGCCTGTCCAACGAACAGGTGCTGAGATT CGTGATGGAAGGCGGCCTGCTGGACAAGCCCGACAACTGTCCTGACATGCTGTTCGAGCT GATGCGGATGTGCTGGCAGTACAACCCCAAGATGAGGCCCAGCTTCCTGGAAATCATCAG CAGCATCAAAGAAGAGATGGAACCCGGCTTCAGAGAGGTGTCCTTCTACTACAGCGAGG AAAACAAGCTGCCCGAGCCTGAGGAACTGGACCTGGAACCTGAGAACATGGAAAGCGTG CCACTGGACCCTAGCGCCAGCTCTAGTTCTCTCCCACTTCCTGATCGGCACTCCGGACACA AGGCCGAGAATGGACCAGGACCTGGCGTGCTGGTTCTGAGAGCCAGCTTCGATGAGAGA CAGCCCTACGCTCACATGAATGGCGGACGGAAGAACGAGAGAGCCCTGCCTCTGCCTCAG TCCAGCACATGCTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCC TAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTA ATAAAAAACATTTATTTTCATTGCGCGGCCGCGGATCCCCGGGTACCGAATTCGATA (SEQ ID NO: 15).

[0038] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1-IGFR1 fusion protein include the nucleic acid sequence comprising, consisting essentially of, or consisting of the mRNA sequence of SEQ ID NO: 16:AUGGGCCUGAGCACAGUUCCUGAUCUGCUGCUGCCUCUGGUGCUGCUGGAACUGCUCG UGGGCAUCUAUCCUAGCGGCGUGAUCGGACUGGUGCCUCACCUGGGCGAUAGAGAAA AGCGGGAUAGCGUGUGCCCUCAGGGCAAGUACAUUCACCCUCAGAACAACAGCAUCUG CUGCACCAAGUGCCACAAGGGCACCUACCUGUACAACGACUGUCCUGGACCAGGCCAG GAUACCGACUGUAGAGAGUGUGAAAGCGGCAGCUUCACCGCCAGCGAGAACCACCUGA GACACUGCCUGAGCUGCAGCAAGUGUCGGAAAGAGAUGGGCCAAGUCGAGAUCAGCA GCUGCACCGUGGACAGAGAUACCGUGUGCGGCUGCCGGAAGAACCAGUACAGACACUA UUGGAGCGAGAAUCUGUUCCAGUGCUUCAACUGCAGCCUGUGCCUGAACGGCACAGU GCACCUGUCCUGCCAAGAGAAGCAGAACACCGUGUGUACCUGCCACGCCGGAUUCUUU CUGCGCGAGAAUGAGUGCGUGUCCUGCAGCAACUGCAAGAAAAGCCUGGAAUGCACC AAGCUGUGCCUGCCUCAGAUCGAGAACGUGAAGGGCACAGAGGAUAGCGGCACCACA GUGCUGCUCCCUCUCGUGAUCUUCUUCGGCCUGUGUCUGCUGAGCCUGCUGUUCAUCG GCCUGAUGUACCGGAAGAGAAACAACAGCCGGCUCGGCAACGGCGUGCUGUACGCUUC UGUGAACCCCGAGUACUUCUCUGCCGCCGAUGUGUACGUGCCCGACGAAUGGGAAGUC GCCCGGGAAAAGAUCACCAUGAGCAGAGAACUCGGCCAGGGCAGCUUCGGCAUGGUG UAUGAAGGCGUGGCCAAGGGCGUCGUGAAGGACGAGCCUGAAACAAGAGUGGCCAUC AAGACCGUGAACGAGGCCGCCAGCAUGAGAGAGAGAAUCGAGUUCCUGAAUGAGGCC AGCGUGAUGAAGGAAUUCAACUGCCACCACGUCGUCCGGCUGCUGGGAGUUGUGUCU CAAGGACAGCCCACACUGGUCAUCAUGGAACUGAUGACACGGGGCGACCUGAAGUCCU ACCUGAGAUCUCUGCGGCCCGAGAUGGAAAACAACCCUGUGCUGGCUCCUCCUAGCCU GAGCAAGAUGAUUCAGAUGGCCGGCGAGAUCGCCGACGGCAUGGCUUAUCUGAACGC CAACAAGUUCGUGCACCGCGACCUGGCCGCCAGAAAUUGCAUGGUGGCCGAGGACUUC ACCGUGAAGAUCGGCGAUUUCGGCAUGACCCGGGACAUCUACGAGACAGACUACUACC GGAAAGGCGGCAAGGGACUGCUGCCCGUCAGAUGGAUGUCUCCCGAGUCUCUGAAGG ACGGCGUGUUCACCACCUACAGCGACGUGUGGUCUUUCGGCGUGGUGCUGUGGGAGA UUGCCACACUGGCCGAGCAGCCUUAUCAGGGCCUGUCCAACGAACAGGUGCUGAGAUU CGUGAUGGAAGGCGGCCUGCUGGACAAGCCCGACAACUGUCCUGACAUGCUGUUCGAG CUGAUGCGGAUGUGCUGGCAGUACAACCCCAAGAUGAGGCCCAGCUUCCUGGAAAUCA UCAGCAGCAUCAAAGAAGAGAUGGAACCCGGCUUCAGAGAGGUGUCCUUCUACUACA GCGAGGAAAACAAGCUGCCCGAGCCUGAGGAACUGGACCUGGAACCUGAGAACAUGG AAAGCGUGCCACUGGACCCUAGCGCCAGCUCUAGUUCUCUCCCACUUCCUGAUCGGCA CUCCGGACACAAGGCCGAGAAUGGACCAGGACCUGGCGUGCUGGUUCUGAGAGCCAGC UUCGAUGAGAGACAGCCCUACGCUCACAUGAAUGGCGGACGGAAGAACGAGAGAGCC CUGCCUCUGCCUCAGUCCAGCACAUGCUAAGCUCGCUUUCUUGCUGUCCAAUUUCUAU UAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGC CUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCGCGGCCGCGG AUCCCCGGGUACCGAAUUCGAUA (SEQ ID NO: 16). B. Chimeric TNFR1-c-MET Fusion Proteins

[0039] In certain aspects, compositions of the disclosure comprise chimeric TNFR1-c-MET fusion proteins. In certain embodiments, the chimeric TNFR1-c-MET fusion protein comprises: (a) a TNFR1 extracellular domain; (b) a TNFR1 transmembrane domain; and (c) a c-MET intracellular signaling domain.

[0040] In certain aspects, the amino acid sequence of the TNFR1 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 8.

[0041] In certain embodiments, the amino acid sequence of the TNFR1 extracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8.

[0042] In certain aspects, the amino acid sequence of the TNFR1 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 9.

[0043] In certain embodiments, the amino acid sequence of the TNFR1 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9.

[0044] In certain embodiments, the coding sequence for the c-MET intracellular signaling domain comprises amino acids 958-1390 of the c-MET coding sequence. In some embodiments, the c-MET intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 11: RKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGS CRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIG RGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRS EGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNC MLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSF GVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSF SELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS (SEQ ID NO: 11).

[0045] In certain embodiments, the amino acid sequence encoding the c-MET signaling domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11.

[0046] In certain embodiments, the coding sequence for the TNFR1 extracellular domain and the TNFR1 transmembrane domain comprise amino acids 1-234 of the TNFR1 coding sequence. In some embodiments, the TNFR1 extracellular domain and transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 7.

[0047] In certain embodiments, the amino acid sequence encoding the TNFR1 extracellular domain and TNFR1 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7.

[0048] In certain aspects, the amino acid sequence of the TNFR1-c-MET fusion protein comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13: MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICC TKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISS CTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAG FFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSLLFIG LMYRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQ FPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVV IGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGII MKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQV AKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTG AKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQ GRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATY VNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS* (SEQ ID NO: 13).

[0049] In certain embodiments, the amino acid sequence encoding the TNFR1-c-MET fusion protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13.

