Chimeric protein compositions for cancer therapy and methods of use thereof

Abstract

Disclosed are useful compositions of mixed lineage kinase domain-like (MLKL) derived fusion constructs and their methods of use in cellular depletion therapies. Embodiments of the present disclosure provide a chimeric cell death protein that induces immunogenic cell death in various types of cancers and also has applications in viral infection. The present disclosure also provides different methods of delivering the disclosed compositions or fusion constructs to a subject with cancer or tumor.

Description

CHIMERIC PROTEIN COMPOSITIONS FOR CANCER THERAPY AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U. S. Provisionai Application No.63 / 716,137, filed November 4, 2024, the entire contents of which are hereby incorporated by reference.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to fusion proteins inducing immunogenic cell death. Particularly, the disclosure relates to useful compositions of mixed lineage kinase domain-like (MLKL) derived fusion protein and methods of use thereof.SEQUENCE LISTING

[0003] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence. xml” (10,000 bytes), which was created on October 31. 2025, which is incorporated by reference herein.BACKGROUND OF THE DISCLOSURE

[0004] Targeted cancer cell death is the chief objective of many cancer therapeutic interventions. While classical chemotherapeutic agents (e.g., alkylating agents, anti-metabolic agents, tubule destabilizing agents, topoisomerase inhibitors, DNA binding agent, etc.) excel at killing ceils they are non-specific, necessitating lower doses to preserve the integrity of non-cancerous tissues. Furthermore, in vivo tumor selection and evolution often results in tumor populations with perturbed cell death pathways, as tumors have spent a lifetime ignoring, circumventing or rewiring classical cell death signaling pathways to survive and jeopardize host mortality. Thus, many tumors are refractory to the lower doses of these classical chemotherapeutic treatments that are necessary to spare host functionality. Yet these doses are still sufficiently potent to drastically reduce functionality of host organ systems causing toxicity. In addition, it has been noted for decades that the classical cell death program of apoptosis intended to be evoked by many of these regimens, can result in immune tolerance, conceptually further hindering the establishment of immunological memory and immune surveillance of residual tumors which may eventually give rise to cancer recurrence.

[0005] The advancement of immunotherapy regimens in the clinic over the past 15 years has demonstrated the great potential in evoking or facilitating anti-tumor immunity for the eradication of established disease and prevention of cancer recurrence. To this end, the development of therapeutic agents that facilitate tumor-specific and immunogenic rather than immunosuppressive cell death has garnered great interest through a wide number of platforms and approaches in the pharmaceutical and biotechnological space. Gene therapy, genomic medicine, and nucleic acid medicine have soared to new heights as a feasible approach to address pressing public health needs. Additionally, a multitude of delivery vehicles for both mRNA and DNA, with a focus on organ-specific and non-toxic delivery of nucleic acids have been reported. However, how the genetic cargo may be manipulated efficiently to fully harness the opportunity of this ever-widening landscape of genetic medicine applications for cancer, remains incompletely understood. This knowledge is important to successfully develop strategies for therapeutic treatments of cancer patients.SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides useful compositions of mixed lineage kinase domain-like (MLKL) derived fusion protein and methods of use thereof in cellular depletion therapies which include applications for cancer, vaccination, and viral infection.

[0007] Embodiments provide for a composition comprising a nucleic acid encoding an isolated mixed lineage kinase domain-like (MLKL) protein for use in inhibition of a tumor in a subject harboring the tumor. In some embodiments, the nucleic acid encoding the isolated MLKL protein further comprises a sequence encoding transferrin receptor. In some embodiments, the isolated MLKL protein induces necroptotic-like death of the tumor in the subject. In some embodiments, the tumor is selected from the group of leukemia, colorectal cancer, breast cancer, and ovarian cancer. In some embodiments, the nucleic acid is delivered to the subject using a nanocarrier. In some cases, the composition is administered to the subject intratumorally.

[0008] Embodiments also provide for a composition comprising a nucleic acid molecule encoding a chimeric protein for use in treatment of a tumor, cancer, or neoplasm in a subject in need thereof, the chimeric protein comprising a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor. In some embodiments, the isolated MLKL protein of the chimeric protein comprises a four helical bundle (4HB) domain and a brace region of a native MLKL protein. In some cases, the isolated MLKL protein of the chimeric protein is devoid of a pseudokinase domain of the native MLKL protein. In some embodiments, the isolated transferrin receptor of the chimeric protein comprises a cytoplasmic domain and a transmembranedomain of a native human transferrin receptor. In some embodiments, the nucleic acid molecule encoding the chimeric protein is DNA, wherein the DNA is plasmid DNA, linear double strand DNA, DNA included in a viral vector, or complexed DNA. In some embodiments, the nucleic acid molecule encoding the chimeric protein is RNA, wherein the RNA is mRNA, RNA included in a viral vector, or complexed (m)RNA. In some embodiments, the nucleic acid molecule encoding the chimeric protein is delivered to the tumor, cancer, or neoplasm of the subject using a lipidpolymeric hybrid nanoparticle system. In some embodiments, an expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof.

[0009] Embodiments provide for a method of treating a tumor, cancer, or neoplasm in a subject experiencing the tumor, cancer, or neoplasm, the method comprising administering to the subject in need thereof a therapeutically effective amount of the composition comprising a nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MIKL) protein and an isolated transferrin receptor. In some embodiments, the nucleic acid composition further comprises a nanocarrier for delivering the composition to the subject. Embodiments also provide for a method of treating a tumor, cancer, or neoplasm in a subject in need thereof, the method comprising: (a) providing a composition comprising a nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor; (b) combining the nucleic acid molecule encoding the chimeric protein with a nanocarrier; and (c) administering an effective amount of the nucleic acid molecule combined with the nanocarrier to the subject for treating the tumor, cancer, or neoplasm. In some embodiments, an expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof. In some embodiments, the nanocarrier or delivery vehicle comprises a system selected from the group of lentivirus, adenovirus, polymeric nanoparticle, lipid-polymeric hybrid nanoparticle, liposome, lipid polyplex, polymersome, dendrimer, metal-based nanoparticle, mesosilicate nanoparticle, exosome, membrane-derived nanovesicle, virus like particle, protein nanocage, proteolipid vehicle, lipid nanoparticle, and stabilized plasmid-lipid particle.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserve the right to present color images of the drawings in later proceedings.

[0011] Figures 1A-1D. Expression of MLKL-TfR in human K562 leukemic cell line induces ZVAD uninhibitable necrotic cell death. Fig. 1A depicts representative flow plots for validation of Annexin V (AV) / Live Dead NIR (LD) apoptosis / necrosis flow cytometric assay in K562 human leukemic cell lines Lenti-virally transduced with Doxycycline (Doxy) inducible MLKL-TfR constructs, treated with apoptosis inducer Mitomycin C (Mme) and pan-Caspase inhibitor peptide ZVAD (Zvad). Fig. 1B depicts representative Annexin V / Live Dead-NIR flow plots after doxycycline induced MLKL-TfR expression. Fig. 1C shows quantitation of apoptotic (AV+, LD-) and necrotic (AV+, LD+) cell frequency 24 hours post treatment (tx) in K562 cell lines. Fig. 1 D shows time course evaluation of apoptotic and necrotic cell frequency post doxycycline treatment in WT K562 (light gray) or cell lines expressing doxycycline induced expression of amino acids 1-210 of MLKL alone (dark gray) or MLKL-TfR chimeric protein (black). For all data N= 4 experimental replicates, error bar = standard error of the mean. Comparison significance was determined by two-way anova for time course experiments and two-tailed student’s T-test for single variable comparisons.

[0012] Figures 2A-2C. Expression of MLKL-TfR induces rapid membrane permeable cell death in phenotypically normal EPH4 mouse mammary epithelial cells. Doxycycline inducible MLKL-TfR genetic constructs were stably integrated into the ROSA26 safe harbor locus utilizing CRISPR-CAS9 genetic recombination strategies in the phenotypically normal, immortalized mouse mammary epithelial cell line EPH4. Fig. 2A shows representative live-cell phase / fluorescence images of MLKL-TfR construct containing EPH4 cell line treated with pan-kinase inhibitor and prototypical apoptosis induced staurosporine (Stauro, 500 nM), necroptosis inducer Nigericin (Ngrcn, 20 pM) or doxycycline (Doxy, 2 pg / mL). All wells contain DNA responsive fluorescence dye Nuc Green Dead (NGD), demonstrating plasma and nuclear membrane permeability. EPH4 cells containing genetic constructs encoding MLKL-TfR under regulation of a doxycycline inducible control element were treated with DMSO (vehicle, black open box), Doxycycline (Doxy, 2 pg / mL, filled black circle), Doxycycline with 30 minute pretreatment of 20 pM ZVAD (Doxy+ Zvad, open gray circle), Staurosporine (Stauro, 500 nm, black up filled triangle) or 20 pM Nigericin (Ngrcn, gray open down triangle) and were evaluated for cell confluence as a fraction of well area over time as indication of cell growth or fraction of cellular phase area positive for NGD signal over time as an indicator of necrotic (nec) cell death (Fig. 2B). EPH4 cells containing ROSA26 recombined genetic constructs containing doxycycline inducible expression of MLKL-TfR-eGFP were likewise treated and evaluated for cell growth and necrotic cell death (Fig. 2C). For all data N= 4 experimental replicates, error bar = standard error of the mean. Comparison significancewas determined by two-way anova for time course experiments and two-tailed student’s T-test for single variable comparisons.

[0013] Figures 3A-3D. MLKL-TfR induced cell death results in canonical immunogenic cell death and Damage Associated Molecular Pattern (DAMP) features. EPH4 cells containing ROSA26 recombined genetic constructs coding for doxycycline inducible MLKL-TfR or MLKL-TfR-eGFP were treated with either DMSO (Vehicle control, black open box), doxycycline (Doxy, 2 pg / mL, filled black circle), staurosporine (Stauro, 500 nm, black up filled triangle) or nigericin (Ngrcn, 20 pM, gray open down triangle) and evaluated for release of extracellular ATP by luciferase detection assay (Fig. 3A), extracellular release of HMGB1 by luciferase assisted ELISA assay (Fig. 3B), caspase 1 activity by phase / fluorescence imaging of fluorescence inhibitor of caspase activity agent (Fig. 3C), and non-endoplasmic reticulum associated cellular localization of Calreticulin (CALR) by phase / fluorescence microscopy using anti-CALR antibodies and nuclear counterstain (nuclear red dead, NRD) (Fig. 3D). For all data N= 4 experimental replicates, error bar = standard error of the mean. Comparison significance was determined by two-way anova for time course experiments and two-tailed student’s T-test for single variable comparisons.

[0014] Figures 4A-4J. Expression of MLKL-TfR in MC38 mouse colorectal cancer cell line induces rapid, necrosis like immunogenic cell death in vitro and in vivo. Utilizing CRISPR-CAS9 genetic recombination approaches genetic constructs containing doxycycline inducible expression of MLKL-TfR-eGFP were introduced into the ROSA26 locus in the MC38 mouse colorectal cancer ceil line. Fig. 4A shows live cell phase fluorescence imaging utilizing Nuc Green Dead (NGD) reagent demonstrated upon doxy treatment indicating rapid shrinking of cells. Fig.4B shows decreased cell growth as indicated by a plot showing cellular confluence overtime from phase imaging. Fig. 4C shows rapid cell permeability and cellular necrosis as indicated by ratio of NGD staining per phase area over time. NOD. SCI D. common gamma chain deficient (NCG) mice were then administered with 1x106MLKL-TfR-eGFP construct containing MC38 cells in the flank and permitted to grow to a group size of 35mm2, upon which time mice received either vehicle (black open circles, n=12) or 200 pg doxycycline (black filled circles, n=13) intraperitoneally, every other day. Mice were monitored for tumor size and survival frequencies. Figs. 4D and 4E depict plots showing tumor size over days post treatment. Tumor size >200mm2is considered terminal size (dashed line). Fig. 4F depicts a plot showing survival frequencies over days post treatment. Syngeneic wildtype C57BL6 (B6) were similarly implanted in the flank with MLKL-TfR-eGFP MC38 tumors and likewise treated with either vehicle (n=10) or doxycycline (n=11) and monitored for tumor size and overall survival frequencies. Figs. 4G and 4H depict plots showing tumor size over days post treatment. Fig. 4I depicts a plot showing survivalfrequencies over days post treatment. In addition, the number of days a mouse survived once their tumor reached 100mm2was determined and plotted for both NCG and 86 hosts in Fig. 4J, illuminating the role of the adaptive immune response in controlling tumor growth after induction of MLKL-TfR-eGFP expression with doxycycline administration. All in vivo experiments were conducted as two separate experimental replicates, all biological replicates are depicted. For in vitro assays N= 4 experimental replicates. For all experiments, error bar = standard error of the mean. Comparison significance was determined by two-way anova for time course experiments and two-tailed student’s T-test for single variable comparisons.