[0050] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1-c-MET fusion protein include the nucleic acid sequence (the mRNA start codon is highlighted in bold and italicized font, and the polyA sequence is shown in lower case letters): aggagacaagcttacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccgccaccATGGGCCTGAGC ACAGTTCCTGATCTGCTGCTGCCTCTGGTGCTGCTGGAACTGCTCGTGGGCATCTA TCCTAGCGGCGTGATCGGACTGGTGCCTCACCTGGGCGATAGAGAAAAGCGGGAT AGCGTGTGCCCTCAGGGCAAGTACATTCACCCTCAGAACAACAGCATCTGCTGCA CCAAGTGCCACAAGGGCACCTACCTGTACAACGACTGTCCTGGACCAGGCCAGG ATACCGACTGTAGAGAGTGTGAAAGCGGCAGCTTCACCGCCAGCGAGAACCACC TGAGACACTGCCTGAGCTGCAGCAAGTGTCGGAAAGAGATGGGCCAAGTCGAGA TCAGCAGCTGCACCGTGGACAGAGATACCGTGTGCGGCTGCCGGAAGAACCAGT ACAGACACTATTGGAGCGAGAATCTGTTCCAGTGCTTCAACTGCAGCCTGTGCCT GAACGGCACAGTGCACCTGTCCTGCCAAGAGAAGCAGAACACCGTGTGTACCTG CCACGCCGGATTCTTTCTGCGCGAGAATGAGTGCGTGTCCTGCAGCAACTGCAAG AAAAGCCTGGAATGCACCAAGCTGTGCCTGCCTCAGATCGAGAACGTGAAGGGC ACAGAGGATAGCGGCACCACAGTGCTGCTCCCTCTCGTGATCTTCTTCGGCCTGTG TCTGCTGAGCCTGCTGTTCATCGGCCTGATGTACCGGAAGCAGATCAAGGACCTG GGCAGCGAACTCGTCAGATACGACGCCAGAGTGCACACCCCACACCTGGACAGA CTGGTGTCTGCCAGAAGCGTGTCCCCTACCACCGAGATGGTGTCCAATGAGAGCG TGGACTACCGGGCCACCTTTCCTGAGGATCAGTTCCCCAACAGCAGCCAGAACGG CTCCTGCCGTCAGGTGCAGTACCCTCTGACAGACATGAGCCCCATCCTGACCAGC GGCGACAGCGATATTTCTAGCCCTCTGCTGCAGAATACCGTGCACATCGACCTGAG CGCCCTGAATCCTGAACTGGTGCAGGCCGTGCAGCACGTTGTGATTGGACCTAGC AGCCTGATCGTGCACTTCAACGAAGTGATCGGCAGAGGCCACTTCGGCTGTGTGTATCACGGCACCCTGCTGGACAACGACGGCAAGAAAATCCACTGCGCCGTGAAGT CCCTGAACCGGATCACAGACATCGGCGAGGTGTCCCAGTTTCTGACCGAGGGCAT CATCATGAAGGACTTCTCTCACCCCAATGTGCTGTCCCTGCTGGGCATCTGTCTGA GATCTGAGGGCTCTCCACTGGTGGTGCTGCCCTATATGAAGCACGGCGACCTGCG GAACTTCATCCGGAACGAGACACACAACCCCACCGTGAAGGACCTGATCGGCTTT GGACTGCAAGTGGCCAAGGGCATGAAGTACCTGGCCAGCAAGAAATTCGTGCAC CGCGATCTGGCCGCCAGAAACTGCATGCTGGACGAGAAGTTCACAGTGAAGGTG GCCGACTTTGGCCTGGCCAGAGATATGTACGACAAAGAGTACTACAGCGTCCACA ACAAGACCGGCGCCAAGCTGCCTGTGAAATGGATGGCCCTGGAAAGCCTGCAGA CCCAGAAGTTTACCACCAAGAGCGACGTGTGGTCCTTCGGCGTTCTGCTGTGGGA GCTGATGACAAGAGGCGCCCCTCCTTATCCTGACGTGAACACCTTCGACATCACC GTCTACCTGCTGCAAGGCAGAAGGCTGCTGCAGCCCGAGTACTGTCCTGATCCTC TGTATGAAGTGATGCTGAAGTGCTGGCACCCCAAGGCCGAGATGAGGCCTAGCTT TAGCGAGCTGGTGTCTCGGATCAGCGCCATCTTCAGCACCTTTATCGGCGAGCACT ATGTGCACGTGAACGCCACCTACGTGAACGTGAAATGCGTGGCCCCTTATCCTAGC CTGCTGAGCAGCGAGGACAACGCCGACGACGAAGTGGATACAAGACCCGCCAGC TTCTGGGAAACCAGCTAAgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaac tgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcgcggccgcggatccccgggtacc gaattcgatatctctatagtgtcacctaaatttaattaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 17).

[0051] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1- c-MET fusion protein include the nucleic acid sequence comprising, consisting essentially of, or consisting of the DNA sequence of SEQ ID NO: 18: atgggcctgagcacagttcctgatctgctgctgcctctggtgctgctggaactgctcgtgggcatctatcctagcggcgtgatcggactg gtgcctcacctgggcgatagagaaaagcgggatagcgtgtgccctcagggcaagtacattcaccctcagaacaacagcatctgctgc accaagtgccacaagggcacctacctgtacaacgactgtcctggaccaggccaggataccgactgtagagagtgtgaaagcggcag cttcaccgccagcgagaaccacctgagacactgcctgagctgcagcaagtgtcggaaagagatgggccaagtcgagatcagcagct gcaccgtggacagagataccgtgtgcggctgccggaagaaccagtacagacactattggagcgagaatctgttccagtgcttcaact gcagcctgtgcctgaacggcacagtgcacctgtcctgccaagagaagcagaacaccgtgtgtacctgccacgccggattctttctgcg cgagaatgagtgcgtgtcctgcagcaactgcaagaaaagcctggaatgcaccaagctgtgcctgcctcagatcgagaacgtgaagg gcacagaggatagcggcaccacagtgctgctccctctcgtgatcttcttcggcctgtgtctgctgagcctgctgttcatcggcctgatgt accggaagcagatcaaggacctgggcagcgaactcgtcagatacgacgccagagtgcacaccccacacctggacagactggtgtc tgccagaagcgtgtcccctaccaccgagatggtgtccaatgagagcgtggactaccgggccacctttcctgaggatcagttccccaac agcagccagaacggctcctgccgtcaggtgcagtaccctctgacagacatgagccccatcctgaccagcggcgacagcgatatttct agccctctgctgcagaataccgtgcacatcgacctgagcgccctgaatcctgaactggtgcaggccgtgcagcacgttgtgattggac ctagcagcctgatcgtgcacttcaacgaagtgatcggcagaggccacttcggctgtgtgtatcacggcaccctgctggacaacgacg gcaagaaaatccactgcgccgtgaagtccctgaaccggatcacagacatcggcgaggtgtcccagtttctgaccgagggcatcatca tgaaggacttctctcaccccaatgtgctgtccctgctgggcatctgtctgagatctgagggctctccactggtggtgctgccctatatgaa gcacggcgacctgcggaacttcatccggaacgagacacacaaccccaccgtgaaggacctgatcggctttggactgcaagtggcca agggcatgaagtacctggccagcaagaaattcgtgcaccgcgatctggccgccagaaactgcatgctggacgagaagttcacagtg aaggtggccgactttggcctggccagagatatgtacgacaaagagtactacagcgtccacaacaagaccggcgccaagctgcctgt gaaatggatggccctggaaagcctgcagacccagaagtttaccaccaagagcgacgtgtggtccttcggcgttctgctgtgggagct gatgacaagaggcgcccctccttatcctgacgtgaacaccttcgacatcaccgtctacctgctgcaaggcagaaggctgctgcagccc gagtactgtcctgatcctctgtatgaagtgatgctgaagtgctggcaccccaaggccgagatgaggcctagctttagcgagctggtgtc tcggatcagcgccatcttcagcacctttatcggcgagcactatgtgcacgtgaacgccacctacgtgaacgtgaaatgcgtggcccctt atcctagcctgctgagcagcgaggacaacgccgacgacgaagtggatacaagacccgccagcttctgggaaaccagctaa (SEQ ID NO: 18).

[0052] In certain embodiments of the disclosure, polynucleotides encoding the chimeric TNFR1-c-MET fusion protein include the nucleic acid sequence comprising, consisting essentially of, or consisting of the mRNA sequence of SEQ ID NO: 19: augggccugagcacaguuccugaucugcugcugccucuggugcugcuggaacugcucgugggcaucuauccuagcggc gugaucggacuggugccucaccugggcgauagagaaaagcgggauagcgugugcccucagggcaaguacauucacccuc agaacaacagcaucugcugcaccaagugccacaagggcaccuaccuguacaacgacuguccuggaccaggccaggauacc gacuguagagagugugaaagcggcagcuucaccgccagcgagaaccaccugagacacugccugagcugcagcaagugucg gaaagagaugggccaagucgagaucagcagcugcaccguggacagagauaccgugugcggcugccggaagaaccaguaca gacacuauuggagcgagaaucuguuccagugcuucaacugcagccugugccugaacggcacagugcaccuguccugcca agagaagcagaacaccguguguaccugccacgccggauucuuucugcgcgagaaugagugcguguccugcagcaacugc aagaaaagccuggaaugcaccaagcugugccugccucagaucgagaacgugaagggcacagaggauagcggcaccacagu gcugcucccucucgugaucuucuucggccugugucugcugagccugcuguucaucggccugauguaccggaagcagauc aaggaccugggcagcgaacucgucagauacgacgccagagugcacaccccacaccuggacagacuggugucugccagaag cguguccccuaccaccgagaugguguccaaugagagcguggacuaccgggccaccuuuccugaggaucaguuccccaaca gcagccagaacggcuccugccgucaggugcaguacccucugacagacaugagccccauccugaccagcggcgacagcgau auuucuagcccucugcugcagaauaccgugcacaucgaccugagcgcccugaauccugaacuggugcaggccgugcagca cguugugauuggaccuagcagccugaucgugcacuucaacgaagugaucggcagaggccacuucggcuguguguaucac ggcacccugcuggacaacgacggcaagaaaauccacugcgccgugaagucccugaaccggaucacagacaucggcgaggu gucccaguuucugaccgagggcaucaucaugaaggacuucucucaccccaaugugcugucccugcugggcaucugucug agaucugagggcucuccacugguggugcugcccuauaugaagcacggcgaccugcggaacuucauccggaacgagacac acaaccccaccgugaaggaccugaucggcuuuggacugcaaguggccaagggcaugaaguaccuggccagcaagaaauuc gugcaccgcgaucuggccgccagaaacugcaugcuggacgagaaguucacagugaagguggccgacuuuggccuggcca gagauauguacgacaaagaguacuacagcguccacaacaagaccggcgccaagcugccugugaaauggauggcccuggaa agccugcagacccagaaguuuaccaccaagagcgacgugugguccuucggcguucugcugugggagcugaugacaagag gcgccccuccuuauccugacgugaacaccuucgacaucaccgucuaccugcugcaaggcagaaggcugcugcagcccgag uacuguccugauccucuguaugaagugaugcugaagugcuggcaccccaaggccgagaugaggccuagcuuuagcgagc uggugucucggaucagcgccaucuucagcaccuuuaucggcgagcacuaugugcacgugaacgccaccuacgugaacgu gaaaugcguggccccuuauccuagccugcugagcagcgaggacaacgccgacgacgaaguggauacaagacccgccagcu ucugggaaaccagcuaa (SEQ ID NO: 19). Delivery of Nucleic Acids with Lipid Nanoparticles A. Lipid Nanoparticles (LNP)

[0053] Polynucleotides encoding TNFR1 fusion proteins as described herein can be delivered to cells using one or more lipid nanoparticle compositions and methods of making the same, as described in International Patent Publication No: WO2023141576 and International Patent Application No. PCT / US2024 / 012245, the contents of which are incorporated herein by reference in its entirety.

[0054] The present disclosure provides compositions comprising at least one nucleic acid and at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one cationic lipid, at least one structural lipid, at least one phospholipid and at least one PEGylated lipid. LNP ComponentsNucleic Acids

[0055] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid molecule of the present disclosure (e.g., a polynucleotide encoding a TNFR1 fusion protein as described herein). In some aspects, a lipid nanoparticle can comprise a plurality of nucleic acid molecules. In some aspects, the plurality of nucleic acid molecules includes at least one nucleic acid of the present disclosure. In some aspects, the at least one nucleic acid molecule or the plurality of nucleic acid molecules can be formulated in a lipid nanoparticle.

[0056] In some aspects, the at least one nucleic acid molecule of the present disclosure (e.g., polynucleotide encoding a TNFR1 fusion protein as described herein) can be an RNA molecule. In some aspects, the RNA molecule is an mRNA molecule. In some aspects, the mRNA molecule further comprises a 5’-CAP. In some aspects, all of the cytidine residues in an mRNA molecule can be 5-methylcytidine.

[0057] In some aspects, the at least one nucleic acid molecule can be a DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA plasmid. In some aspects, the at least one DNA molecule is a DNA nanoplasmid.

[0062] In some aspects, the at least one nucleic acid can comprise at least one RNA molecule and at least one DNA molecule. That is, the LNPs of the present disclosure can comprise both RNA molecules and DNA molecules.

[0063] In some aspects, the LNPs of the present disclosure can comprise both RNA molecules and DNA molecules. In some embodiments the RNA molecules comprise at least one nucleic acid sequence encoding a TNFR1 fusion protein as described herein and at least one nucleic acid sequence that encodes a transposase; and the DNA molecules comprise at least one nucleic acid sequence that comprises a transposon. In some aspects, the transposase can be any of the transposases described herein. In some aspects, the transposon can be a transposon comprising at least one nucleic acid sequence encoding a FVIII polypeptide. In some aspects, the transposon can be a transposon comprising at least one nucleic acid sequence encoding a human propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide.

[0064] In some aspects wherein the LNPs of the present disclosure comprise both RNA (e.g. mRNA) and DNA, the ratio of RNA to DNA (RNA:DNA) in the LNPs can be about 1:2, or about 1:3, or about1:4, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1 or about 10:1.

[0065] In some aspects, a lipid nanoparticle can comprise lipid and nucleic acid at a specified ratio (weight / weight).

[0066] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1 or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1, lipid:nucleic acid, weight / weight.

[0067] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, lipid:nucleic acid, weight / weight. Cationic Lipid

[0068] In some aspects, the cationic lipid is HMA-404, comprising the following structure:.

[0069] In some aspects, the cationic lipid is COMPOUND NO. 37, comprising the following structure:.

[0070] The synthetic route for COMPOUND NO. 37 is shown in General Scheme E.1. This two-step sequence begins with an esterification reaction between trans-4-pentylcyclohexane carboxylic acid and hydroxy substituted alkyl bromides of different lengths (C3, C5, and C7)catalyzed by N-Ethyl-N -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl)and N,N-Dimethylpyridin-4-amine (DMAP). The corresponding ester which bears a bromide as a functional handle reacts with hydroxy substituted amines (Hn, where n = 2, 3, or 4) to give the target compounds.

[0071] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (22 mg) was combined with BC6B5C (442 mg) and DIPEA (200 L) in THF / CH3CN (1:1, 0.8 mL). After the reaction, the crude was purified by 6% MeOH / DCM eluants. Brown oil, 127 mg (36%); 1H NMR (499 MHz, CDCl3) 4.12 (t, J = 4.8 Hz, 12H), 3.81 – 3.75 (m, 2H), 2.65 (s, 2H), 2.39 (td, J = 12.8, 6.8 Hz, 5H), 2.32 (t, J = 7.5 Hz, 4H), 2.22 (tt, J = 12.2, 3.6 Hz, 4H), 1.98 – 1.91 (m, 8H), 1.84 – 1.76 (m, 8H), 1.72 – 1.60 (m, 7H), 1.50(s, 4H), 1.44 – 1.13 (m, 49H), 0.95 – 0.84 (m, 20H); LC-MS: Rt 9.504 min, m / z calculated [M+H]: 1200.92, found 1200.75.

[0072] In some aspects, the cationic lipid compound is the followingstructure:. Structural Lipids

[0073] In some embodiments, the LNP comprises a structural lipid. In some aspects, a structural lipid can be a steroid. In some aspects, a structural lipid can be a sterol. In some aspects, a structural lipid can comprise cholesterol. In some aspects, a structural lipid can comprise ergosterol. In some aspects, a structural lipid can be a phytosterol. Phospholipid

[0074] In some embodiments, the LNP comprises a phospholipid. As used herein, the term “phospholipid” is used in its broadest sense to refer to any amphiphilic molecule that comprises a polar (hydrophilic) headgroup comprising phosphate and two hydrophobic fatty acid chains.

[0075] In some aspects of the lipid nanoparticles of the present disclosure, a phospholipid can comprise dioleoylphosphatidylethanolamine (DOPE).

[0076] In some aspects of the lipid nanoparticles of the present disclosure, a phospholipid can comprise DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine).

[0077] In some aspects of the lipid nanoparticles of the present disclosure, a phospholipid can comprise DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine). PEGylated Lipid

[0078] Ins some embodiments, the LNP comprises a PEGylated lipid. As used herein, the term “PEGylated lipid” is used to refer to any lipid that is modified (e.g., covalently linked to) at least one polyethylene glycol molecule. In some aspects, a PEGylated lipid can comprise l,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000, hereafter referred to as DMG- PEG2000. Targeting Ligand

[0079] In some aspects, an LNP of the present disclosure can further comprise at least one targeting ligand.

[0080] A targeting ligand may be any ligand that provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

[0081] In some aspects, a composition comprising a targeting lipid is well-tolerated and provides an adequate therapeutic index, such that patient treatment with an effective dose of the composition is associated with an improved toxicity and / or risk profile to the patient, compared to patient treatment with an effective dose of a composition that does not comprise a targeting ligand.

[0082] In some aspects, a targeting ligand provides an enhanced affinity for the liver or liver cells, such as hepatocytes. A non-limiting example of a targeting ligand with enhanced affinity for the liver or liver cells is GalNac (n-acetyl-galactosamine). Thus, in some embodiments, the invention provides LNP compositions comprising a targeting ligand comprising GalNac.

[0083] In some aspects, a targeting ligand comprising GalNac is GalNac-DSPE-PEG.

[0084] In some aspects, a targeting ligand can comprise a multivalent GalNac compound of the following structure:

[0085] Exemplary LNP Compositions

[0086] In some aspects, the at least one nucleic acid is encapsulated in at least one lipid nanoparticle comprising: about 40% of HMA-404 by moles, about 52.5% of cholesterol by moles, about 5% of DOPC by moles, about 2% of DMG-PEG2000 by moles, and about 0.5 mol% of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g. mRNA molecule), and wherein the ratio of lipid to RNA molecule in the at least one nanoparticle is about 50:1 (w / w). Pharmaceutical Compositions of the Present Disclosure

[0092] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle as described herein. In some embodiments, the pharmaceutical composition comprises at least one lipid nanoparticle as described herein, and at least one pharmaceutically-acceptable excipient or diluent. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle comprising at least one nucleic acid molecule encoding a TNFR1 fusion protein as described herein. In some aspects, the at least one nucleic acid molecule is an RNA molecule. In some aspects, the at least one lipid nanoparticle further comprises at least one nucleic acidmolecule encoding at least one transposase and at least one nucleic acid molecule encoding at least one transposon. In some aspects, the at least one nucleic acid molecule encoding the at least one transposase can be an RNA molecule (e.g. mRNA molecule) and the at least one nucleic acid molecule encoding the at least one transposon can be a DNA molecule (e.g. a DoggyBone DNA molecule or a DNA nanoplasmid).