[0015] Figures 5A-5G. PTTG1 expression correlates with poor survival. PTTG1 promoter enables PPDP2 nanoparticle MLKL-TfR-eGFP cancer therapy in vivo. Fig. 5A shows FPKM Upper Quartile (UQ) normalized PTTG1 expression in human normal (black circles, n) and cancer (gray circles, t) tissues (Pancreatic = Pancr, Mesothelioma = Meso) from TCGA dataset. Fig. 5B shows that Hi PTTG1 expression (black line) predicts poorer overall survival 5 years post primary diagnosis compared to low PTTG1 expression (grey line). Fig. 5C shows lipofection of CT26 cell line utilizing 4K commercial transfection reagents containing plasmid DNA encoding PTTG1 promoter driven GFP expression in a commercially available DNA vaccine backbone (NTC7482) demonstrating successful GFP expression. Fig. 5D shows representative digital light scattering (dis) distribution of plasmid incubated, self-assembled, dialyzed PPDP2 self-assembling lipid coblock polymeric nanoparticles. Peak ~ 160nm represents pDNA containing nanoparticles while peak at 29nm represents PPDP2 only self-assembled nanoparticles. Average size determination derived from assessment of separate nanoparticle assemblies (PPDP2 alone, n=7, PPDP2 with Plasmid, n=20) and error is reported as standard deviation. Wild type Balb / c mice were implanted unilaterally in the flank with 4 x105wild-type CT26 mouse colorectal cancer cells. Tumors were grown to an average size of 35mm2upon which mice received 15 pg of plasmid DNA encoding constructs CMV-GFP (black open circles, n=9) or PTTG1-MLKL-TfR-eGFP (black filled circles, n=10) encapsulated by the PPDP2 nanoparticles or equivalent amounts of nanoparticles consisting of polymer alone (grey open circles, n~7) or equivalent vehicle volume alone (grey open squares, n=7) every 4 days. Mice were monitored for tumor growth and overall survival calculated based upon time post treatment tumors reached 200mm2(dashed line). Figs. 5E and 5F depict plots showing tumor size over days post treatment. Fig. 5G depicts a plot showing survival frequencies over days post treatment. All in vivo experiments were conducted as 2 separate experimental replicates, with all biological replicates quantified. Differences in tumor growth over time were determined by two-way anova, while survival significance was determinedby Log-rank (Mantel-Cox) test, with Hazard Ratios (HR) calculated based upon log-rank determination.REFERENCE TO SEQUENCE LISTING

[0016] The nucleic acid and / or amino acid sequences described herein are shown using standard letter abbreviations, as defined in 37 C. F. R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate. Each sequence herein has been given a unique sequence ID number (SEQ ID NO), as shown below. Each nucleic acid sequence has a 5’-end and a 3’-end. The change in uppercase nucleotide residues to lowercase nucleotide residues and vice versa indicate different genetic elements. The numbering of nucleotide residues in each nucleic acid sequence begins from 5’-end and ends at 3’-end. Similarly, the numbering of amino acid residues in each protein sequence starts from N-terminus (amino acid number 1) and goes to C-terminus.

[0017] SEQ ID NO: 1 is the nucleic acid sequence for human MLKL-TfR chimeric construct: 5’ATGgaaaacctgaaacatattattaccctgggccaggtgattcataaacgctgcgaagaaatgaaatattgcaaaaaacagtgc cgccgcctgggccaCcgcgtgctgggcctgattaaaccgctggaaatgctgcaggatcagggcaaacgcagcgtgccgagcgaa aaactgaccaccgcgatgaaccgctttaaagcggcgctggaagaagcgaacggcgaaattgaaaaatttagcaaccgcagcaac atttgccgctttctgaccgcgagccaggataaaattctgtttaaagatgtgaaccgcaaactgagcgatgtgtggaaagaactgagcct gctgctgcaggtggaacagcgGatgccggtgagcccgattagccagggcgcgagctgggcgcaggaagatcagcaggatgcgg atgaagatcgccgcgcgtttcagatgctgcgccgcgataacgaaaaaattgaagcgagcctgcgccgcctggaaattaacatgaaa gaaattaaagaaaccctgcgccagtatctgccgccgaaatgcatgcaggaaattccgcaggaacagattaaagaaattaaaaaag aacagctgagcggcagcccgtggattctgctgcgcGATCAAGCTAGATCAGCATTCTCTAACTTGTTTGGTG GAGAACCATTGTCATATACCCGGTTCAGCCTGGCTCGGCAAGTAGATGGCGATAACAGTC ATGTGGAGATGAAACTTGCTGTAGATGAAGAAGAAAATGCTGACAATAACACAAAGGCCAA TGTCACAAAACCAAAAAGGTGTAGTGGAAGTATCTGCTATGGGACTATTGCTGTGATCGTC TTTTTCTTGATTGGATTTATGATTGGCTACTTGGGCTATTGTAAAGGGGTAGAACCAAAAAC TGAGTGTGAGAGACTGGCAGGAACCGAGTCTCCAGTGAGGGAGGAGCCAGGAGAGGACT TCCCTGCAtaa3’. The first three uppercase nucleotide residues (residue 1, 2, and 3) at the 5’-end indicates the translational start codon. The following lowercase nucleotide residues (residue 4 - residue 630 of SEQ ID NO: 1) code for MLKL (amino acids 2-210 of SEQ ID NO: 2). The following uppercase nucleotide residues (residue 631 - residue 978 of SEQ ID NO: 1) code for TfR (amino acids 211-326 of SEQ ID NO: 2). The last three lowercase nucleotide residues (residue 979, 980, and 981) at the 3’-end indicates the translational stop codon. Additionally, anyrapid shift within a given uppercase or lowercase nucleotide sequence notates changes made for codon optimization.

[0018] SEQ ID NO: 2 is the amino acid sequence for MLKL-TfR human chimeric protein: MENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHRVLGLIKPLEMLQDQGKRSVPSEKLTTAMNR FKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQ GASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQ IKEIKKEQLSGSPWILLRDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENA DNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPG EDFPA. Amino acid residue 1 at N-terminus to amino acid residue 210 of SEQ ID NO: 2 indicates the MLKL (amino acids 1-210) protein sequence. Amino acid residue 211 to amino acid residue 326 at C-terminus of SEQ ID NO: 2 indicates the TfR (amino acids 3-118 (https: / / www.uniprot.org / uniprotkb / P02786 / entry as of November 04, 2025)) protein sequence.

[0019] SEQ ID NO: 3 is the nucleic acid sequence for MLKL-TfR-eGFP chimeric construct: 5’ATGgaaaacctgaaacatattattaccctgggccaggtgattcataaacgctgcgaagaaatgaaatattgcaaaaaacagtgc cgccgcctgggccaCcgcgtgctgggcctgattaaaccgctggaaatgctgcaggatcagggcaaacgcagcgtgccgagcgaa aaactgaccaccgcgatgaaccgctttaaagcggcgctggaagaagcgaacggcgaaattgaaaaatttagcaaccgcagcaac atttgccgctttctgaccgcgagccaggataaaattctgtttaaagatgtgaaccgcaaactgagcgatgtgtggaaagaactgagcct gctgctgcaggtggaacagcgGatgccggtgagcccgattagccagggcgcgagctgggcgcaggaagatcagcaggatgcgg atgaagatcgccgcgcgtttcagatgctgcgccgcgataacgaaaaaattgaagcgagcctgcgccgcctggaaattaacatgaaa gaaattaaagaaaccctgcgccagtatctgccgccgaaatgcatgcaggaaattccgcaggaacagattaaagaaattaaaaaag aacagctgagcggcagcccgtggattctgctgcgcGATCAAGCTAGATCAGCATTCTCTAACTTGTTTGGTG GAGAACCATTGTCATATACCCGGTTCAGCCTGGCTCGGCAAGTAGATGGCGATAACAGTC ATGTGGAGATGAAACTTGCTGTAGATGAAGAAGAAAATGCTGACAATAACACAAAGGCCAA TGTCACAAAACCAAAAAGGTGTAGTGGAAGTATCTGCTATGGGACTATTGCTGTGATCGTC TTTTTCTTGATTGGATTTATGATTGGCTACTTGGGCTATTGTAAAGGGGTAGAACCAAAAAC TGAGTGTGAGAGACTGGCAGGAACCGAGTCTCCAGTGAGGGAGGAGCCAGGAGAGGACT TCCCTGCAgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggcc acaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct gcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacg acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgcc gaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctgggg cacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaag atccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctg ctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagTTAtacaagtaa3’. The first three uppercase nucleotide residues (residue 1, 2, and 3) at the 5’-end indicates the translational start codon. The following lowercase nucleotide residues (residue 4 - residue 630 of SEQ ID NO: 3) code for MLKL (amino acids 2-210 of SEQ ID NO: 4). The following uppercase nucleotide residues (residue 631 -residue 978 of SEQ ID NO: 3) code forTfR (amino acids 211-326 of SEQ ID NO: 4). The following lowercase nucleotide residues (residue 979 - residue 1692 of SEQ ID NO: 3) code for GFP. The last three lowercase nucleotide residues (residue 1693, 1694, and 1695) at the 3’-end indicates the translational stop codon. Additionally, any rapid shift within a given uppercase or lowercase nucleotide sequence notates changes made for codon optimization.

[0020] SEQ ID NO: 4 is the amino acid sequence for MLKL-TfR-eGFP chimeric protein: MENLKHIITLGQVIHKRCEEMKYCKKQCRRLGHRVLGUKPLEMLQDQGKRSVPSEKLTTAMNR FKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKELSLLLQVEQRMPVSPISQ GASWAQEDQQDADEDRRAFQMLRRDNEKIEASLRRLEINMKEIKETLRQYLPPKCMQEIPQEQ IKEIKKEQLSGSPWILLRDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENA DNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPG EDFPAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK. Amino acid residue 1 at N-terminus to amino acid residue 210 of SEQ ID NO: 4 indicates the MLKL (amino acids 1-210) protein sequence. Amino acid residue 211 to amino acid residue 326 of SEQ ID NO: 4 indicates the TfR (amino acids 3-118 (https: / / www.uniprot.org / uniprotkb / P02786 / entry as of November 04, 2025)) protein sequence. Amino acid residue 327 to amino acid residue 564 at C-terminus of SEQ ID NO: 4 indicates the GFP protein sequence.

[0021] SEQ ID NO: 5 is the nucleic acid sequence for PTTG1 promoter (561 base pairs): 5’ATTGGTTCTACTTGGTGACCACGCCCACGGCCCCGCCTCCTGGGCGGAAGAGCCAATTG GGCCGCGAGTTGTGGTTTAAACCAGGAGTGCGCCGCGTCCGTTCACCGCGGCCTCAGAT GAATGCGGCTGTTAAGACCTGCGTGAGTGAATGGGAGGGTCGCGGGTGGTTAGTTGAGC CGGCTCCGGCGGGGAAGGAGGCGGGCTGCGGCTGCGGCTGGGGCTGAAGCTGGGGCT GGGGTTGGGGGACTGCCCGGGGCTTAGATGGCTCCGAGCCCGTTTGAGCGTGGTCTCGG ACTGCTAACTGGACCAACGGCAACTGTCTGATGAGTGCCAGCCCCAAACCGCGCGCTGCT CGGGACCTTAGAGCCTCTGACTCAGGCTGGAAGATTTGAGAGCTGGATTAAGTACTTGTTG GCTCACGCCCGTGACTGTTCCGCTGTTTAGCTCTTGTTTTTTGTGTGGACACTCCTAGGAT AGAAAGTTTGGTATGTTGCTATACCTTTGCTTCTCCCACCTTCCCCAATATCTAATATGTATTTCTCATTCTTAGAATAATCCAGA3’. The nucleic acid sequence shown in SEQ ID NO: 5 is a selected portion of the human promoter for PTTG1 based upon detailed analysis of transcription factor binding regions and sequence conservation across species, predicted to confer positive transcriptional activity.DETAILED DESCRIPTION

[0022] Treatment modalities seek to achieve the cancer free state by inducing tumor cell death whether through small molecule inhibitors, cellular, radiation, immuno, or chemotherapies. Tumors evade the induction of cell death through competing survival pathways. Even when executed to completion, the classical programmed cell death of apoptosis induces immune tolerance impairing further immune mediated tumor cell removal. Leveraging advances in gene therapy vehicles, gene expression data, and immune and bioengineering principals, the present disclosure provides a chimeric cell death protein, MLKL-TfR that induces unavoidable, immunogenic cell death. Therapeutically, expression of this potent cell death molecule is achieved through plasmid DNA encoded tumor specific promoter control of the construct, which is delivered to the tumor by a self-assembling polymeric lipid nanoparticle PPS-PPDP2.

[0023] Thus, the present disclosure provides novel compositions and methods of use of the genetic construct platform (MLKL-TfR) for initiating immunogenic cell death. Through utilization of multiple species (mouse, human) and multiple cell lines in vivo and in vitro the present disclosure has robustly demonstrated the efficacy of this construct in killing cells. Further, it has been demonstrated that expression of the construct in vitro generates many hallmark features previously characterized for immunogenic, necrotic cell death. The current in vivo results in NCG and wildtype B6 mice with the identical construct containing MC38 cell line highlight the importance of an intact immune system for maximizing the effect of controlling tumor growth downstream of construct expression and thus speaks to the in vivo immunogenic nature of the cell death elicited. Intended as a platform technology, the impact of additional genetic cargo (eGFP, MLKL-TfR-eGFP) on construct performance was assessed and it was found to be as designed, unperturbed and indistinguishable from the parental MLKL-TfR construct, likely due to the rationale placement of MLKL and GFP cargo on opposite positions of the TfR transmembrane sequence. Firmly established as a tractable platform, some embodiments of the present disclosure may include the addition of biologically functional extracellular cargo including molecular encoded adjuvants, cytokines and antigens to further enhance specific immune milieus. In addition, the TFRC sequence itself represents fertile ground for additional platform development, containing well annotated regions impactful in modulating membrane associationdynamics. One skilled in the art may evaluate gains in potency and immunogenicity through additional construct optimization.