[0093] In some aspects, the present disclosure provides a composition comprising at least one cell that has been contacted by at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using any method of the present disclosure. Methods of the Present Disclosure

[0094] The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0095] The present disclosure provides a method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of genetically modifying at least one cell comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0096] In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell otherwise would not normally express, or such that the at least one cell expresses at least one protein at a level that is higher than the level that the cell would otherwise normally express the at least one protein, or such that the cell expresses at least one protein at a level that is lower than the level that the cell would otherwise normally express the at least one protein. In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic to the cell such that at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[0097] In some aspects, the methods of the present disclosure can yield a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, orat least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cell in the plurality express at least one protein that was encoded in at least one nucleic acid that was delivered to the plurality of cells via a nanoparticle of the present disclosure.

[0098] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0099] In some methods, compositions and / or kits of the present disclosure, the at least one cell can be a liver cell. A liver cell can include, but is not limited to, a hepatocyte, a hepatic stellate cell, a Kupffer cell or a liver sinusoidal endothelial cell.

[0100] In some aspects of the methods of the present disclosure, a cell can be in vivo, ex vivo or in vitro. In some aspects, any of the methods of the present disclosure can be applied in vivo, ex vivo or in vitro.

[0101] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0102] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of cells, wherein the cells have been contacted by at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein. The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of cells, wherein the cells have been genetically modified using the compositions and / or methods of the present disclosure.

[0103] The disclosure provides methods for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, comprising administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of a composition disclosed herein. In one aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0104] The disclosure provides methods of treating at least one disease or disorder in a subject, comprising administering to the subject at least one therapeutically effective amountof at least one composition disclosed herein comprising at least one nucleic acid encoding a therapeutic protein.

[0105] The disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. In some aspects, the at least one disease can be a malignant disease, including, but not limited to, cancer. In some aspects, the at least one disease can be Hemophilia A or Hemophilia B. In some aspects, the at least one disease can be a metabolic liver disorder (MLD). In some aspects, the at least one disease can be a urea cycle disorder (UCD). An MLD and / or UCD can include, but is not limited to, N- Acetylglutamate Synthetase (NAGS) Deficiency, Carbamoylphosphate Synthetase I Deficiency (CPSI Deficiency), Ornithine Transcarbamylase (OTC) Deficiency, Argininosuccinate Synthetase Deficiency (ASSD) (Citrullinemia I), Citrin Deficiency (Citrullinemia II), Argininosuccinate Lyase Deficiency (Argininosuccinic Aciduria), Arginase Deficiency (Hyperargininemia), Ornithine Translocase Deficiency (HHH Syndrome), methylmalonic acidemia (MMA) or any combination thereof.

[0106] Methods of the disclosure may be used to treat a disease or disorder by use of a therapeutic transgene encoding for an exogenous nucleic acid sequence or exogenous amino acid sequence. In such methods, the transgene is delivered to a target cell to replace or repair a mutated gene. Diseases that may be treated with such methods are generally caused by a mutation in a gene that results in no protein being expressed or non-functional proteins being expressed. Examples of therapeutic transgenes that can be delivered using the compositions disclosed herein include: Beta-Thalassemia (HBB T87Q, BCL11A shRNA, IGF2BP1), Sickle Cell Disease (HBB T87Q, BCL11A shRNA, IGF2BP1), Hemophilia A (Factor VIII), Hemophilia B (Factor IX), X-linked Severe Combined Immunodeficiency (Interleukin 2 receptor gamma (IL2RG)), Hypophosphatasia (Tissue Non-specific Alkaline Phosphatase (TNAP)), Osteopetrosis (TCIRG1), Glycogen Storage Disease Type II (Pompe Disease) (Alpha Glucosidase (GAA)), Alpha-Galactosidase A Deficiency (Fabry disease) (Alpha- galactosidase A (GLA)), Mucopolysaccharidosis Type I (MPS I) (Alpha-L-iduronidase (IDUA)), Mucopolysaccharidosis Type II (MPS II) (Iduronate 2-sulfatase (IDS)), Mucopolysaccharidosis Type IIIA (MPS IIIA) (sulfoglycosamine-sulfohydrolase (SGSH)), Mucopolysaccharidosis Type IIIB (MPS IIIB) (N-alpha-acetylglucosaminidase (NAGLU)), Mucopolysaccharidosis Type IV A (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)), Mucopolysaccharidosis Type IV B (MPS IVB) Beta-galactosidase (GLB1 (Beta-galactosidase (GLB1)), Cholesteryl Ester Storage Disease (CESD) (Lysosomal acid lipase (LIPA)), Cystinosis (Cystinosin lysosomal cystine transporter (CTNS)), X-linkedchronic granulomatous disease (X-CGD) (CYBB), Wiskott-Aldrich Syndrome (WAS) (WAS), X-linked Adrenoleukodystrophy (X-ALD) (ABCD1), Metachromatic leukopdystrophy (MLD) (ARSA), Phenylketonuria (PAH), Methylmalonic academia (MMUT), Propionic Acidemia (PCCA, PCCB), Retinitis Pigmentosa (RPE65), Usher Syndrome (MYO7A), and Gaucher Disease (GBA).

[0107] Methods of the present disclosure can optionally further comprise co-administration or combination therapy for treating such diseases or disorders, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one chemotherapeutic agent (e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical).

[0108] The present disclosure provides methods for reducing TNF -mediated cytokine levelsin a cell, the method comprising contacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein,wherein the TNF -mediated cytokine levels in the cell are reduced at least 2-fold comparedto TNF -mediated cytokine levels in a cell that is contacted with a composition that does notcomprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein. In certain embodiments, the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a fusion protein of the present disclosure. In certain embodiments, the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a polynucleotide of the present disclosure. In some embodiments, a composition comprising a polynucleotide is encapsulated in at least one lipid nanoparticle composition (LNP), for example in an LNP of the present disclosure.

[0109] The present disclosure provides methods for reducing transaminase expression in a cell, comprising contacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, wherein the transaminase expression in the cell is reduced at least 2-fold compared to transaminase expression in a cell that is contacted with a composition that does not comprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein. In certain embodiments, the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a fusion protein of the present disclosure. In certain embodiments, the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is a polynucleotide of the present disclosure. In some embodiments, a composition comprising a polynucleotide is encapsulated in at least one lipid nanoparticle composition (LNP), for example in an LNP of the present disclosure.

[0110] In some embodiments, administration of the compositions provided herein thatcomprise a TNFR1 fusion protein results in less TNF -mediated cytokine release and lesstransaminase expression than administration of the same composition not comprising theTNFR1 fusion protein. In certain embodiments, the TNF -mediated cytokine release andtransaminase expression after administration of the composition comprising the TNFR1 fusion protein is at least 2-fold less. Cytokine release may be measured using any suitable method known in the art or described herein. For example, cytokine levels may be determined in the blood of a subject receiving the LNP composition comprising the targeting ligand using enzyme-linked immunosorbent assays (ELISAs). The cytokine levels may then be compared to pre-treatment baseline levels. Nucleic Acid Molecules

[0111] In some aspects, a nucleic acid molecule can be a synthetic nucleic acid molecule. In some aspects, a nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some aspects, a non-naturally occurring nucleic acid molecule can comprise at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some aspects, a nucleic acid molecule can be a modified nucleic acid molecule. In some aspects, a modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0112] In some aspects, an mRNA molecule can be capped using any method and / or capping moiety known in the art. An mRNA molecule can be capped with m7G(5’)ppp(5’)G moiety. A m7G(5’)ppp(5’)G moiety is also referred to herein as a “Cap0”. An mRNA molecule can be capped with a CleanCap® moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. An mRNA molecule can be capped with an anti-reverse cap analog (ARCA®) moiety. An ARCA® moiety can comprise a m7(3’-O- methyl)G(5’)ppp(5’)G moiety. An mRNA molecule can be capped with a CleanCap® 3’OMe moiety (CleanCap®+ARCA®).

[0113] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0114] The at least one modified nucleic acid can comprise 5-methoxyuridine (5moU). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA molecule are 5-methoxyuridine bases. In some aspects, all of the uridine bases in an mRNA molecule are 5-methoxyuridine bases. Without wishing to be bound by theory, 5-methoxyuridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem.2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy – Nucleic Acids, 2018, 12, 530-542).

[0115] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0116] The at least one modified nucleic acid can comprise N1-methylpseudouridine (me1). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA N1-methylpseudouridine bases. In some aspects, all of the uridine bases in an mRNA molecule are N1-methylpseudouridine bases. Without wishing to be bound by theory, N1-methylpseudouridine can improve protein expression (see Li et al., Bioconjugate Chem.2016, 27, 3, 849-853).

[0117] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0118] The at least one modified nucleic acid can comprise pseudouridine ( ). In someaspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA pseudouridine bases. In some aspects, all of the uridine bases in an mRNA molecule are pseudouridine bases. Without wishing to be bound by theory, pseudouridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy – Nucleic Acids, 2018, 12, 530-542).

[0119] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid. The at least one modified nucleic acid can comprise 5-methylcytidine (5-MeC). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or atleast about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the cytidine bases in an mRNA 5-MeC bases. In some aspects, all of the cytidine bases in an mRNA molecule are 5-MeC bases.

[0120] In some aspects, a nucleic acid molecule can comprise a DNA molecule. Thus, in some aspects, a lipid nanoparticle can comprise a DNA molecule. In some aspects, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid or DNA nanoplasmid. Thus, in some aspects, a lipid nanoparticle can comprise a circular DNA molecule. In some aspects, a lipid nanoparticle can comprise a Doggybone DNA molecule. In some aspects, a lipid nanoparticle can comprise a DNA plasmid. In some aspects, a lipid nanoparticle can comprise a DNA nanoplasmid. In some aspects, a DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0121] A DNA plasmid or DNA nanoplasmid can comprise can be at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb in length.

[0122] In some aspects, a nucleic acid molecule formulated in a lipid nanoparticle of the present disclosure can comprise at least one transgene sequence. In some aspects, a transgenesequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposase. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposon. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, wherein the at least one therapeutic protein is operatively linked to the at least one promoter sequence.

[0123] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform. In some aspects, the microfluidic-mixing platform can be a non-turbulent microfluidic mixing platform.

[0124] In some aspects, a microfluidic-mixing platform can produce the lipid nanoparticles of the present invention by combining a miscible solvent phase comprising the lipid components of the nanoparticle and an aqueous phase comprising the lipid nanoparticle cargo (e.g. nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some aspects, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. As the two phases move under laminar flow in a microfluidic channel, microscopic features in the channel can allow for controlled, homogenous mixing to produce the lipid nanoparticles of the present disclosure.

[0125] In some aspects, the microfluidic-mixing platform can include, but are not limited to the NanoAssemblr® Spark (Precision NanoSystems), the NanoAssemblr® Ignite™ (Precision NanoSystems), the NanoAssemblr® Benchtop (Precision NanoSystems), the NanoAssemblr® Blaze (Precision NanoSystems) or the NanoAssemblr® GMP System (Precision NanoSystems).

[0126] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0127] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform mixes a miscible solvent phase and an aqueous phase at a ratio of about 10:1, or about 9:1, or about8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solvent:aqueous, v:v. piggyBac ITR sequences

[0128] In some aspects, a nucleic acid can comprise a piggyBac ITR sequence. In some aspects, a nucleic acid can comprise a first piggyBac ITR sequence and a second piggyBac ITR sequence. In some embodiments the first piggyBac ITR sequence comprises the nucleic acid sequence: ccctagaaagatagtctgcgtaaaattgacgcatg (SEQ ID NO: 20). In some embodiments the second piggyBac ITR sequence comprises the nucleic acid sequence: ccctagaaagataatcata (SEQ ID NO: 21).

[0129] In some aspects, a piggyBac ITR sequence can comprise any piggyBac ITR sequence known in the art.

[0130] In some aspects of the methods of the present disclosure, a piggyBac ITR sequence (such as a first piggyBac ITR sequence and / or a second piggyBac ITR sequence in an AAV piggyBac transposon) can comprise, consist essentially of, or consist of a Sleeping Beauty transposon ITR, a Helraiser transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR or any combination thereof. Transposition systems

[0131] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon.

[0132] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon, and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR, wherein the length of the inter-ITR sequence is equal to or less than 700 nucleotides.

[0133] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some aspects when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBac™ (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0134] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No.6,218,182; U.S. Patent No.6,962,810; U.S. Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296.

[0135] The PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and inserts the contents between the ITRs at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence) or at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence). The target sequence of the PB or PBL transposon can comprise or consist of 5’-CTAA-3’, 5’-TTAG-3’, 5’-ATAA-3’, 5’-TCAA-3’, 5’AGTT-3’, 5’-ATTA-3’, 5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’-TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’, 5’-CTAG-3’, 5’-TGAA-3’, 5’-AGGT-3’, 5’-ATCA- 3’, 5’-CTCC-3’, 5’-TAAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’- ACAA-3’, 5’-ACAT-3’, 5’-ACTC-3’, 5’-AGTG-3’, 5’-ATAG-3’, 5’-CAAA-3’, 5’-CACA-3’, 5’-CATA-3’, 5’-CCAG-3’, 5’-CCCA-3’, 5’-CGTA-3’, 5’-GTCC-3’, 5’-TAAG-3’, 5’-TCTA- 3’, 5’-TGAG-3’, 5’-TGTT-3’, 5’-TTCA-3’5’-TTCT-3’ and 5’-TTTT-3’. The PB or PBL transposon system has no payload limit for the genes of interest that can be included between the ITRs.

[0136] Exemplary amino acid sequences for one or more PB, PBL and SPB transposases are disclosed in U.S. Patent No.6,218,185; U.S. Patent No.6,962,810 and U.S. Patent No. 8,399,643, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1. In some embodiments, the PB transposases comprises the amino acid sequence of SEQ ID NO: 1

[0137] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, at three or more or at each of positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 1. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 1 wherein the amino acid substitution at position 30 can be a substitution of a valine (V) for anisoleucine (I), the amino acid substitution at position 165 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 can be a substitution of a lysine (K) for an asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 2. In some embodiments, the SPB transposase comprises the amino sequence set forth in SEQ ID NO: 2.

[0138] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the PB, PBL and SPB transposases can further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0139] In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 3. In some embodiments, the PB transposase comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0140] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, at three or more, or at each of positions 29, 164, 281, and / or 537 of the sequence of SEQ ID NO: 3. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 3 wherein the amino acid substitution at position 29 can be a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 164 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 281 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 537 can be a substitution of a lysine (K) for an asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4. In some embodiments, the SPB transposase comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0141] In certain aspects wherein the transposase comprises the above-described mutations at positions 29, 164, 281, and / or 537, the PB, PBL and SPB transposases can further comprise an amino acid substitution at one or more of positions 2, 45, 81, 102, 118, 124, 176, 179, 184, 186, 199, 206, 208, 225, 234, 239, 240, 242, 257, 295, 297, 310, 314, 318, 326, 327, 339, 420, 435, 455, 469, 485, 502, 551, 569 and 590 of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Exemplary transposases are described in more detail in PCT Publication No. WO 2019 / 173636 and No. WO 2020 / 051374 , each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0142] The PB, PBL or SPB transposases can be isolated or derived from an insect, vertebrate, crustacean or urochordate as described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816. In preferred aspects, the PB, PBL or SPB transposases is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0143] A hyperactive PB or PBL transposase is a transposase that is more active than the endogenous transposase from which it is derived. In a preferred aspect, a hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No.6,218,185; U.S. Patent No.6,962,810, U.S. Patent No.8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No.10,041,077, which is incorporated herein by reference in its entirety for examples of amino acid substitutions that may be introduced into the transposases described herein. A transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U.S. Patent No.9,228,180, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0144] In some aspects, the PB or PBL transposase is integration deficient. An integration deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. Examples of integration deficient PB or PBL transposases are disclosed in U.S.Patent No.6,218,185; U.S. Patent No.6,962,810, U.S. Patent No.8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. A list of integration deficient amino acid substitutions is disclosed in US patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of amino acid substitutions that may be introduced into transposases described herein

[0145] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No.6,218,185; U.S. Patent No.6,962,810, U.S. Patent No.8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0146] A transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U.S. Patent No. 9,228,180, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0147] A transposon or nanotransposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibat1. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, as disclosed in WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein).

[0148] A transposon or nanotransposon of the present disclosure can be a Tol2 transposon. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019 / 173636).

[0149] A transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (for example, as disclosed in WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein). The TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding aTcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0150] In some aspects, a mutant TcBuster transposase comprises one or more sequence variations when compared to a wild type TcBuster transposase as described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0151] The cell delivery compositions (e.g., transposons) disclosed herein can comprise a nucleic acid molecule encoding a therapeutic protein or therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0152] In some aspects, a therapeutic protein can comprise a FVIII polypeptide. An exemplary nanoplasmid encoding an FVIII polypeptide is provided in SEQ ID NO: 5. Accordingly, a nucleic acid formulated in a nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 5.

[0153] In some aspects, a therapeutic protein can comprise a propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide. An exemplary transposon encoding a PCCA polypeptide is provided in SEQ ID NO: 6. Accordingly, a nucleic acid formulated in a nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 6. Formulations, Dosages and Modes of Administration

[0154] The present disclosure provides formulations, dosages and methods for administration of the compositions described herein.

[0155] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed.,Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the “Physician's Desk Reference”, 52nd ed., Medical Economics (Montvale, N.J.) 1998. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and / or stability of the composition as well known in the art or as described herein.

[0156] For example, the disclosed LNP compositions of the present invention can further comprise a diluent. In some compositions, the diluent can be phosphate buffered saline (“PBS”).

[0157] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / protein components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0158] The compositions can also include a buffer or a pH-adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate. In some aspects, the buffer can include sucrose.

[0159] Many known and developed modes can be used for administering therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed herein. Non- limiting examples of modes of administration include bolus, buccal, infusion, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal means.

[0160] A composition of the disclosure can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms, such as, but not limited to, creams and suppositories; for buccal, or sublingual administration, such as, but not limited to, in the form of tablets or capsules; or intranasally, such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally, such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, D. S., Eds., pp.59-90 (Marcel Dekker, Inc. New York 1994,), or applications of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (U.S. Pat. Nos.4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference).

[0161] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Agents for injection can be a non-toxic, non-orally administrable diluting agent, such as aqueous solution, a sterile injectable solution or suspension in a solvent. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent or suspending solvent, sterile involatile oil can be used. For these purposes, any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No.5,839,446, each ofwhich is incorporated herein by reference in its entirety for examples of injection devices that may be used in conjunction with the compositions and methods described herein.