[0024] The present disclosure approaches cancer gene therapy as a representative therapeutic strategy for which immunogenic cellular depletion is anticipated to provide benefit, as would also be the case for vaccination and diseases associated with viral and bacterially burdened cells, and cellular depletion is approached through the administration or expression of pore-forming proteins. With the more recent increase in understanding of mechanisms and diversity of programmed cell death pathways, there has been a strong resurgence in both chemical and genetic approaches to induce immunogenic cancer cell death. The sparse contemporary genetic approaches to induce ceil death have focused on the expression of the active domains of Gasdermin family members, with varying degrees of preclinical therapeutic success. Distinct from the molecular mechanism employed by the 4HB domain of MLKL, Gasdermin D requires additional molecular mediators to orchestrate oligomerization and subsequent pore formation. As such, pore-formation and cell death through Gasdermin expression remains interruptible through disruption of these secondary mediators. In contrast, by focusing on the properties of the 4HB domain of MLKL to self-oligomerize and generate pores, even in acellular assays, and by further completely separating this domain from the regulatory protein domains in the pseudokinase region (regulated by RIPK cascade), the present disclosure provides a genetic construct that is uninhibitable by the pan Caspase inhibitor ZVAD, the RIPK inhibitor necrostatin-1s. Despite its ability to activate Caspase 1, it acts independently of both Caspase 1 and Caspase 9 activity. Thus, the present disclosure shows application of these properties of the 4HB domain of MLKL in a cancer gene therapy platform, which represents a significant biotechnological advancement in both intellectual approach and implementation of pore-forming, immunogenic cell death therapy.

[0025] The present disclosure demonstrates potential translational relevance of the MLKL-TfR construct. Transferring the construct in vivo validated commercial “vaccine” plasmid DNA backbone (NTC-7482), identification and validation of a tumor specific promoter element (PTTG1) to control the immunogenic cell death activity of the construct, and the employment of a suitable delivery vehicle. Self-assembling PPS based nanoparticles are effective at delivering both RNA and DNA to immune cells in vitro and can also be modified to provide targeted delivery of cargo to specific cancer cell subtypes in vivo. More recently the PPS based PPDP2 nanoparticle system demonstrated efficacy in delivery of RNA for intratumoral expression of a genetically encoded RAS degrader. Combined with the results of the present disclosure, this places the PPDP2 system amongst a much smaller number of platforms that have demonstrated effective delivery in vivo and in vitro for both RNA and DNA genetic cargo. Furthermore, PPS nanoparticles showremarkably high and repeat dose tolerability. This aligns with the data of the present disclosure, where administrations of nanoparticle encapsulated irrelevant plasmid (CMV-GFP, 15 pg), nanoparticle encapsulated experimental plasmid (PTTG1-MLKL-TfR-eGFP, 15 pg), and empty nanoparticles intratumorally every four days throughout the duration of the study. Both tumor growth and survival plots for the irrelevant plasmid and empty nanoparticles was observed to overlap with vehicle controls, consistent with no delivery or vector-based conferred toxicity or immunogenicity. Some embodiments of the present disclosure may include modifying the PEG domain of PPDP2 to include cancer targeting ligands (i.e. folate, peptide) by further optimization and characterization of this nanoparticle system. This may allow subsequent determination of the impacts these modifications have on dose efficacy for both intra-tumoral and systemic administration.

[0026] While in vivo deployment of the PTTG1-MLKL-TfR-eGFP construct resulted in significant control of tumor growth, prolonging overall survival, the majority of mice succumbed to their disease, demonstrating a need for additional improvements. Careful consideration applied to the observed kinetics of tumor growth and the results of the NCG mouse experiments lead to two important conclusions. First, “construct ignorance” occurs as a consequence of heterogeneous tumor populations, and second, immune activity provides prolonged survival. Here, the term “construct ignorance" is defined as to refer to the continued outgrowth of tumors due to either a lack of induction of construct expression or tumors devoid of construct presence. This term may be preferred over the broader term “therapeutic resistance” which lacks specificity with regards to what is the underlying resistance. In the present disclosure, it is highly probable that both forms of construct ignorance occur. With respect to ROSA26 dataset, it is likely that tumor growth continues as a tumor subpopulation devoid of construct replaces tumor cells that die as a result of expressing construct. With respect to the in vivo dataset, in the absence of higher doses or tumor targeting it is likely that not every cell either takes up or permits effective nuclear localization of pDNA, resulting in impaired construct expression. Lastly, for the cells that do effectively take up the nanoparticles and have appropriate accessibility to the pDNA, only a fraction will express a transcriptome aligned with utilization of the PTTG1 derived promoter. In order to address this, some embodiments of the effective therapeutic strategies may include either additional promoters within the construct (programming “OR” type expression) or a mixture of pDNA with distinct promoter activities, driving expression of MLKL-TfR. Promoters that not only provide robust expression in diverse tumor subtypes, but also provide spatial temporal control of genetic expression may be used.

[0027] Provided herein are useful compositions of mixed lineage kinase domain-like (MLKL) derived fusion constructs and their methods of use in cancer therapy. Embodiments of the present disclosure provide a chimeric cell death protein that induces immunogenic cell death in various types of cancers as well as non-transformed cells. The present disclosure also provides different methods of delivering the disclosed compositions or fusion constructs to subjects with cancer or tumor. MLKL-TfR construct of the present disclosure potently induces unavoidable, immunogenic tumor cell death and represents a novel platform for cancer gene therapy and other immunogenic cellular depletion applications. The present disclosure also provides the sequence of the PTTG1 derived promoter that enables tumor enhanced expression of the MLKL-TfR construct, reducing systemic effects and can be utilized in other DNA constructs for likewise directing tumor enhanced DNA expression activity.

[0028] Aspects of the current disclosure are now described with additional details and options as follows: (I) Definitions; (II) Compositions and Methods of Use; and (III) Examples. These sections do not limit the interpretation of the disclosure and are provided for organizational purposes only.(I) Definitions

[0029] Cancer: A disease or condition in which abnormal cells divide without normal regulation or control, and are able to invade other tissues. Cancer cells spread to other body parts through the blood and lymphatic systems. Cancer is a term for many diseases. There are more than 100 different types of cancer in humans. Most cancers are named after the organ in which they originate. For instance, a cancer that begins in the colon can be termed a colon cancer. However, the characteristics of a cancer, especially with regard to the sensitivity of the cancer to therapeutic compounds, are not limited to the organ in which the cancer originates. A cancer cell is any cell derived from any cancer, whether in vitro or in vivo. A tumor is characterized by abnormal or uncontrolled cell growth. The terms tumor and cancer are sometimes used interchangeably but can be distinguished from each other. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

[0030] Chimeric or fusion construct: As used herein, the term “chimeric construct” or “fusion construct” refers to an engineered protein or a gene construct that is inserted into a vector to produce an engineered protein e.g., a fusion protein). In embodiments disclosed herein, thefusion construct comprises a fusion between mixed lineage kinase domain-like (MLKL) protein and human transferrin receptor to form a MLKL-TfR chimeric construct.

[0031] Mixed lineage kinase domain-like (MLKL) protein: MLKL protein is a protein that plays a role in programmed cell death (necroptosis) and other processes. MLKL is a key component of the necrosome, which mediates necroptosis. It interacts with receptor-interacting protein 3 (RIP3), a key signaling molecule in the necroptosis pathway. MLKL is an important factor in cancer cell death. MLKL protein in humans is encoded by the MLKL gene. MLKL protein comprises a pseudokinase domain, a four helical bundle (4HB) domain and a brace region or bridge domain (RCSB PDB ID: 4BTF).

[0032] Transferrin receptor: The transferrin receptor is a membrane glycoprotein whose function is to mediate cellular uptake of iron from a plasma glycoprotein, transferrin. Transferrin receptor is expressed by all nucleated cells of the human body. The expression level of transferrin receptor corresponds to the cellular iron requirements and proliferative status. Transferrin receptor protein 1 (TfR1) in humans is encoded by the TFRC gene. Transferrin receptor comprises a large extracellular domain, a single transmembrane region, and a small cytoplasmic domain at the N-terminus (RCSB PDB ID: 1SUV).

[0033] Immunotherapy: As used herein, the term “immunotherapy” refers to a treatment that uses substances to activate or suppress the immune system to help fight a disease. It can be used to treat cancer, infections, and other diseases.

[0034] Immunogenic cell death (ICD): As used herein, the term “immunogenic cell death” refers to a type of cell death that triggers an immune response. It occurs when dying cells release damage-associated molecular patterns (DAMPs) that activate the immune system. ICD can be caused by several anticancer treatments, including chemotherapeutic drugs, oncolytic viruses, photodynamic therapy, radiotherapy, and other targeted therapies. ICD is a potential cancer treatment because it can provide antitumor immunity, act as a cancer vaccine, and overcome the immunosuppressive tumor microenvironment. Some DAMPs released during ICD include Calreticulin (CRT), Heat-shock proteins (HSP70 and HSP90), Adenosine triphosphate (ATP), High-mobility group box-1 (HMGB1), Type I IFNs, and members of the IL-1 cytokine family. ICD can be measured in real time using the real-time glow extracellular ATP assay. This assay measures the amount of extracellular ATP to determine when and how ICD is induced. Immunogenic cell death involves changes in the composition of the cell surface as well as the release of soluble mediators, occurring in a defined temporal sequence. Such signals operate on a series of receptors expressed by dendritic cells to stimulate the presentation of tumor antigens to T cells.

[0035] Necroptosis or necroptotic-like death: As used herein, the term “necroptosis” or “necroptotic-like death” refers to a form of programmed cell death that is a genetically regulated, inflammatory, and lytic process that involves cell swelling and rupture of the plasma membrane. It is distinct from, but related to, apoptosis (programmed cell death) and necrosis (unregulated cell death). This pathway is mediated by a cascade of proteins, including RIPK1 (receptorinteracting protein kinase 1), RIPK3 (receptor-interacting protein kinase 3), and MLKL (mixed-lineage kinase domain-like protein), and its dysregulation is linked to various diseases, such as cancer, inflammation, and neurodegeneration. Necroptosis is triggered by certain stimuli, such as death receptor ligands. When the cell's apoptotic pathway is blocked, the cell can instead be directed toward necroptosis. The process involves the activation of a "necrosome" complex made of RIPK1, RIPK3, and MLKL. MLKL is then phosphorylated and forms pores in the plasma membrane, causing it to rupture and release the cell's contents. This release of cellular content triggers an inflammatory response. Thus, necroptosis is a regulated form of necrosis that is inflammatory and involves cell swelling and membrane rupture.

[0036] Immune response or cytotoxic T cell immune response: As used herein, the term “immune response” or “cytotoxic T cell immune response” refers to a process where T cells recognize and destroy infected or cancerous cells to prevent the spread of disease. The cytotoxic (CD8) T cell immune response is a key part of the adaptive immune system that kills infected or cancerous cells by recognizing specific antigens presented on their surface via MHC class I molecules. Thus, this response involves activation by antigen-presenting cells and clonal expansion to create more T cells. CDS T cells proliferate and differentiate into effector cells that release cytotoxic molecules like perforin and granzymes, inducing apoptosis in the targeted cell. This response is important for controlling viruses and eliminating tumors.

[0037] Engineered: As used herein, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences, that are not linked together in that order in nature, are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. Those of skill in the art will appreciate that an “engineered” nucleic acid or amino acid sequence can be a recombinant nucleic acid or amino acid sequence and can be referred to as “genetically engineered.” In some embodiments, an engineered polynucleotide includes a coding sequence that is found in nature operably linked with a first sequence but is not found in nature operably linked with a second sequence, which is, in the engineered polynucleotide, operably linked in with the second sequence by the hand of man. In some embodiments, a cell or organism is considered to be “engineered” or “genetically engineered” if it has been manipulated so that its geneticinformation is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution, deletion, or mating). As is common practice and is understood by those of skill in the art, progeny or copies, perfect or imperfect, of an engineered polynucleotide or cell are typically still referred to as “engineered" even though the direct manipulation was of a prior entity.

[0038] Expression: As used herein, “expression” of a polynucleotide (for example, a gene or a transgene) refers to the process by which the coded information of a transcriptional unit (including, e.g., gDNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, northern blot, RT-PCR, western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0039] Sample: "Sample" or "biological sample" refers to a biological material isolated from or derived from a subject. Embodiments of “derived from” refer to a biological sample being obtained from a subject or other source and including any modification to the sample, addition to the sample, or removal from the sample. The biological sample can contain any biological material including fluid, tissue, cellular and / or non-cellular material. In embodiments disclosed herein, a biological sample can include cells, non-cancerous cells, tumor cells, etc.

[0040] Isolated: An “isolated” biological component (such as a polynucleotide, polypeptide, or small molecules (e.g., hormones)) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component originated or was made or naturally occurs (ie., other chromosomal and extra-chromosomal DNA and RNA, and proteins), while effecting a chemical or functional change in the component (e.g., a nucleic acid may be isolated from a chromosome by breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome; ora chemical compound may be converted to a purified form that is effective or more effective for some use(s) because it is removed from the presence of other components, which may be viewed as contaminants). Polynucleotides andsmall molecules that have been isolated specifically include nucleic acid molecules purified by standard purification methods. The term also embraces biological components (such as nucleic acid molecules) prepared by recombinant expression or production in a host organism or host cell, as well as chemically-synthesized versions, including when they are substantially separated or purified away from other biological components in that product milieu.

[0041] Agent: An “agent" or a “therapeutic agent” refers to any biologically active compound capable of treating or inhibiting at least one disease state or condition. The agent or therapeutic agent is sufficient to yield a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic response. A therapeutic agent may comprise a wide variety of compounds such as nucleic acids, polypeptides, drugs, inhibitors, small-molecule compounds, antibodies, antibody fragments, nanobodies, and the like. In embodiments disclosed herein, an agent may also comprise a compound complexed with a delivery vehicle, e.g., a nucleic acid molecule encapsulated in a nanoparticle.