[0162] For pulmonary administration, preferably, a composition or pharmaceutical composition described herein is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. The composition or pharmaceutical composition can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers (e.g., jet nebulizer, ultrasonic nebulizer), dry powder generators, sprayers, and the like. All such devices can use formulations suitable for the administration for the dispensing of a composition or pharmaceutical composition described herein in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles. In a metered dose inhaler (MDI), a propellant, a composition or pharmaceutical composition described herein, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations and related devices is disclosed in PCT Publication No. WO 2019 / 049816, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0163] For absorption through mucosal surfaces, compositions include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (see, e.g., U.S. Pat. No. 5,514,670, which is incorporated herein by reference in its entirety for examples). Mucous surfaces suitable for application of the emulsions of the disclosure can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, e.g., suppositories, can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (see, e.g., U.S. Pat. No.5,849,695, which is incorporated herein by reference in its entirety for examples). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816, eachof which is incorporated herein by reference in its entirety for examples of formulations that may be used in conjunction with the compositions and methods described herein.

[0164] For transdermal administration, a composition or pharmaceutical composition disclosed herein is encapsulated in a delivery device, such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (see, e.g., U.S. Pat. No.5,814,599, each of which is incorporated herein by reference in its entirety for examples). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816, which is incorporated herein by reference in its entirety for examples of formulations and devices that may be used in conjunction with the compositions and methods described herein.

[0165] It can be desirable to deliver the disclosed compounds to the subject over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized.

[0166] Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value or fraction thereof, or to achieve a serum concentration of about 0.1-5000 g / ml serum concentration per single or multiple administration, or any range, value or fraction thereof. A preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg / kg, up to about 3, about 6 or about 12 mg / kg of body weight of the subject.

[0167] Alternatively, the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.

[0168] As a non-limiting example, treatment of humans or animals can be provided as a one- time or periodic dosage of the compositions or pharmaceutical compositions disclosed herein about 0.1 to 100 mg / kg or any range, value or fraction thereof per day, on at least one of day 1-40, or, alternatively or additionally, at least one of week 1-52, or, alternatively or additionally, at least one of 1-20 years, or any combination thereof, using single, infusion or repeated doses.

[0169] In aspects where the compositions to be administered to a subject in need thereof are modified cells as disclosed herein, the cells can be administered between about 1x103and 1x1015cells; 1x103and 1x1015cells, about 1x104and 1x1012cells; about 1x105and 1x1010cells; about 1x106and 1x109cells; about 1x106and 1x108cells; about 1x106and 1x107cells; or about 1x106and 25x106cells. In an aspect the cells are administered between about 5x106and 25x106cells.

[0170] A more detailed description of pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 04981, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0171] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of the composition or pharmaceutical composition. In an aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0172] The disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. Non-limiting examples of a malignant disease or disorder include cancer and liver diseases or disorders.

[0173] Any method can comprise administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal or subject in need of such modulation, treatment or therapy. Such a method can optionally further comprise co- administration or combination therapy for treating such diseases or disorders, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one chemotherapeutic agent (e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical).

[0174] In some aspects, the subject does not develop graft vs. host (GvH) and / or host vs. graft (HvG) following administration. In an aspect, the administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is by an intravenous injection or an intravenous infusion. In an aspect, the administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, local administration is by intra-tumoral injection or infusion, intraspinal injection or infusion, intracerebroventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0175] In some aspects, the therapeutically effective dose is a single dose. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic cells, the dose is an amount sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0176] In some aspects of the methods of treatment described herein, the treatment can be modified or terminated. Specifically, in aspects where the composition used for treatment comprises an inducible proapoptotic polypeptide, apoptosis may be selectively induced in the cell by contacting the cell with an induction agent. A treatment may be modified or terminated in response to, for example, a sign of recovery or a sign of decreasing disease severity / progression, a sign of disease remission / cessation, and / or the occurrence of an adverse event. In some aspects, the method comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (for example, when a sign or symptom of the disease reappear or increase in severity and / or an adverse event is resolved). Construction of Nucleic Acids

[0177] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as well-known in the art.

[0178] The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present disclosure. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequenceprovides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of a polynucleotide of the disclosure.

[0179] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). Recombinant Methods for Constructing Nucleic Acids

[0180] The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some aspects, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). Synthetic Methods for Constructing Nucleic Acids

[0181] The isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences. Recombinant Expression Cassettes

[0182] The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure. A nucleic acid sequence of the disclosure can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.

[0183] In some aspects, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and / or substitution. Expression Vectors and Host Cells

[0184] The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure and host cells that are genetically engineered with the recombinant vectors, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.

[0185] The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0186] The DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

[0187] Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos.5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E.coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0188] Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood.2014 Aug 21; 124(8):1277-87).

[0189] Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0190] Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid molecule encoding a protein of the disclosure. Definitions

[0191] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0192] 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 onhow the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or 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. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0193] In the chemical formulas shown herein, the markingindicates the position where a functional group bonds to another portion of a molecule. Definitions of specific functional groups and chemical terms are described in more detail below.

[0194] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0195] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group," as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.

[0196] The term "aliphatic," as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limitedto, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.

[0197] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-18 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, --CH2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, --CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, --CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, --CH2-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0198] The term "alkyl" as used herein refers to saturated, straight- (e.g., unbranched) or branched-chain aliphatic groups having from 1 to 18 carbon atoms, As such, "alkyl" encompasses C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n- pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0199] The term “alkylene” refers to a divalent alkyl radical. Any of the above mentioned monovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. As herein defined, alkylene may also be a C1-C18 alkylene. An alkylene may further be a C1-C12 alkylene. Typical alkylene groups include, but are not limited to, -CH2-, - CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, - CH2CH2CH2CH2-, and the like.

[0200] The term "alkenyl" refers to an unsaturated straight or, when applicable, branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 18 carbon atoms. As such, "alkenyl" encompasses C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 groups. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1- yl, and the like.

[0201] The term "alkynyl" refers to an unsaturated straight or, when applicable, branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 18 carbon atoms. As such, "alkynyl" encompasses C2, C3, C4, C5, C6, C7, C8, C9, C10, C11and C12groups. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0202] As used herein, the term "aryl" group is a C6 - C14 aromatic moiety comprising one to three aromatic rings, which is optionally substituted. As such, "aryl" includes C6, C7, C8, C9, C10, C11, C12 C13, and C14 cyclic hydrocarbon groups. An exemplary aryl group is a C6-C10 aryl group. Particular aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl.

[0203] As used herein, the term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons. As such, "cycloalkyl" includes C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 cyclic hydrocarbon groups. Representative cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0204] As used herein, the term “hydroxyalkyl” refers to -alkyl-OH or an alkyl chain substituted with at least one -OH.

[0205] As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0206] It will be understood that the compounds of any one of the Formulae disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers, mixtures of stereoisomers, polymorphs of all isomeric forms of said compounds.

[0207] The term "independently selected" is used herein to indicate that the R groups can be identical or different.

[0208] The term "substituted," whether preceded by the term "optionally" or not, and "substituent," as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may also be further substituted (e.g.,an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions).

[0209] The disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0210] Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and / or repeat units in target vectors that can subsequently be assembled into a final destination vector. Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector. The disclosure provides polypeptide produced by this method as well nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced this modular approach.

[0211] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope hereof to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the antibodies hereof as defined herein. Thus, according to anaspect hereof, the term “antibody hereof” in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and / or added, compared to the antibodies hereof as defined herein.

[0212] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

[0213] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0214] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[0215] “Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0216] The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.

[0217] Non-covalently linked components and methods of making and using non-covalently linked components, are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where suchassociation is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

[0218] A method for directing proteins to a specific locus in a genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.

[0219] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0220] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a depicted single strand. A nucleic acid of the disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode for the same protein.

[0221] Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

[0222] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or syntheticnucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non- naturally occurring.

[0223] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.

[0224] As used throughout the disclosure, the term "promoter" refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[0225] As used throughout the disclosure, the term "substantially identical" refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0226] As used throughout the disclosure, the term "variant" when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0227] As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterialartificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0228] As used throughout the disclosure, the term "variant" when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.

[0229] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No.4,554,101, incorporated fully herein by reference.

[0230] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0231] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables 1, 2, or 3 below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced bymodification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1.

[0232] Table 1 – Conservative Substitutions I

[0233] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set forth in Table 2.

[0234] Table 2 - Conservative Substitutions II

[0235] Alternately, exemplary conservative substitutions are set out in Table 3. Table 3 - Conservative Substitutions III

[0236] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.

[0237] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally- occur, rendering the entire amino acid sequence non-naturally occurring.

[0238] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[0239] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non- naturally occurring genome location.

[0240] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" is intended presenting to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. EXAMPLES

[0254] The Examples in this section are provided for illustration and are not intended to limit the invention.

[0255] Example 1: Construction of Chimeric Stimulatory Receptors Comprising Tumor Necrosis Factor Receptor 1 (TNFR1) Extracellular Binding Domains

[0256] The coding sequence of the extracellular domain and transmembrane domain of TNFR1 receptor (amino acids 1-234; SEQ ID NO: 7) was fused in-frame to the intracellular domain of a) the Insulin Like Growth Factor 1 receptor (IGF1R) (amino acids 930-1337; SEQ ID NO: 10) or b) the intracellular domain of cMet receptor (amino acids 958-1390; SEQ ID NO: 11) to generate a TNFR1-IGF1R fusion protein (SEQ ID NO: 12) or a TNFR1-cMET fusion protein (SEQ ID NO: 13), respectively.