[0042] Amount: An “amount” or “effective amount” or “pharmaceutically effective amount” of an agent is an amount that upon administration to a subject in need thereof is sufficient to generate a desired response such as reducing or eliminating a sign or symptom of a condition or a disease. An effective amount also encompasses an effective amount of a first agent and an effective amount of a second agent administered in combination with the first agent. In some examples, the effective amount of the two combined agents is less than that of either agent when administered alone. In some examples, the effective amount of the two combined agents is more than that of either agent when administered alone.

[0043] Subject: A “subject” or a “patient” refers to a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. A "mammal" includes both human and non-human mammals, such as mice, rats. In some embodiments of the present disclosure, a subject is a patient, such as a patient diagnosed with cancer. In some embodiments of the present disclosure, a subject is a patient yet to be diagnosed.

[0044] Treatment: Treatment refers to any therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, a decrease in tumor volume, an increase in life expectancy, an improvement in the overall health or well-being of the subject, or by other clinical or physiological parameters associated with aparticular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. A “therapeutic” treatment is a treatment administered after the development of significant signs or symptoms of the disease. The terms “for treatment of”, “for use in the treatment of’, “for treating”, “for use in treating” and the like are understood to be synonymous and may be interchanged in describing and claiming the methods herein.

[0045] Tumor A “tumor” is a lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A “tumor cell” is an abnormal cell divided by a rapid, uncontrolled cellular proliferation and continues to divide after the stimuli that initiated the new division ceases. T umors show partial or complete lack of structural organization and functional coordination with the normal tissue. Usually, they form a distinct mass of tissue, either benign, pre-malignant, or malignant. Neoplastic cell growth and proliferation, whether malignant or benign, including all pre-cancerous and cancerous cells and tissues. Tumor markers include polynucleotides and polypeptides expressed by tumors to a greater extent than they are expressed by non-tumor cells, including cell surface or cytoplasmic or nuclear tumor antigens.

[0046] Examples of types of tumors include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma cerebellar or cerebral; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem glioma; brain tumor; brain tumor, cerebellar astrocytoma; brain tumor, cerebral astrocytoma / malignant glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumors; brain tumor, visual pathway and hypothalamic glioma; breast cancer; bronchial adenomas / carcinoids; Burkitt lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal; carcinoma of unknown primary; central nervous system lymphoma, primary; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; cervical cancer; childhood cancers; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; endometrial cancer; ependymoma; esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); germ cell tumor: extracranial, extragonadal, or ovarian; gestational trophoblastic tumor; glioma of the brain stem; glioma cerebral astrocytoma; glioma visual pathway and hypothalamic; gastric carcinoid; hairy cell leukemia; head and neck cancer: heart cancer;hepatocellular (liver) cancer; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi sarcoma; kidney cancer (renal cell cancer); laryngeal cancer; leukemias; acute lymphoblastic leukemia (also called acute lymphocytic leukemia); acute myeloid leukemia (also called acute myelogenous leukemia); leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); chronic myelogenous leukemia (also called chronic myeloid leukemia); hairy cell leukemia; lip and oral cavity cancer; liver cancer (primary); non-small cell lung cancer; small cell lung cancer; lymphomas; AIDS-related lymphoma; Burkitt lymphoma; cutaneous t-cell lymphoma; Hodgkin lymphoma; lymphomas, non-Hodgkin lymphoma (an old classification of all lymphomas except Hodgkin’s); primary central nervous system lymphoma; Marcus whittle, deadly disease; malignant fibrous histiocytoma of bone / osteosarcoma; medulloblastoma; melanoma; intraocular (eye) melanoma; Merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia acute; myeloid leukemia acute; myeloma, multiple (cancer of the bone-marrow); chronic myeloproliferative disorders; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung cancer; oral cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer (surface epithelial-stromal tumor); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; islet cell pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary adenoma; plasma cell neoplasia / multiple myeloma; pieuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); renal pelvis and ureter, transitional cell cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma, Ewing family of tumors; Kaposi sarcoma; soft tissue sarcoma; uterine sarcoma; Sezary syndrome; skin cancer (nonmelanoma); skin cancer (melanoma); skin carcinoma, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer with occult primary, metastatic; stomach cancer; supratentorial primitive neuroectodermal tumor; cutaneous T-Cell lymphoma; testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; gestational trophoblastic tumor; ureter and renal pelvis, transitional cell cancer; urethral cancer; uterine cancer, endometrial; uterinesarcoma; vaginal cancer; visual pathway and hypothalamic glioma; vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.

[0047] Identical: As used herein, the term “identical” or “substantial identity” with regard to a reference polynucleotide or polypeptide, refers to a polynucleotide or polypeptide having contiguous nucleotides or amino acids that is substantially identical to polynucleotides or polypeptides herein sharing at least 80% sequence identity. In examples herein, substantially identical polynucleotides or polypeptides have between 80% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 85% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 86% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 87% and 00% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 88% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 89% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 90% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 91% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 92% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 93% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 94% and 100% sequence identity. In other examples, substantially identical polynucleotides or polypeptides have between 95% and 100% sequence identity. In yet other examples, substantially identical polynucleotides or polypeptides have between 96% and 100% sequence identity.

[0048] Sequence Identity: The term “sequence identity” or “identity,” as used herein in the context of two nucleotide sequences or two amino acid sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. The term “percentage of sequence identity” may refer to the value determined by comparing two optimally aligned nucleotide sequences over a comparison window, wherein the portion of the nucleotide sequence in the comparison window may include additions or deletions ( / .e., gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0049] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-44, 1988: Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet eta / ., Nucleic Acids Res. 16:10881-90, 1988; Huang et al., Comp. Appl. Biosci. 8:155-65, 1992; Pearson et al., Methods Moi. Biol. 24:307-31, 1994; Tatiana et al., FEMS Microbiol. Lett. 174:247-50, 1999. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al., J. Mol. Biol. 215:403-10, 1990.

[0050] The National Center for Biotechnology information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLAST™. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleotide sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.

[0051] Polypeptide: Any chain of amino acids, regardless of length or posttranslational modification (such as glycosylation, methylation, ubiquitination, phosphorylation, or the like). The term polypeptide is used interchangeably with peptide or protein, and is used to refer to a polymer of amino acid residues. The term residue refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.

[0052] Operably linked: A first polynucleotide is operably linked with a second polynucleotide when the first polynucleotide is in a functional relationship with the second polynucleotide. When recombinantly produced, operably linked polynucleotides are generally contiguous, and, where necessary to join two coding regions, in the same reading frame (e.g., in a translationally fused ORF). However, polynucleotides need not be contiguous to be operably linked. The term, “operably linked,” when used in reference to a regulatory genetic element and a polynucleotide, means that the regulatory element affects the expression of the linked polynucleotide. “Regulatory elements,” “control elements,” or “regulatory sequences” refer to polynucleotides that influence the timing and level / amount of transcription (or RNA processing or stability) of the operably linkedpolynucleotide. Regulatory sequences include, for example and without limitation promoters, translation leaders, introns, enhancers, stem-loop structures, repressor binding sequences, termination sequences, and polyadenylation recognition sequences. Particular regulatory elements may be located upstream and / or downstream of a polynucleotide operably linked thereto. Also, particular regulatory elements operably linked to a polynucleotide may be located on the associated complementary strand of a double-stranded nucleic acid molecule.

[0053] Nucleic acid molecule: As used herein, the term “nucleic acid molecule” refers to a polymeric form of nucleotides, which includes in specific examples both or either of sense and anti-sense strands of RNA, cDNA, genomic DNA, plasmid DNA, and synthetic forms and mixed polymers of the foregoing. The term includes single- and double-stranded forms of DNA and RNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. A nucleotide may be a ribonucleotide, deoxyribonucleotide, or modified form of either. A “polynucleotide” refers to a physical contiguous nucleotide polymer, such as may be included in a larger nucleic acid molecule. A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. By convention, the nucleotide sequence of a nucleic acid molecule is read from the 5' to the 3' end of the molecule. The “complement” of a nucleic acid molecule refers to a polynucleotide having nucleobases that may form base pairs with the nucleobases of the nucleic acid molecule (i.e., A-T / U, and G-C).

[0054] Some embodiments include nucleic acids including a template DNA that is transcribed into an RNA molecule that includes a polyribonucleotide that hybridizes to a mRNA molecule. In some examples, the template DNA is the complement of the polynucleotide transcribed into the mRNA molecule, present in the 5’ to 3’ orientation, such that RNA polymerase (which transcribes DNA in the 5’ to 3’ direction) will transcribe the polyribonucleotide from the complement that can hybridize to the mRNA molecule. Unless explicitly stated otherwise, or it is clear to be otherwise from the context, the term “complement” therefore refers to a polynucleotide having nucleobases, from 5’ to 3’, that may form base pairs with the nucleobases of a reference nucleic acid. In some examples, the template DNA is the reverse complement of the polynucleotide transcribed into the mRNA molecule. Thus, unless it is explicitly stated to be otherwise (or it is clear to be otherwise from the context), the “reverse complement” of a polynucleotide refers to the complement in reverse orientation. The foregoing is demonstrated in the following illustration:ATGATGATG polynucleotideTACTACTAC “complement” of the polynucleotideCATCATCAT “reverse complement" of the polynucleotide

[0055] Two polynucleotides are said to exhibit “complete complementarity" when every nucleotide of a polynucleotide read in the 5' to 3' direction is complementary to every nucleotide of the other polynucleotide when read in the 5' to 3' direction. Similarly, a polynucleotide that is completely reverse complementary to a reference polynucleotide will exhibit a nucleotide sequence where every nucleotide of the polynucleotide read in the 5’ to 3' direction is complementary to every nucleotide of the reference polynucleotide when read in the 3' to 5' direction. These terms and descriptions are recognized in the art and are understood by those of ordinary skill in the art.

[0056] “Nucleic acid molecules” include all polynucleotides, for example: single- and doublestranded forms of DNA; single-stranded forms of RNA; and double-stranded forms of RNA (dsRNA). The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex. The term “ribonucleic acid” (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNAs, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). The term “deoxyribonucleic acid” (DNA) is inclusive of cDNA, gDNA, plasmid DNA, and DNA-RNA hybrids. The terms “polynucleotide" and “nucleic acid,” and “fragments” thereof will be understood by those in the art as a term that includes both gDNAs, ribosomal RNAs, transfer RNAs, messenger RNAs, operons, and smaller engineered polynucleotides that encode or may be adapted to encode, peptides, polypeptides, or proteins.

[0057] A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc. charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc. chelators: alkylators; and modified linkages; for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.

[0058] With respect to DNA, the term “coding polynucleotide,” “structural polynucleotide,” or “structural nucleic acid molecule” refers to a polynucleotide that is ultimately transcribed into an RNA; for example, when placed under the control of appropriate regulatory elements (e.g., a promoter). The boundaries of a coding polynucleotide are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding polynucleotides include, but are not limited to, gDNA, cDNA, ESTs, and recombinant polynucleotides. As used herein, “transcribed non-coding polyribonucleotide” refers to segments of mRNA molecules such as 5'UTR, 3'UTR, and intron segments that are not translated into a polypeptide. For example, a transcribed non-coding polyribonucleotide may be a polyribonucleotide that natively exists as an intragenic “spacer” in an RNA molecule.

[0059] Label: Any substance capable of aiding a machine, detector, sensor, device, column, or enhanced or unenhanced human eye from differentiating a labeled composition from an unlabeled composition. Labels may be used for any of a number of purposes and one skilled in the art will understand how to match the proper label with the proper purpose.

[0060] Examples of uses of labels include purification of biomolecules, identification of biomolecules, detection of the presence of biomolecules, detection of protein folding, and localization of biomolecules within a cell, tissue, or organism. Examples of labels include: radioactive isotopes or chelates thereof; dyes (fluorescent or non-fluorescent), stains, enzymes, nonradioactive metals, magnets, protein tags, fluorescent proteins (e.g., GFP), any antibody epitope, any specific example of any of these; any combination between any of these, or any label now known or yet to be disclosed. A label may be covalently attached to a biomolecule or bound through hydrogen bonding, Van Der Waals or other forces. A label may be covalently or otherwise bound to the N-terminus, the C-terminus or any amino acid of a polypeptide or the 5' end, the 3' end or any nucleic acid residue in the case of a polynucleotide.

[0061] A particular example of a label is a protein tag. A protein tag includes a sequence of one or more amino acids that may be used as a label as discussed above. In some examples, the protein tag is covalently bound to the polypeptide. It may be covalently bound to the N-terminal amino acid of a polypeptide, the C-terminal amino acid of a polypeptide or any other amino acid of the polypeptide. Often, the protein tag is encoded by a polynucleotide sequence that is immediately 5' of a nucleic acid sequence coding for the polypeptide such that the protein tag is in the same reading frame as the nucleic acid sequence encoding the polypeptide. Protein tags may be used for all of the same purposes as labels listed above and are well known in the art. Examples of protein tags include chitin binding protein (CBP), maltose binding protein (MBP), Green fluorescent protein (GFP), glutathione-S-transferase (GST), poly-histidine (His),thioredoxin (TRX), FLAG™, V5, c-Myc, HA-tag, and so forth. A His-tag facilitates purification and binding to on metal matrices, including nickel matrices, including nickel matrices bound to solid substrates such as agarose plates or beads, glass plates or beads, or polystyrene or other plastic plates or beads. Other protein tags include BCCP, calmodulin, Nus, Thioredoxin, Streptavidin, SBP, and Ty, or any other combination of one or more amino acids that can work as a label described above.

[0062] Targeted Gene Editing for genetic recombination: In particular embodiments, gene editing is mediated by a gene editing system such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system. The widely popular CRISPR / Cas9 system is one method by which targeted editing is performed. The CRISPR nuclease system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPRs are DNA loci containing short repetitions of base sequences. In the context of a prokaryotic immune system, each repetition is followed by short segments of spacer DNA belonging to foreign genetic elements that the prokaryote was exposed to. This CRISPR array of repeats interspersed with spacers can be transcribed into RNA. The RNA can be processed to a mature form and associate with a Cas (CRISPR-associated) nuclease. A CRISPR-Cas system including an RNA having a sequence that can hybridize to the foreign genetic elements and Cas nuclease can then recognize and cut these exogenous genetic elements in the genome.

[0063] A single Cas enzyme can be programmed by a gRNA molecule to site-specifically cleave a specific target nucleic acid. Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 includes two distinct endonuclease domains (HNH and RuvC / RNase H-like domains), one for each strand of the target nucleic acid. RuvC and HNH together produce double-stranded breaks (DSBs); separately each domain can produce single-stranded breaks. Base-pairing between the gRNA and target nucleic acid causes double-stranded breaks (DSBs) due to the endonuclease activity of Cas9. Binding specificity is determined by both gRNA-target nucleic acid base pairing and the PAM juxtaposed to the DNA complementary region. In particular embodiments, the CRISPR system only requires a minimal set of two molecules — the Cas protein and the gRNA.

[0064] A large number of Cas9 orthologs are known in the art (Fonfara et al., NAR, 42:2577-2590, 2014; Chylinski et al., NAR, 42:6091-6105, 2014; Esvelt et al., Nature Methods, 10:1116-1121, 2013). A number of orthogonal Cas9 proteins have been identified including Cas9 proteins from Neisseria meningitidis, Streptococcus thermophilus and Staphylococcus aureus. Other Class 2 Cas proteins that can be used include Cas12a (Cpf1), Cas13a (C2c2), and Cas13B (C2c6). The Cpf1 nuclease particularly can provide added flexibility in target site selection bymeans of a short, three base pair recognition sequence (TTN), known as the protospacer-adjacent motif or PAM. CpfTs cut site is at least 18 bp away from the PAM sequence, thus the enzyme can repeatedly cut a specified locus after indel (insertion and deletion) formation. Exemplary engineered Cpfls are described in US 2018 / 0030425, US 2016 / 0208243, WO / 2017 / 184768 and Zetsche et al., Cell 163: 759-771, 2015; and single gRNAs in Jinek et al., Science 337:816-821, 2012; Jinek etal., eLife 2:e00471, 2013; Segal, eLife 2:e00563, 2013.

[0065] In particular embodiments, polynucleotide sequences encoding mutant forms of Cas9 nuclease can be used in genetic constructs of the disclosure. For example, a Sniper Cas9, a variant of Cas9 with optimized specificity (minimal off-target effects) and retained on-target activity can be used (Lee et al., J Vis Exp. (144), 2019; Lee et al., Nat Commun. 9(1):3048, 2018; WO 2017 / 217768). As another example, a mutant Cas9 nuclease containing a D10A amino acid substitution can be used. This mutant Cas9 has lost double-stranded nuclease activity present in the wild type Cas9 but retains partial function as a single-stranded nickase. This mutant Cas9 generates a break in the complementary strand of DNA rather than both strands. This allows repair of the DNA template using a high-fidelity pathway rather than non-homologous end joining (NHEJ). The higher fidelity pathway prevents formation of insertions / deletions at the targeted locus while maintaining ability to undergo homologous recombination (Cong et al., Science 339(6121 ):819-823, 2013). Paired nicking has been shown to reduce off-target activity by 50- to 1,500- fold in cell lines (Ran et al., Cell 154(6): 1380-1389, 2013).

[0066] In particular embodiments, a Cas protein can include one or more degrons to selfinactivate the Cas protein by accelerating degradation of expressed Cas protein. A degron can include a portion of a polypeptide that is important in regulation of protein degradation. In particular embodiments, a degron includes short amino acid sequences, structural motifs, and / or exposed amino acids (e.g., a lysine or arginine) located anywhere in a protein. In particular embodiments, a degron can be ubiquitin-dependent or ubiquitin-independent.

[0067] In particular embodiments, a Cas protein can be fused to a heterologous polypeptide that provides for subcellular localization. Such heterologous peptides include, for example, a nuclear localization signal (NLS) such as the SV40 NLS for targeting to the nucleus (e.g., see Lange et al., J. Biol. Chem. 282:5101-5105, 2007). Such subcellular localization signals can be located at the N-terminus, the C-terminus, or anywhere within the Cas protein. An NLS can include a stretch of basic amino acids and can be a monopartite sequence or a bipartite sequence.

[0068] In particular embodiments, a Cas protein can also include a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of tags include green fluorescent protein (GFP), glutathione-S-transferase (GST), myc,Flag, hemagglutinin (HA), Nus, Softag 1, Softag 3, Strep, polyhistidine, biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP), and calmodulin.

[0069] Additional information regarding CRISPR-Cas systems and components thereof are described in US Patents No. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233, 8,999,641, and applications related thereto; and International Patent Publications WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473, WO2015 / 089486, W02016 / 205711, WO2017 / 106657, WO2017 / 127807, and applications related thereto.

[0070] Teachings of the disclosure in relation to CRISPR can be applied to other gene editing systems that similarly utilize nucleases.

[0071] Delivery vehicle or nanocarrier: As used herein, the term “delivery vehicle” or “nanocarrier” refers to agents used to transport and deliver bioactive molecules to specific locations in the body. For example, these vehicles may use the outer coat of viruses to deliver gene therapies. In some examples, administration of a nucleic acid may be aided by specific formulation of the nucleic acid e.g. in liposomes (lipoplexes) or polymersomes (synthetic variants of liposomes), as polyplexes (nucleic acid complexed with polymers), carried on dendrimers, in inorganic (nano)particles (e.g. containing iron oxide in case of magnetofection), or combined with a cell penetrating peptide (CPP) to increase cellular uptake. Tumor-, cancer- or neoplasmtargeting strategies may also be applied to the nucleic acid (nucleic acid combined with tumor-, cancer-, or neoplasm-targeting moiety); these include passive targeting (mostly achieved by adapted formulation) or active targeting (e.g. by coupling a nucleic acid-comprising nanoparticle with folate or transferrin, or with an aptamer or antibody binding to a target cell-specific antigen) (e.g., Steichen et al. 2013, Eur J Pharm Sci 48:416-427). In embodiments herein, delivery systems that may employ the disclosed constructs include lentivirus, adenovirus (e.g., AAV), polymeric nanoparticles, lipid-polymeric hybrid nanoparticles, metal based (e.g., Gold) nanoparticle, mesosilicate nanoparticles (MSN), exosome delivery systems, membrane derived nanovesicles, virus like particles (VLP), protein nanocages and proteolipid vehicles (PLV), lipid nanoparticles, and stabilized plasmid-lipid particles (SPLP). In embodiments herein, lipid-polymeric hybrid nanoparticles have been used to deliver the disclosed constructs, and these nanoparticles are a subset of nanocarrier described herein.(II) Compositions and Methods of Use

[0072] Disclosed are compositions comprising a nucleic acid encoding an isolated mixed lineage kinase domain-like (MLKL) protein for use in therapeutic inhibition of a tumor in a subject harboring the tumor. In embodiments, the nucleic acid encoding the isolated MLKL protein further comprises a sequence encoding transferrin receptor. In embodiments, the isolated MLKL protein induces necroptotic-like death of the tumor in the subject.

[0073] Existing “suicide gene” therapies rely largely on induction of apoptosis through signaling cascades which are frequently non-functional in cancer cells, limiting the utility of these therapies. Additionally, induction of cell death through apoptotic pathways does not result in a robust inflammatory response and abundant antigen availability to drive anti-tumor immunity. However, other programmed cell death pathways, such as necroptosis, have these features. Necroptosis is a form of programmed cell death which is frequently triggered in acute viral infection and results in rapid cell death, release of abundant cellular antigen, and release of abundant inflammatory mediators into the microenvironment. The present disclosure provides compositions comprising a novel fusion construct which incorporates the functional unit from the end effector molecule of necroptosis, human mixed lineage kinase domain-like molecule (hMLKL) with the transmembrane portion of the human transferrin receptor (hTFR). This fusion protein retains the pore-forming functionality of hMLKL while removing the regulatory domain from this molecule. A portion of the hTFR protein was used in the fusion to both facilitate trafficking of the hMLKL to the plasma membrane and to facilitate the addition of additional cargo to the fusion protein on the opposite side of the plasma membrane with consistent orientation. In embodiments, the disclosed chimeric protein contains hMLKL on the amino terminus of the fusion protein (intracellular domain), the hTFR transmembrane domain, and optionally a green fluorescent protein (GFP) on the carboxy terminus of the fusion protein (extracellular domain). In some embodiments, the addition of GFP to the fusion / chimeric protein was to demonstrate that a functional fusion protein could be constructed with both intracellular and extracellular domains without impairing the function of the hMLKL domain.

[0074] In some embodiments, the tumor targeted by the disclosed fusion construct is selected from the group of leukemia, colorectal cancer, mammary cancer, and ovarian cancer. In examples of the present disclosure, data demonstrated that the fusion protein induces rapid cell death in a variety of both human and murine tumor and non-tumor cell lines. The data also demonstratedthat the mechanism of cell death induced by the fusion protein is consistent with necrotic cell death, releasing multiple danger associated molecular pattern markers (DAMPS). Exemplary dataset also demonstrated that the function of the fusion protein cannot be inhibited using caspase inhibition, unlike suicide genes targeting apoptotic pathways. It was also demonstrated that this fusion protein can be encoded within a genetic construct that can be delivered to tumor cells both in vitro and in vivo, resulting in tumor cell death, control of tumor growth, and increased survival of mammals receiving functional construct compared to a control construct.

[0075] The disclosed fusion construct is effective in killing every cell that expresses the fusion protein. Embodiments show that the MLKL-TFR chimeric / fusion protein effectively cause immunogenic cell death in any mammalian cell in which it is expressed. In some examples, the fusion protein induces immunogenic cell death in phenotypically normal mammary epithelial cells. In other examples, the disclosed fusion protein induces immunogenic cell death in a wide variety of cells varying in p53 and DNA repair status including, but not limited to, leukemic cancer cells, colorectal cancer cells, normal mammary cancer cells, ovarian cancer, brain cancer, head & neck cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, skin cancer, prostate cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), among many others. In yet other examples, the disclosed fusion protein may potentially induce immunogenic cell death in cellular reservoirs of viral or bacterial infection, wherein lysis of the pathogen-containing cell could eliminate the reservoir and / or induce in situ vaccination or immune boosting against the pathogen. Utilizing pathogen specific promoters, this fusion construct can eliminate these reservoirs, in some examples. Thus, the disclosed fusion construct can be used for infectious disease applications as well, such as eliminating cellular viral reservoirs (i.e., cells containing virus) that perpetuate diseases such as the case in chronic viral infection. This may include diseases such as Hepatitis B, Hepatitis C, HIV, among many others. Therefore, when targeted at the correct viral life cycle, cell lysis may be an effective means of eliminating virus using the disclosed fusion construct.

[0076] The disclosed fusion protein may be used as a direct cell death protein as well as for therapeutic inhibition of tumor. A key feature of the disclosed fusion construct is that it is a (I) specialized “suicide gene”, that can kill any cell (including tumor cells) that expresses it without the involvement of the immune system, and (ii) it may also allow a cell to die in an immunogenic cell death manner, so that if an immune system is around it can be engaged and promote a therapeutic antitumor response. Both of these features are evident in various embodiments of the present disclosure. Using the EPH4 cells (non-cancer, epithelial cell lines), in some examples of the present disclosure, it was demonstrated that any cell expressing the disclosed fusion construct dies. Embodiments covering the in vitro data / results with colorectal tumor, mammaryepithelial and leukemic cell lines in Examples 2-4, no functional immune system is present in the culture conditions. Hence, all tumor control is necessarily by direct cell death, independent of immune stimulating features. In contrast, embodiments covering the in vivo data / results with colorectal tumor cells in mouse models described in Examples 5 and 6 highlight that if an immune system is present, death of construct-expressing cells confers enhanced immunity against nonconstruct expressing cells, evident by the delayed tumor growth. Thus, the disclosed fusion construct exhibits a direct cell death mechanism as well as an immunotherapeutic inhibition of cells expressing the construct.

[0077] In some embodiments, the disclosed composition is delivered to the subject using lentiviral / adenoviral system or using a nanoparticle system (e.g., neutral lipid, cationic lipid, or polymeric nanoparticle systems). In some cases, the composition is administered to the subject intratumorally. Administration of a MLKL-TFR fusion construct to a mammal harboring a tumor, cancer or neoplasm may for instance be by intra-tumor, intra-cancer or intra-neoplasm delivery. In some examples, the administration of MLKL-TFR fusion construct may alternatively be remote (administration remotely from the tumor, cancer or neoplasm); in this case the MLKL-TFR fusion construct may optionally be combined with or (recombinantly or non-recombinantly) fused to for instance a tumor-, cancer- or neoplasm-targeting moiety, including enhancing delivery specifically through similar modifications to the delivery vehicle enabling systemic administration but targeted delivery.

[0078] The administration of an agent can be for prophylactic or therapeutic purposes. For prophylactic and therapeutic purposes, the treatments can be administered to the subject in a single bolus delivery, via continuous delivery over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the treatment for a disease can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a disease or condition.

[0079] Therapeutic treatments can be distinguished from effective amounts based on the presence or absence of a research component to the administration. As will be understood by one of ordinary skill in the art, however, in human clinical trials effective amounts, prophylactic treatments and therapeutic treatments can overlap.