[0257] Briefly, a nucleic acid molecule encoding amino acids 1-234 of the TNFR1 receptor and amino acids 930-1337 of the IGF1R receptor was synthesized (GeneArt™, Thermo Fisher) to generate the nucleic acid sequence encoding the TNFR1-IGF1R fusion protein (SEQ ID NO: 14). Similarly, the nucleic acid encoding amino acids 1-234 of the TNFR1receptor and amino acids 958-1390 of the cMet receptor was synthesized to generate the nucleic acid sequence encoding the TNFR1-cMET fusion protein (SEQ ID NO: 17).

[0258] Each of the nucleic acids was cloned into a T7 in vitro transcription vector for the production of recombinant mRNA for use in cell-based assays.

[0259] Example 2: Expression of TNFR1-Comprising Chimeric Stimulatory ReceptorsResults in a Dominant Negative Effect on NF B p65 Subunit Activation

[0260] This Example illustrates that the expression of TNFR1 comprising chimeric stimulatory receptors of the present disclosure in hepatocyte cell lines diminishes activation of cytokine stimulatory proteins.

[0261] On Day 1, approximately 62,500 Huh7 cells were seeded into 24 well plates in DMEM medium supplemented with 10% fetal bovine serum (FBS), GlutaMax™ (Thermo Fisher Scientific) and Pen / Strep and incubated overnight at 37°C in 5.0% CO2 atmosphere. On Day 2, cells were transfected with either 0 ng, 25 ng, 50 ng or 100 ng / well of an mRNA encoding the TNFR1-IGFR1 or TNFR1-cMET chimeric receptors comprising 5-methyl- cytosine modifications, a CleanCap® AG 5’ cap and an enzymatically added poly(A) tail, using lipofectamine MessengerMax™ (Thermo Fisher) in accordance with the manufacturer’s instructions. The transfected cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS), GlutaMax™ (Thermo Fisher) and Pen / Strep at 37°C in 5.0% CO2atmosphere for 24 hours. At 24 hours post-transfection, cellswere stimulated by incubating with 20 ng / ml tumor necrosis factor alpha (TNF ), 50 ng / ml ofinsulin-like growth factor 1 (IGF1), or 50 ng / ml of hepatocyte growth factor (HGF), respectively, for 20 minutes. Stimulated cells were harvested, lysed in RIPA Lysis and Extraction buffer (Thermo Fisher) in accordance with the manufacturer’s instructions to prepare cell lysates.

[0262] The cell lysates were subjected to electrophoresis through denaturing polyacrylamide gels, analyzed by Western blotting using a primary antibody targeting phosphorylated IGF1R (Cell Signaling Technology), a primary antibody targeting phosphorylated cMET (Cell Signaling Technology), or a primary antibody targetingphosphorylated NF B p65 subunit, and detected using an HRP-labeled secondary antibody.Beta-actin expression was used as an internal control to adjust for any well-to-well variance. The results are shown in FIGs.1A and 1B.

[0263] As shown in FIGs.1A and 1B, increasing amounts of mRNA encoding the TNFR1- IGF1R (FIG.1A) or TNFR1-cMET (FIG.1B) chimeric receptors resulted in a dose-dependent phosphorylation of each of the intracellular chimeric receptor domains. Exogenoustreatment with recombinant TNF did not further increase phosphorylation of the TNFR1-IGF1R or TNFR1-cMET chimeric receptors. Example 3: Expression of TNFR1 Comprising Chimeric Receptors Results in a Dominant Negative Down Regulation of Endogenous Cytokine Receptor Binding

[0264] This Example illustrates that the expression of TNFR1-comprising chimeric receptors of the present disclosure in hepatocyte cell lines results in a dominant negative down regulation of cytokine binding to endogenous receptors.

[0265] In a first experiment, 100 ng of mRNA encoding the TNFR1-IGF1R or TNFR1- cMET chimeric receptors was transfected into Huh7 hepatocytes as described in Example 3. Transfected cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS), GlutaMax™ and Pen / Strep at 37°C in 5.0% CO2 atmosphere.

[0266] Approximately 100,000 transfected cells expressing the TNFR1-IGF1R receptor, transfected cells expressing the TNFR1-cMet receptor or untransfected (mock) cell (positive control) were incubated in the absence or presence of 20 ng / ml of tumor necrosis factor alpha(TNF ; R&D Systems) in DMEM medium supplemented with 10% fetal bovine serum(FBS), GlutaMax™ and Pen / Strep culture medium for 6 hours. At 6 hours post-transfection, cells were harvested, lysed and total mRNA was isolated from cells using the RNeasy® Plus Kit (Qiagen) kit in accordance with the manufacturer’s instructions.

[0267] Isolated total RNA was subject to quantitative PCR (qPCR) to determine expressionlevels of the CXCL1 gene, which is positively upregulated by TNF , from cell samples.Quantitated mRNA levels were normalized relative to the HPRT housekeeping gene. The results are shown in Table A.

[0268] Table A: Relative CXCL1 mRNA fold expression (normalized to HPRT)

[0269] As shown in Table A, the expression of TNFR1-cMet or TNFR1-IGF1R chimericreceptors down regulated TNF -dependent induction of the CXCL1 gene approximately a 2-or 7-fold, respectively, by endogenous TNF receptors compared to positive control, untransfected cells. These results demonstrate that expression of the TNFR1-cMET or TNFR1-IGF1R chimeric receptors have a dominant negative effect on endogenous TNFR1receptors by competing for TNF binding. In addition, increasing doses of TNFR1-IGF1R orTNFR1-cMET chimeric receptor expression resulted in decreased phosphorylation of theNF B p65 subunit (see Fig. 1).

[0270] Example 4: Expression of TNFR1-Comprising Chimeric Stimulatory Receptors Induces Pro-Survival Cell Signaling

[0271] This Example illustrates that the expression of TNFR1-comprising chimeric receptors of the present disclosure in hepatocyte cell lines induces pro-survival cell signaling independent of the absence or presence of cytokines.

[0272] On Day 1,100,000 Hepa 1-6 cells were seeded into 24 well plates in DMEM medium supplemented with 10% fetal bovine serum (FBS), GlutaMax™ and Pen / Strep and incubated overnight at 37°C in 5.0% CO2atmosphere. On Day 2, cells were transfected with 100 ng / well of an mRNA encoding the TNFR1-IGF1R or TNFR1-cMET chimeric receptors comprising 5-methyl-cytosine modifications, a CleanCap® AG 5’ cap and an enzymatically added poly(A) tail, using a lipofectamine MessengerMax™ (Thermo Fisher) in accordance with the manufacturer’s instructions. The transfected cells were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS), GlutaMax™ and Pen / Strep at 37°C in 5.0% CO2 atmosphere for five hours, the culture medium was changed to low-serum DMEM medium supplemented with 0.1% fetal bovine serum (FBS), GlutaMax™ and Pen / Strep and the cells incubated for an additional 14 hours. At 19 hours post-transfection, cells were stimulated by incubating with 50 ng / ml of insulin-like growth factor 1 (IGF1), 50ng / ml of hepatocyte growth factor (HGF), or 20 ng / ml TNF respectively, for 20 minutes.Stimulated cells were harvested, lysed in RIPA Lysis and Extraction buffer (ThermoFisher) in accordance with the manufacturer’s instructions to prepare cell lysates.

[0273] The cell lysates were subjected to electrophoresis through denaturing polyacrylamide gels, analyzed by Western blotting using a primary antibody targeting phosphorylated IGF1R (Cell Signaling Technology), a primary antibody targeting phosphorylated cMET (Cell Signaling Technology), a primary antibody targeting phosphorylated protein kinase B (AKT; Cell Signaling Technology), or a primary antibodytargeting phosphorylated ribosomal protein S6 (Cell Signaling Technology), and detected using an HRP-labeled secondary antibody. Beta-actin expression was used as an internal control to adjust for any well-to-well variance. The results are shown in FIG.2.

[0274] As shown in FIG.2, the expression of TNFR1-IGF1R (FIG.2B) or TNFR1-cMET (FIG.2C) chimeric receptors resulted in phosphorylation and activation of the AKT and S6 proteins, with expression of the TNFR1-IGFR1 receptor eliciting a more robust activation than expression of the TNFR1-cMET receptor. Moreover, in each instance, the degree of activation of AKT and S6 was similar in the absence or presence of the cognate cytokinesTNF . These results demonstrate that expression of the TNFR1-IGF1R or TNFR1-cMETchimeric stimulatory receptors elicit a cytokine-independent activation of pro-survival signaling represented by phosphorylation of AKT and S6.

[0275] Example 5: Expression of TNFR1 -Comprising Chimeric Receptors Reduces In Vivo Cytokine Expression and Transaminase Levels

[0276] This Example illustrates that the expression of TNFR1-comprising chimeric receptors of the present disclosure in vivo reduces cytokine.