[0080] For administration, therapeutically effective amounts can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administeredto a particular subject can be determined by the subject, a physician, veterinarian, or researcher taking into account parameters such as physical, physiological and psychological factors including target, body weight, condition, previous or concurrent therapeutic interventions, and / or idiopathy of the subject.

[0081] An effective amount or concentration of an agent can be any amount administered alone or in combination with additional therapeutic agents, is sufficient to achieve a desired effect in a subject. The effective amount of the agent will be dependent on several factors, including, but not limited to, the subject being treated and the manner of administration of the agent. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease or condition, or which is capable of reducing symptoms caused by any disease or condition.

[0082] In one example, a desired effect is to reduce or inhibit one or more symptoms associated with a disease or condition. The one or more symptoms do not have to be completely eliminated for the therapeutic agent to be effective. For example, an agent can decrease the sign or symptom by a desired amount, for example by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to how the sign or symptom would have progressed in the absence of the agent or in comparison to currently available treatments.

[0083] The actual effective amount will vary according to factors such as the type of disease to be protected against / therapeutically treated and the particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like) time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of treatments for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.

[0084] In some aspects, embodiments of the present disclosure provide a composition comprising a nucleic acid molecule encoding a chimeric protein for use in therapeutic treatment of a tumor, cancer, or neoplasm in a subject in need thereof, the chimeric protein comprising a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor. In some embodiments, the isolated MLKL protein of the chimeric protein comprises a four helical bundle (4HB) domain and a brace region of a native MLKL protein In some cases, the isolated MLKL protein of the chimeric protein is devoid of a pseudokinase domain of the native MLKL protein. In some embodiments, the isolated transferrin receptor of the chimeric protein comprises a cytoplasmic domain and a transmembrane domain of a native human transferrinreceptor. In some examples, the key domain of the human transferrin receptor within the fusion construct is the transmembrane domain which comprises proximal amino acid residues that may be further modified to alter membrane dynamics or attach an intracellular / extracellular cargo.

[0085] In some embodiments, the nucleic acid molecule encoding the chimeric protein is DNA, wherein the DNA is plasmid DNA, linear double strand DNA, DNA included in a viral vector, or complexed DNA. In some embodiments, the nucleic acid molecule encoding the chimeric protein is RNA, wherein the RNA is mRNA, RNA included in a viral vector, or complexed (m)RNA. In some embodiments, the mRNA further comprises a 5' cap and / or a 3' poly(A)tail and / or a 5' untranslated region and / or a 3' untranslated region.

[0086] In particular examples, human MLKL protein sequence (https: / / www.uniprot.org / uniprotkb / Q8NB16 / entry as of November 04, 2025) overlaps 100% with human reference amino acid sequence 1-210 (out of 471 total amino acids residues). In embodiments, human MLKL DNA sequence(https: / / www. ncbi. nlm.nih.gov / nucleotide / NM_152649.4?report=genbank&!og$=nuclali gn&blast_rank=1& RID=F98T2X7J013) and the codon optimized sequence used in the present disclosure, aligns with nucleotide residues 416-1038 of NM_152649.4. The DNA sequence was derived from (1) reverse engineering the WT protein sequence and performing codon optimization for expression (2) further selecting codon usage to minimize alternative transcripts from alternative reading frames when considered in conjunction with the plasmid backbone and in frame with the TfR and TfR-eGFP sequences. Consequently, while the protein sequence is 100% identical, the DNA sequence shares 76% sequence overlap with human MLKL DNA sequence.

[0087] Human transferrin receptor protein sequence is provided in https: / / www.uniprot.org / uniprotkb / P02786 / entry as of November 04, 2025. In particular embodiments, the DNA construct sequence of transferrin receptor used in the present disclosure codes for amino acid residues 3-118 of the reference amino acid sequence. This sequence incorporates (1) the majority of the intracellular tail which contains (1a) amino acids motifs known to impact membrane recycling features (impacting retention of protein construct at plasma membrane, these are modifiable to enhance membrane accumulation (AA20-23), Lys39, Lys53, Lys58, Lys60) (1b) disulfide bonds which drive homodimerization (for facilitating oligomerization, Cys89, Cys98) as well as (2) the transmembrane domain (AA65-91), which acts as an ER signal peptide directing plasma membrane association. Amino acid sequences 92-118 contain extracellular residues that act as a scaffold for extracellular attachment and contain a protease sensitive site (R 100), that can be modified to allow for membrane anchored (when not present) or membrane cleavable / releasable extracellular genetically encoded cargo. All of these variantsprovide opportunities for cargo optimization depending on the intended membrane retention time and intended extracellular membrane association of encoded cargo. DNA sequence of human transferrin receptor (https: / / www.ncbi. nlm.nih.gov / nucleotide / NM_003234.4?report=genbank&log$=nuclalign&blast_ rank=1& RID=FB61AXWC013) shows 100% nucleotide alignment with reference genome sequence (NM003234.4) representing nucleotide residues 290-637 in reference genome sequence.

[0088] In the present disclosure, a gene therapy construct has been created utilizing the NTC7482 plasmid backbone (Williams JA, Carnes AE, and Hodgson CP. (2009) Plasmid DNA vector design; impact on efficacy, safety and upstream production. Biotechnology Advances 27:353-370). The original use of this plasmid backbone was the generation of immune responses to genetically encoded constructs driven by a proprietary CMV promoter. In the usage of this plasmid backbone for the fusion constructs of the present disclosure, some of the intended immunogenic features have been eliminated as well as the CMV promoter has been removed altogether (which would be incompatible with using a cell death construct) and the Poly A tail has also been removed (replacing it with a shorter Poly A, SVPLA). In essence, this plasmid backbone has been used here for its minimal bacterial backbone size (-2.5Kb) and robust plasmid production properties, along with demonstrated expression permissiveness (independent of promoter) in mammalian cells in vivo and in vitro.

[0089] To drive expression of the disclosed fusion protein, PTTG1 promoter has been used in particular embodiments. PTTG1 has been identified as a gene which is overexpressed in a wide range of solid tumors and is associated with more severe disease and decreased overall survival and had little to no expression in most normal mammalian tissues. In embodiments, a highly conserved 561 base pair regulatory sequence from this promoter has been identified (SEQ ID NO.: 5) and that expression of this regulatory domain is highly associated with neoplastic gene regulatory pathways. In disclosed embodiments, delivery of the fusion construct with this regulatory element results in increased tumor control with no observable off target effects in the animal models. However, it is to be noted that PTTG1 promoter sequence disclosed herein has uses beyond the disclosed MLKL-TfR fusion construct. For example, the PTTG1 promoter sequence can be used specifically with cell depletion therapies of cancer or to drive expression of other therapeutic proteins (adjuvants, antibodies, cytokines, antigens, antigenic peptides) enabling greater spatial and tissue (tumor) specific expression of DNA encoded agents.

[0090] In embodiments, translational start site proximal nucleotides of the human PTTG1 gene have been selected for possessing 7 distinct highly conserved domains and containing at leasttwo well described E2F1 binding sites (Cuiqi Zhou, Kolja Wawrowsky, Serguei Bannykh, Shiri Gutman, Shlomo Melmed, E2F1 Induces Pituitary Tumor Transforming Gene (PTTG1) Expression in Human Pituitary Tumors, Molecular Endocrinology, Volume 23, Issue 12, 1 December 2009, Pages 2000-2012). The promoter element used in the embodiments of the present disclosure has 100% sequence identity to human reference sequence (https: / / ncbi.nlm.nih.gov / gene / 9232) for the included nucleotide bases. The promoter binding evaluation has been conducted for -3000BP upstream of the translation start site identifying various transcription factor binding regions (including those of AP1 and hormone receptors > 1000BP upstream.) Two of the many E2F1 binding domains in the native PTTG1 promoter fall within the selected region, and KLF6 binding site also fall within the included sequence (406-246) that is responsible for the inhibition of PTTG1 expression in monocyte differentiation. Thus, this promoter element enables DNA based cancer gene therapy in tumor cells.

[0091] The nucleic acid sequence shown in SEQ ID NO: 5 is a selected portion of the human promoter for PTTG1 based upon detailed analysis of transcription factor binding regions and sequence conservation across species, predicted to confer positive transcriptional activity. Epigenetically modified accessible regions upstream of PTTG1 containing genetic elements that determine PTTG1 expression begins at location designated by Ensembl (https: / / www.ensembl.org / Homo_sapiens / lnfo / lndex) genomic assembly location GRCh38:5: 160417784. The beginning of the PTTG1 protein coding region (methionine start codon) occurs at GRCh38:5: 160422314, yielding a 4,530 bp region upstream of the protein coding sequences in PTTG1 that could be involved in regulation of PTTG1 expression. The isolated and identified promoter elements of PTTG1 utilized to promote construct expression in tumor tissue is located at GRCh38:5:160,421,752 and continues to the methionine start codon at GRCh38:5:160422314 yielding a 561 base pair promoter element in the present disclosure which demonstrates tumor biological activity through controlling gene expression.

[0092] In various embodiments, the efficiency and features of the disclosed MLKL-TfR construct has been evaluated by utilizing ROSA26 CRISPR / CAS9 and doxycycline inducible DNA construct approaches to control the expression of MLKL-TfR in phenotypically normal (EPH4) epithelial cells as well as MC38 colorectal tumors. These cell lines have been employed in Damage Associated Molecular Pattern (DAMP) assays along with live cell dual phase / fluorescence imaging to determine the features of MLKL-TfR induced cell death. Utilizing Lentiviral constructs, in various embodiments, LKL-TfR potency was confirmed in a human tumor cell line (K562). In vitro, extracellular release assays demonstrated induction of MLKL-TfR leading to rapid and significant release of the DAMP molecules extracellular ATP and HMGB1. In embodiments, livecell dual phase / fluorescence microscopy approaches characterized the plasma and nuclear membrane features. In embodiments, the kinetics of induced cell death demonstrated that MLKL-TfR expression induces rapid necrosis like cell death, uninhibitable by any Caspase or RIPK inhibitors.

[0093] In additional embodiments, Nod. Scid Common Gamma Deficient Mice (NCG, n=12) and WT mice were employed as subjects to demonstrate construct expression induced immune mediated tumor control. In some examples, to model therapeutic deployment, Balb / c mice bearing established (35mm2) CT26 colorectal tumors were utilized and pDNA encoding a tumor specific promoter controlling expression of MLKL-TfR was delivered through the PPS-PPDP2 nanoparticle delivery system. These further embodiments demonstrated an enhancement of tumor cell control through collaboration between the immune system and construct expression. Nanoparticle delivery of pDNA encoding MLKL-TfR (n =10) to established CT26 tumors under the control of a tumor specific promoter demonstrated significant control of tumor growth (p=.0027) and overall survival (p=. O156) compared to delivery of control plasmid (CMV- GFP).

[0094] In some embodiments, an expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof. In some embodiments, the nucleic acid molecule encoding the chimeric protein comprises a sequence at least 95% identical to SEQ ID NO: 1. In some cases, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1. In some embodiments, the disclosed chimeric protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2. In some cases, the disclosed chimeric protein comprises the amino acid sequence of SEQ ID NO: 2.

[0095] In some aspects, the present disclosure provides a composition comprising a nucleic acid molecule with a first sequence encoding a tumor-specific promoter and a second sequence encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor for treating a tumor, cancer, or neoplasm in a subject. In one embodiment, the chimeric protein comprises a fusion of a portion of mixed lineage kinase domain-like (MLKL) protein (amino acid residues 1-210 of SEQ ID NO. 2) and a portion of transferrin receptor (amino acid residues 211-326 of SEQ ID NO. 2). In some embodiments, the first sequence encoding the tumor-specific promoter is positioned immediately upstream of the second sequence encoding the chimeric protein. In some embodiments, the second sequence encoding the chimeric protein is operably linked to the first sequence encoding the tumor-specific promoter. In some embodiments, the tumor-specific promoter comprises PTTG1. In some cases, the first sequence encoding the tumor-specificpromoter PTTG1 further comprises a sequence at least 95% identical to SEQ ID NO: 5. In some cases, the second sequence encoding the chimeric protein comprises a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, this composition comprising the nucleic acid molecule encoding the tumor-specific promoter and the chimeric protein may be combined with a nanocarrier for delivering to the subject.

[0096] In some embodiments, the nucleic acid molecule encoding the chimeric protein is delivered to the tumor, cancer, or neoplasm of the subject using a lipid-polymeric hybrid nanoparticle system or nanocarriers. In particular embodiments herein, PPS system (e.g., PPS-PPDP2 nanoparticle delivery system) has been used, which is a self-assembling lipid-coblock polymeric system. This nanoparticle delivery system comprises both lipid moieties and peptide conjugated lipid moieties. This system comprises a synthetic PEG-b-PPS-linker-DP polymer for producing nanostructures comprising a polyethylene glycol-b-poly(propylene sulfide) copolymer (PEG-b-PPS) conjugated with a dendritic-specific branched cationic peptide (DP). This system provides a non-toxic method of delivering a polynucleotide to a cell. In embodiments, the size of the nanoparticles when associated with the plasmid are approximately 165nm. Suitable preparation methods of the PEG-b-PPS-linker-DP nanostructure disclosed herein can be prepared via known methods, e.g., Du, F., et al., (2019): Homopolymer Self-Assembly via Polypropylene Sulfone) Networks. ChemRxiv. Preprint; Du F. et al., Sequential intracellular release of water-soluble cargos from Shell-crosslinked polymersomes. J Control Release. 2018; 282:90-100; and Yi S., et al, Tailoring Nanostructure Morphology for Enhanced Targeting of Dendritic Cells in Atherosclerosis. ACS Nano. 2016; 10(12): 11290-11303, each of which are incorporated herein by reference in their entirety. The PEG-b-PPS polymers provide both hydrophobic moieties of PPS to stabilize the nanostructure and hydrophilic PEG corona to enhance cellular uptake and decrease toxicity. The integration of a bioreducible disulfide bond between PPS and DP improves gene delivery efficiency, due to the improved endosomal escape and cargo release in the reductive intracellular environment. In embodiments herein, lipidpolymeric hybrid nanoparticles have been used to deliver the disclosed constructs, and these nanoparticles are a subset of nanocarrier described herein.

[0097] In some embodiments, other delivery vehicles or nanocarrier systems may be used to deliver the disclosed fusion construct to a subject that may include without limitation: lentiviral system, adenoviral system, polymeric nanoparticle system, liposome system, lipid polyplex system, polymersome system, dendrimer system, metal-based nanoparticle system, mesosilicate nanoparticle system, exosome delivery system, membrane-derived nanovesicle system, virus like particle (VLP) system, protein nanocages, proteolipid vehicles, lipid nanoparticle system, andstabilized plasmid-lipid particle system. In embodiments, the disclosed fusion constructs / genetic cargo may be encapsulated within the nanocarrier system, or conjugated to the nanocarrier system, or attached to a surface of the nanocarrier via covalent / non-covalent interactions. In some embodiments herein, the genetic cargo or nucleic acid molecule encoding the chimeric protein has been encapsulated within the disclosed nanocarrier for delivering to the subject.

[0098] Suitable doses for encapsulated genomic material or genetic cargo may be used, and it may depend on the intended therapeutic effect, body weight of the subject, age of the subject, and the like. In general, suitable dosages or effective amounts of the genetic cargo may comprise dose ranging from about 0.5 pg to about 50 pg per 25mm2tumor volume for intra-tumoral injections. Exemplary embodiments may include a dose or quantity of genetic cargo ranging from about 0.5-5 pg, 0.5-10 pg, 0.5-20 pg, 0.5-30 pg, 0.5-40 pg, 5-10 pg, 5-15 pg, 5-20 pg, 5-30 pg, 5-40 pg, 5-50 pg, 10-15 pg, 10-20 pg, 10-30 pg, 10-40 pg, 10-50 pg, 15-20 pg, 15-25 pg, 15-30 pg, 15-40 pg, 15-50 pg, 20-30 pg, 20-40 pg, 20-50 pg, 30-40 pg, 30-50 pg, or 40-50 pg per 25mm2tumor volume. For example, suitable dosages of the genetic cargo may be about 0.5 pg, 0.8 pg, 1 pg, 2 pg, 3 pg, 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, or 50 pg per 25mm2tumor volume. For intramuscular / intravenous / subcutaneous injections, the suitable doses or effective amounts of the genetic cargo may range from about 0.0001 mg / Kg to about 3.0 mg / Kg. Exemplary embodiments may include a dose or quantity of genetic cargo ranging from about 0.0001-0.001 mg / Kg, 0.0001-0.01 mg / Kg, 0.0001-0.1 mg / Kg, 0.0001-1 mg / Kg, 0.0001-2 mg / Kg, 0.001-0.01 mg / Kg, 0.001-0.1 mg / Kg, 0.001-1 mg / Kg, 0.001-2 mg / Kg, 0.001-3 mg / Kg, 0.01-0.1 mg / Kg, 0.01-1 mg / Kg, 0.01-2 mg / Kg, 0.01-3 mg / Kg, 0.1-1 mg / Kg, 0.1-2 mg / Kg, 0.1-3 mg / Kg, 1-2 mg / Kg, 1-3 mg / Kg, or 2-3 mg / Kg. For example, suitable dosages of the genetic cargo may be about 0.0001 mg / Kg, 0.001 mg / Kg, 0.01 mg / Kg, 0.1 mg / Kg, 0.5 mg / Kg, 0.75 mg / Kg, 1 mg / Kg, 1.25 mg / Kg, 1.5 mg / Kg, 1.75 mg / Kg, 2 mg / Kg, 2.25 mg / Kg, 2.5 mg / Kg, 2.75 mg / Kg, or 3 mg / Kg. The relative amounts of nanocarrier to genetic cargo may vary based upon the genomic delivery system.