[0277] Adult male C57 mice (4 mice / group) were divided into 3 treatment groups and 1 control group. Control group mice were administered PBS, whereas the three treatment groups were intravenously administered 1.0 mg / kg of a lipid nanoparticle composition (40 mol% terpene lipidoid HMA-404 (e.g., see International Patent Application Publication No: WO2023141576); 52.5 mol% cholesterol; 5 mol% DOPC, 2 mol% DMG-PEG2k and 0.5 mol% GalNac-DSPE-PEG; lipid:NA 50:1) comprising:

[0278] (Group 2) a) 0.67 mg / kg of a DNA piggyBac transposon comprising the Factor VIII X5 variant gene under the control of the TTRe promoter and b) 0.33 mg / kg of an mRNA encoding a catalytically dead Super PiggyBac transposase;

[0279] (Group 3) a) 0.67 mg / kg of a DNA piggyBac transposon comprising the Factor VIII X5 variant gene under the control of the TTRe promoter and b) 0.33 mg / kg of an mRNA encoding the TNFR1-IGF1R chimeric receptor; or

[0280] (Group 4) a) 0.67 mg / kg of a DNA piggyBac transposon comprising the Factor VIII X5 variant gene under the control of the TTRe promoter and b) 0.33 mg / kg of an mRNA encoding the TNFR1-cMet chimeric receptor.

[0281] At 4 hours post LNP administration, blood samples were retro-orbitally collected from mice for cytokine level analysis. At 24 hours, mice were weighed, terminally bled in accordance with standard laboratory protocols, and the livers were perfused and collected.

[0282] The four-hour serum concentration of the cytokines IL6, interferon-gamma (IFN- ),TNF or MCP-1 were determined by MSD assay for the control and treatment groups. Theresults (pg / ml serum) are shown in Table B.

[0283] Table B: Serum cytokine concentration (pg / ml) 4h post LNP delivery

[0284] As shown in Table B, TNF , IL6 and IFN- concentrations in serum were unaffectedby the delivery and expression of TNFR1-comprising chimeric receptors (Group 3 and Group4 compared to control Group 2), whereas MCP-1 expression, regulated by TNF and NF B,was reduced in mice expressing the TNFR1-IGF1R (Group 3) and TNFR1-cMet chimeric receptors (Group 4).

[0285] The serum concentrations of the liver transaminases alanine transaminase (ALT), and aspartate transferase (ASP) were determined from control and treated animals by IDEXX. The results are shown in Table C.

[0286] Table C: Serum transaminase concentration (U / L) 24h post LNP delivery

[0287] As shown in Table C, the expression of the TNFR1-IGF1R (Group 3) and TNFR1- cMet (Group 4) chimeric receptors resulted in 2-fold and 3-fold reduction, respectively, in the liver transaminase ALT compared to control group (Group 2), and a 2-fold decrease in AST serum concentrations for TNFR1-cMet expressing mice (Group 4).

[0288] These results demonstrate that the TNFR1-comprising chimeric receptors arecapable in vivo of reducing TNF -dependent cytokine levels and serum transaminases inmice resulting from DNA / RNA LNP delivery.

Claims

We Claim:

1. A chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, comprising: a. a TNFR1 extracellular domain comprising the amino acid sequence of SEQ ID NO: 8; b. a TNFR1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 9; and c. an insulin-like growth factor receptor 1 (IGFR1) intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:

10.

2. A chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein, comprising: a. a TNFR1 extracellular domain comprising the amino acid sequence of SEQ ID NO: 8; b. a TNFR1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 9; and c. a c-MET receptor intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:

11.

3. The chimeric TNFR1 fusion protein of claim 1 or claim 2, wherein the TNFR1 extracellular domain and the TNFR transmembrane domain comprise the amino acid sequence of SEQ ID NO:

7.

4. The chimeric TNFR1 fusion protein of claim 1, wherein the TNFR1 fusion protein comprises the amino acid sequence of SEQ ID NO:

12.

5. The chimeric TNFR1 fusion protein of claim 2, wherein the TNFR1 fusion protein comprises the amino acid sequence of SEQ ID NO:

13.

6. A composition comprising a chimeric TNFR1 fusion protein of any one of claims 1-5.

7. A polynucleotide encoding a chimeric TNFR1 fusion protein of any one of claims 1-5.

8. The polynucleotide of claim 7, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:

19.

9. The polynucleotide of claim 7, wherein the polynucleotide is DNA.

10. The polynucleotide of claim 7, wherein the polynucleotide is RNA.

11. The polynucleotide of claim 10, wherein the RNA is an mRNA.

12. The polynucleotide of claim 11, wherein the mRNA comprises a 5’-CAP.

13. The polynucleotide of claim 12, wherein the 5’-CAP is a 5’ CleanCap®.

14. A vector comprising a polynucleotide of any one of claims 7-13.

15. A composition comprising a polynucleotide of any one of claims 7-13.

16. A lipid nanoparticle composition (LNP) comprising the composition of claim 15.

17. The LNP of claim 16, wherein the LNP comprises at least one RNA molecule and / or at least one DNA molecule.

18. The LNP of claim 17, wherein the LNP comprises about 40% of HMA-404 by moles, about 52.5% of cholesterol by moles, about 5% of DOPC by moles, about 2% of DMG-PEG2000 by moles, and about 0.5 mol% of a targeting ligand comprising GalNac; wherein the at least one RNA molecule comprises a polynucleotide encoding a chimeric TNFR1 fusion protein; wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w);and wherein HMA-404 comprises the following structure:.

19. The LNP of claim 18, wherein the RNA molecule is an mRNA molecule, preferably wherein the mRNA molecule further comprises a 5’-CAP.

20. The LNP of any one of claims 17-19, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, preferably wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a helitron transposase, a Tol2 transposase, a TcBuster transposase or a mutant TcBuster transposase.

21. The LNP of any one of claims 17-19, wherein the DNA molecule is a circular DNA molecule, a DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule.

22. The LNP of any one of claims 17-19, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

23. The LNP of any one of claims 17-22, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding at least one therapeutic protein.

24. The LNP of any one of claims 17-23, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding at least one transposon, wherein the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.

25. A pharmaceutical composition comprising a composition of any one of the preceding claims and at least one pharmaceutically-acceptable excipient or diluent.

26. A method of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with the composition of any one of claims 6, 15, or 25.

27. A method of genetically modifying at least one cell comprising contacting the at least one cell with the composition of claim 6, claim 15, or claim 25.

28. The method of claim 26 or claim 27, wherein the at least one cell is a liver cell.

29. The method of claim 28, wherein the liver cell is a hepatocyte, a hepatic stellate cell, a Kupffer cell or a liver sinusoidal endothelial cell.

30. A method of treating at least one disease or disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of claim 6, claim 15, or claim 25.

31. The method of claim 30, wherein the at least one disease or disorder is a liver disease or disorder.

32. A method for reducing TNF -mediated cytokine levels in a cell, comprisingcontacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein; wherein the TNF -mediated cytokine levels in the cell are reduced at least 2-fold compared to TNF -mediated cytokine levels in a cell that is contacted with acomposition that does not comprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein.

33. A method for reducing transaminase expression in a cell, comprising contacting the cell with a composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein; wherein the transaminase expression in the cell is reduced at least 2-fold compared to transaminase expression in a cell that is contacted with a compositionthat does not comprise a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein.

34. The method of claim 32 or claim 33, wherein the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises: a. a TNFR1 extracellular domain; b. a TNFR1 transmembrane domain; and c. an insulin-like growth factor receptor 1 (IGFR1) intracellular signaling domain.

35. The method of claim 32 or claim 33, wherein the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises: a. a TNFR1 extracellular domain; b. a TNFR1 transmembrane domain; and c. a c-MET receptor intracellular signaling domain.

36. The method of claim 34, wherein the IGFR1 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

10.

37. The method of claim 35, wherein the c-MET intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

11.

38. The method of any one of claims 33-37, wherein the TNFR1 extracellular domain comprises the amino acid sequence of SEQ ID NO:

8.

39. The method of any one of claims 33-37, wherein the TNFR1 transmembrane domain comprises the amino acid sequence of SEQ ID NO:

9.

40. The method of any one of claims 33-39, wherein the TNFR1 extracellular domain and the TNFR transmembrane domain comprise the amino acid sequence of SEQ ID NO:

7.

41. The method of any one of claims 33-34, wherein the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises the amino acid sequence of SEQ ID NO:

12.

42. The method of claim 32, claim 33 or claim 35, wherein the chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises the amino acid sequence of SEQ ID NO:

13.

43. The method of claim 32 or claim 33, wherein the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

44. The method of claim 32 or claim 33, wherein the polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein comprises the nucleic acid sequence of SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO:

19.

45. The method of any one of claims 32-44, wherein the cell is a liver cell.

46. The method of claim 45, wherein the liver cell is a hepatocyte, a hepatic stellate cell, a Kupffer cell or a liver sinusoidal endothelial cell.

47. The method of claim 32 or claim 33, wherein the composition comprising a polynucleotide encoding a chimeric tumor necrosis factor receptor 1 (TNFR1) fusion protein is encapsulated in at least one lipid nanoparticle composition.

48. The method of claim 47, wherein the at least one LNP comprises about 40% of HMA- 404 by moles, about 52.5% of cholesterol by moles, about 5% of DOPC by moles, about 2% of DMG-PEG2000 by moles, and about 0.5 mol% of a targeting ligand comprising GalNac; wherein the polynucleotide encoding a chimeric TNFR1 fusion protein is RNA, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w); and wherein HMA-404 comprises the following structure:.

Images