[0099] In some aspects, embodiments of the present disclosure provide a pharmaceutical composition comprising a pharmaceutically effective amount of the compositions described above and a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 23rd Ed. Academic Press, 2020. Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E, preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders,disintegration agents, and / or lubricants. Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts. Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol. Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.[000100] In some aspects, the compositions may include one or more adjuvants including a mineral adjuvant, gel-based adjuvant, tensoactive agent, oil emulsion, particulated adjuvant, fusion protein, and lipopeptide (Jennings R, Simms JR, Heath AW. Adjuvants and Delivery Systems for Viral Vaccines-Mechanisms and Potential).[000101] In some aspects, the present disclosure provides a composition comprising a nanocarrier encapsulating a nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor for treating a tumor, cancer, or neoplasm in a subject. In some embodiments, the isolated MLKL protein of the chimeric protein comprises a four helical bundle (4HB) domain and a brace region of a native MLKL protein. In some embodiments, the isolated transferrin receptor of the chimeric protein comprises a cytoplasmic domain and a transmembrane domain of a native human transferrin receptor. In some embodiments, the nucleic acid molecule encoding the chimeric protein comprises a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the disclosed chimeric protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2. In some cases, the nucleic acid molecule encoding the chimeric protein is operably linked to a tumor-specific promoter. In embodiments, the tumor-specific promoter comprises PTTG1. In some cases, PTTG1 comprises a sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the nanocarrier may include lipidpolymeric hybrid nanoparticle system, lentiviral system, adenoviral system, polymeric nanoparticle system, liposome system, lipid polyplex system, polymersome system, dendrimer system, metal-based nanoparticle system, mesosilicate nanoparticle system, exosome delivery system, membrane-derived nanovesicle system, virus like particle (VLP) system, proteinnanocages, proteolipid vehicles, lipid nanoparticle system, or stabilized plasmid-lipid particle system.[000102] In some aspects, the present disclosure provides a method of treating a tumor, cancer, or neoplasm in a subject experiencing the tumor, cancer, or neoplasm, the method comprising administering to the subject in need thereof a therapeutically effective amount of the composition comprising a nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor. In some embodiments, the nucleic acid composition further comprises a nanocarrier for delivering the composition to the subject. In some aspects, the present disclosure provides a method of treating a tumor, cancer, or neoplasm in a subject in need thereof, the method comprising: (a) providing a composition comprising a nucleic acid molecule encoding a chimeric protein, wherein the chimeric protein comprises a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor; (b) combining the nucleic acid molecule encoding the chimeric protein with a nanocarrier; and (c) administering an effective amount of the nucleic acid molecule combined with the nanocarrier to the subject for treating the tumor, cancer, or neoplasm. In some embodiments, the nucleic acid molecule encoding the chimeric protein may be operably linked to a promoter, e.g., PTTG1 promoter within a plasmid backbone. The PTTG1 promoter is a tumor-specific promoter. In some examples, the PTTG1 promoter comprises a sequence at least 95% identical to the disclosed SEQ ID NO: 5. Although the present disclosure shows the use of the PTTG1 promoter with the disclosed MLKL-TfR fusion construct, it is to be noted that this tumor-specific promoter sequence can be used in any construct beyond the context of LKL-TfR by one of ordinary skill in the art. For example, the disclosed PTTG1 promoter sequence can be used with other cell depletion therapies of cancer or to drive expression of other therapeutic proteins (adjuvants, antibodies, cytokines, antigens, antigenic peptides) enabling greater spatial and tissue (tumor) specific expression of DNA encoded agents.[000103] In some examples, the nucleic acid molecule encoding the chimeric protein may comprise an mRNA. In some embodiments, an expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof. In some embodiments, the nanocarrier system or delivery vehicle comprises a lipid-polymeric hybrid nanoparticle system.[000104] Thus, the methods described in the present disclosure can be used for treating a subject in need of gene therapy, comprising administering to the subject an effective amount of the systemcomprising of a polynucleotide, wherein the polynucleotide contains the disclosed fusion construct (MLKL-TFR) for DNA based cancer gene therapy.[000105] As described above, an effective amount is one in which any toxic or detrimental side effects of the compound and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. Determination of effective amount is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease or condition symptoms in the subject. Suitable models in this regard include, for example, murine, rat, rabbit, porcine, feline, non-human primate, and other accepted animal model subjects known in the arts. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the disclosed compositions for the treatment of cancer, tumor, or neoplasm in human patients.[000106] Overall, the disclosed fusion construct has potential for use in cellular therapy as a suicide gene, vaccine development, gene therapy in general, and cancer therapy. The disclosed fusion protein can be used in any instance where immediate and potentially immunogenic cell death is required. The examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the scope of the disclosure.(Ill) Examples[000107] Example 1: Materials and Methods[000108] K562, MC38, EpH4, HEK293T cells were purchased from ATCC. K562 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, Thermofisher), 5% heat inactivated fetal bovine serum (FBS), 2mM glutamine, 0.1mM nonessential amino acids, 1mM sodium pyruvate, and 10mM HEPES buffer at 37°C under 5% CO2conditions. Single colony selected MLKL clones were generated by lentiviral mediated transfection into the parental K562, MC38, and EpH4 cells. Briefly, the lentiviral pLVX vectors were transfected into the Lenti-X HEK293T (Takara) cells together with the single shot packaging plasmids provided by Takara. Cells were selected using puromycin (Invivogen) over the course of 10 days and then subjected to limiting dilution in order to isolate monoclonal populations.[000109] Reagents: Where indicated, the following drugs were used at the listed concentration: 1pM staurosporine (Millipore Sigma), 25pg / ml Mitomycin C in ultra-pure molecular biology water(Sigma), 50pM Z-VAD in DMSO (InvivoGen), 1pg / ml Doxycycline in PBS, 0.05% Tween 20 in PBS.[000110] Detection of cell death type by flow cytometry with Annexin V-amine reactive LIVE / DEAD staining: Cells were plated at 1x10A5 cells / well and treated with the indicated reagents. Samples were harvested at specified timepoints, washed, and then stained on ice with LIVE / DEAD NearIR (ThermoFisher) at a dilution of 1:6000 for 10 minutes. The reaction was quenched with neutral buffer containing 0.5% BSA and washed before staining with Annexin V fluorescently conjugated to Alexa Fluor 594 (ThermoFisher) at a dilution of 1:75 for 15 minutes at room temperature, and afterwards fixed in 4% paraformaldehyde in Hank’s balanced salt solution for 20 minutes. Cells were then washed with Annexin V binding buffer enriched in calcium and maintained in the same solution to minimize loss of Annexin V binding. Counting beads (Invitrogen) were added after sample processing to quantitatively determine absolute count of whole cells. Data were acquired on the Cytek Aurora using Spectroflo acquisition software and analyzed with FloJo (BD).[000111] Incucyte: Cells were plated at 1.5k / weil overnight and cultured in puromycin. The culture media was replaced with fresh media containing 1% NucGreen Dead (Invivogen). Cells were treated with the indicated reagents. Cells were placed into Incucyte SX5 (Sartorius). Phase and fluorescence data were captured at 2hour intervals over the course of 96hours. Data were processed using Incucyte basic analysis software and analyzed on Excel & Prism.[000112] Quantification of extracellular ATP release by bioluminescence: Cells were plated at 40K / well overnight and cultured in puromycin. The culture media was replaced with 1x RealTime Gio Extracellular ATP reagent in imaging media containing HEPES (Promega). Cells were allowed to equilibrate at room temperature for 2hrs. Cells were then treated with the indicated reagents. Cells were immediately placed into plate reader for continuous scanning at 15min intervals for 24hrs. Data were analyzed on Excel & Prism.[000113] Human Transferrin receptor protein 1 (TFR1, TFRC, https: / / www.uniprot.org / uniprotkb / P02786 / entry cytoplasmic region amino acids 3(D) through 118 (A), including the transmembrane region signal peptide. Binds with HLAII-DR1. 68-88 type II transmembrane domain signal sequence. 89-118 is the extracellular space including disulfide binding domains at residues 89, 98 - Homodimer.[000114] Ribbon structure was generated utilizing Phyre 2 predictive protein folding server (http: / / www.sbg.bio.ic.ac.uk / ~phyre2 / html / ) in putting the indicated amino acid sequences. K562 cells (Human Leukemic Cell Line) originally obtained from ATCC (American Type Culture Collection, Cat#CCL-243) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco,Thermofisher), 5% heat inactivated fetal bovine serum (FBS,), 2mM glutamine, 0.1mM nonessential amino acids, 1mM sodium pyruvate, and 10mM HEPES buffer at 37CC under 5% CO2 conditions. On day of treatment, live K562 cells were separated from dead cells and debris by layering cell culture supernatant containing non-adherent K562 cells on top of an equal volume of Ficoll Paque PLUS (Cytvia, Cat# 17144002) and performing differential centrifugation at 1200xG for 30mins at 4C. Cells at the aqueous / Ficoll interface were collected, washed with media and counted. K562 Cells were plated at 1x10A5 cells / well in 200pl of media in a round bottom 96 well tissue culture treated plates and treated with the indicated reagents from the designated vendors at the following concentrations: Staurosporine (Stauro, EMD Millipore, Cat# 569396, 1pM), Mitomycin C (Mme, Sigma-Aldrich, Cat#M4287, 25pg / mL), Z-VAD (Zvad, InvivoGen, Cat #tlrl-vad, 50pM), Doxycyline Hydrochloride (Doxy, ThermoFisher Scientific, Cat #BP26531, 1 pg / mL). Samples were incubated in media and conditions as described above and harvested at specified timepoints, washed, and then stained on ice with LIVE / DEAD Fixable Near IR (ThermoFisher Scientific, Cat# L10119) at a dilution of 1:6000 for 10 minutes. The reaction was quenched with neutral buffer containing 0.5% BSA and washed before staining with Annexin V fluorescently conjugated to Alexa Fluor 594 (ThermoFisher Scientific, Cat#A13203) at a dilution of 1:75 for 15 minutes at room temperature, and afterwards fixed in 4% paraformaldehyde in Hank’s balanced salt solution for 20 minutes. Cells were then washed with Annexin V binding buffer enriched in calcium and maintained in the same solution to minimize loss of Annexin V binding. Counting beads (Invitrogen, CountBrite Absolute Counting Beads, Cat#C36950) were added after sample processing to quantitatively determine absolute count of cells. Data were acquired on the Aurora Spectral Cytometer (Cytek) using Spectroflo acquisition software and analyzed with FlowJov10.8.1 (BD Biosciences). Bar and line graphs of data along with statistical analyses were generated using Prism software (GraphPad, v10.3.1). To generate inducible construct expressing K562 cell lines, we cloned the indicated constructs into the bicistronic, doxycycline inducible, puromycin resistant pLVX vector backbone (pLVX-TetOne-Puro, Takara, Cat#631849). To generate competent Lentivirus following manufacturer’s instructions, 7pg of construct containing vector plasmid was mixed with thawed VSV-G pseudotype Lenti-X Packaging Single Shots (Takara, Cat#631275) and 150 l / well of prescribed mixture was added to 75% confluent Lenti-X HEK293T (Takara, Cat#632180) in tissue culture treated 6 well plates containing 2mLs of antibiotic free serum containing media. Supernatants were tested every 24 hours for presence of virus (Lenti-X GoStix Plus, Takara, Cat#631280). Supernatants containing virus were filtered (0.45pM Super Membrane, Pall Life Sciences, Cat#4614) re-titered and frozen at-80C. For lenti-transduction of K562 cells, healthy K562 cells were isolated as described above,250,000 cells were resuspended in 2.5mLs of antibiotic-free serum containing media and treated with Polybrene (Sigma Aldrich, Cat# TR-1003, 4pg / mL) in a 6-well plate. 250pL of viral supernatant was added to the media containing cells, and plates were spun at 32C at 1200xG for 90min, after which time cells were placed into BSL2 designated incubator. 72 hours after introduction of viral supernatant, construct containing cells were isolated by administration of Puromycin (Invivogen, Cat# ant-pr, 10pg / mL) and clonal populations selected by 3 passages of limiting dilution. Construct enriched populations were determined by examining cell death activity after doxycycline administration. Construct enriched populations were expanded and maintained in Puromycin and utilized in the assays.[000115] Phenotypically normal, spontaneously immortalized, nontumorigenic, Balb / c mouse mammary epithelial cell line, EPH4, originally derived and characterized by Rober Friis laboratory and presently commercially available (Millipore, Cat#SCC284) were subjected to transgene insertion in the ROSA26 safe harbor locus of the doxycycline inducible construct utilizing CRISPR / CAS9 genome engineering approaches. The doxycycline inducible, puromycin resistant and MLKL construct containing regions from the PLVX lenti-viral vectors were cloned into a previously validated ROSA26 targeting vector. 1250 EPH4 cells / well were seeded in a 96-well plate and incubated overnight. Following day media was removed and replaced with 200pL of Opti-MEM (ThermoFisher Scientific, Cat# 31985070). 10pl per well containing 2pmol of recombinant CAS9 Nuclease (GenScript, Cat#Z03469), 4pmol of ROSA26 synthetic guide RNA, 37ng of construct containing ROSA26 targeting vector, 0.5pL of CAS9 Plus Reagent (Invitrogen Cat# 100035634), 0.3pL of CRISPRMAX Transfection Reagent (Invitrogen, Cat# 100035629, Lipofectamine CRISPRMAX, Cat#CMAX00001) were added per well. 48 hours after treatment media was replaced and 96 hours of treatment, cells were subjected to puromycin selection (Invivogen, Cat# ant-pr, 10pg / mL) and clonal populations were selected by 3 passages of limiting dilution. Construct enriched populations were determined by examining cell death activity after doxycycline administration. Construct enriched populations were expanded and maintained in Puromycin and utilized in the assays. For live cell imaging assays, construct containing EPH4 cells were plated 1500 cells / well in a flat bottom 96-well tissue culture treated plastic plate in puromycin free, phenol free DMEM media supplemented with 10% Fetal Bovine Serum, 2mM glutamine, 0.1mM nonessential amino acids, 1mM sodium pyruvate, and 10mM HEPES buffer at 37°C under 5% CO2conditions. The following day the plate was flicked to remove media and replaced with 200pL of fresh media / well containing 2 pl / well of NucGreen Dead 488 reagent (Invitrogen, Cat#R37109). After addition of indicated treatments, the 96-well plate was placed into an Incucyte SX5 (Sartorius) residing in a cell culture incubator for automated acquisition of phaseand green fluorescence (400ms acquisition) images every 2 hours for 72 hours utilizing 10x objectives and 2-4 scanning windows per well, 4 wells per treatment group. Staurosporine (Stauro, EMD Millipore, Cat# 569396), Nigericin (Ngrcn, ImmunoChemistry Technologies, Cat#6698). Bar and line graphs of data along with statistical analyses were generated using Prism software (GraphPad, v10.3.1).[000116] 40,000 construct containing EPH4 cell lines were seeded per 96-well plate in puromycin free, phenol free DMEM media supplemented with 10% Fetal Bovine Serum, 2mM glutamine, 0.1 mM nonessential amino acids, 1mM sodium pyruvate, and 10mM HEPES buffer at 37°C under 5% CO2conditions. The following day the plate was flicked to remove media and replaced with 200pL of fresh media / well, treated with the above-described conditions and subjected to a real time extracellular ATP release assay following manufacturer’s instructions (Promega, RealTime-Glo Extracellular Release Assay, GA5010). In this assay, the presence of extracellular ATP was detected as luminescence every 30 minutes over the course of 24 hours at room temperature utilizing a GloMax Multi-*- Detection System (Promega). In another assay, 3000 cells / well of construct containing EPH4 cells were seeded in a flat bottom 96-well plate overnight. The following day media was removed and replaced with fresh 200pl media. Cells were treated as indicated and supernatants were collected, centrifuged at the indicated time points, and centrifuged at 450xG for 5 min to remove any cell or cell debris and then stored at -80C. Once all specimens were collected, supernatants were thawed on ice and subjected to HMGB1 ELISA utilizing the Lumit HMGB1 Human / Mouse Immunoassay (Promega, W6110), providing absolute quantification of extracellular HMGB1. In another assay, 3000 cells / well of construct containing EPH4 cells were seeded in a flat bottom 96-well plate overnight. The following day media was removed and replaced with fresh 100pl media. Cells were treated as indicated. To assess Caspase 1 activity at discrete time points, 60 minutes prior to observation time points, cells were incubated with the Caspase 1 FLICA (Fluorescence Inhibitor of Caspase Activity) FAM-YVAD-FMK (ImmunoChemsitry Technologies, Cat# 655, 1 / 75hvial per well). After FLICA incubation, cells were centrifuged (450xG for 5 min), FLICA containing media was removed and replaced with 150pl / well of 4C media free of FLICA. Cells were then centrifuged again (450xG for 5 minutes), media was removed, and cells were fixed with 4% Paraformaldehyde 3% Sucrose 1 x PBS solution for 10 min, after which cells were centrifuged at 550xG for 5 min, solution was removed and replaced with 1 x PBS and placed into the Incucyte SX5 for fluorescence (450ms acquisition) and phase imaging and quantification. In another assay, 3000 cells / well of construct containing EPH4 cells were seeded in a flat bottom 96-well plate overnight. The following day media was removed and replaced with fresh 100pl media. Cells were treated as indicated. Toassess Calreticulin localization at discrete time points, 60 minutes prior to observation time points, cells were incubated with Calreticulin Antibody (Invitrogen, Cat# MA5-32131, Recombinant Rabbit Monoclonal, 1 / 500). After 60 minutes, cells were washed twice with 1xPBS by centrifuging at 450 x G for 5 minutes, 4C and flicking off media. Then cells were fixed with 4% Paraformaldehyde 3% Sucrose 1 x PBS solution for 10 min (room temperature), after which time cells were centrifuged at 550xG for 5 min, solution was removed and replaced with 1 OOJJI of 1 X PBS, 2mM EDTA, 0.5% BSA staining solution. To detect calreticulin antibody serum crossabsorbed FITC-Donkey anti-Rabbit IgG antibody (Biolegend, Cat# 406403, 1 / 1000) was added to each well and incubated at room temperature for 30 min, during which time NucRed Dead 647 (Invitrogen, Cat# R37113, 1 / 400) was added as a nuclear counterstain. After incubation, cells were washed two more times (550 x G) with staining solution, then cells were fixed with 4% Paraformaldehyde 3% Sucrose 1 x PBS solution for 10 min, after which cell were centrifuged at 550xG for 5 min, solution was removed and replaced with 1 x PBS and placed into the Incucyte SX5 for green fluorescence (450ms acquisition), far red fluorescence (550ms acquisition), and phase imaging and quantification.[000117] MC38 Colorectal Cancer cell line derived from C57BL6 mice (Kerafast, Cat# ENH204-FP), underwent ROSA26 loci recombination to generate stable puromycin resistant, doxycycline inducible MLKL-TfR-eGFP construct containing cell lines utilizing the CRIPSR-CAS9 approach, puromycin selection and limiting dilution approaches described above. To validate construct activity in MC38 cell line, 3500 cells / well were seeded in flat bottom 96-well plate in puromycin free, phenol free DMEM media supplemented with 10% Fetal Bovine Serum, 2mM glutamine, 0.1mM nonessential amino acids, 1mM sodium pyruvate, and 10mM HEPES buffer at37°C under 5% CO2 conditions. The following day the plate was flicked to remove media and replaced with 200pL of fresh media / well containing 2 pl / well of NucGreen Dead 488 reagent (Invitrogen, Cat#R37109). After addition of indicated treatments, the 96-well plate was placed into an Incucyte SX5 (Sartorius) residing in a cell culture incubator for automated acquisition of phase and green fluorescence (400ms acquisition) imaged every 2 hours for 72 hours utilizing 10x objectives and 2-4 scanning windows per well, 4 wells per treatment group. Subsequently, 1x10A6 ROSA26 doxycycline inducible MLKL-TfR-eGFP construct containing MC38 cell lines were implanted unilaterally in the flank of female immunodeficient NOD-Prkdcem26Cd52ll2rgem26Od22 / NjuCrl mice (NCG, Charles River, Cat# 572). After tumors were established and reached the indicated size, mice were administered doxycycline (200pg / injection / 100pl water) or vehicle as indicated and monitored for tumor growth kinetics. The day after a single intra-peritoneal administration of anti-CD8 (BioXcell, Cat# BE0004-1, Clone 53-6.7, 200pg), 1x10A6 ROSA26 doxycycline inducibleMLKL-TfR-eGFP construct containing MC38 ceil lines were implanted unilaterally in the flank of syngeneic female C57BL6 mice (Charles River, C57BL / 6NCr, Cat# 556). After tumors were established and reached the indicated size, mice were administered doxycycline (200pg / injection / 100pl water) or vehicle as indicated and monitored for tumor growth kinetics. Tumor size was determined by caliper measurements.[000118] PPDP2 nanoparticles containing plasmid DNA were assembled by reconstituting lypholized PPDP2 polymer to a 10mg / mL stock in25mM Sodium Acetate Buffer solution. Plasmid DNA diluted to 0.1pg / pL was reacted with equal volume of polymer stock diluted in molecular grade water as to result in a 60:1 polymer to pDNA weight ratio. After gentle mixing by pipette for 30 seconds, the polymer / DNA is incubated at room temperature to allow for nanoparticle selfassembly for 40 minutes. After 40 minutes, 1 / 10 volume of 1M HEPES was used to neutralize the pH of the solution, then the whole solution was placed in a pre-rinsed centrifugation dialysis cassette (Sartorius, Vivaspin 15R, MWCO 30KDa, HY membrane, Cat# VS15RH22) and centrifuged for 12 minutes at 700 x G at 15C. The nanoparticies were retrieved from the cassette by gentle pipetting and quantified based upon absorbance 230, 260, 280 nm absorbance determined on a Nanodrop One (Thermo Fischer Scientific, ND-ONE-W) as well as particle density, DNA concentration, and size determined and confirmed on Stunner dynamic light scattering analyzer (Unchained Labs). For in vitro applications, PPDP2 nanoparticies were used at a concentration of 200ng pDNA / well / 200pl in a 96-well plate, while for in vivo applications, 15pg of pDNA was delivered intratumorally per injection. For determination of in vivo efficacy of pDNA / PPDP2 nanoparticle combination, 400,000 WT CT26 Balb / c derived colorectal tumor cell line (ATCC, Cat# CRL-2638) cells were implanted unilaterally in the flank of syngeneic Balb / c mice (Charles River, BALB / cAnNCr, Cat#555), permitted to grow to indicated size and then treated as described above and monitored for tumor growth by caliper.[000119] Example 2: Rationally designed chimeric MLKL-TfR protein results in rapid necrotic cell death in K562 human leukemic cell line

[0020] Several “suicide genes” have been previously developed and explored as tools for targeted cell removal, for both basic sciences investigations and therapeutic applications. Commonly employed cellular deletion tools include genetic constructs for inducible Caspase 9, Diptheria Toxin A and expression of the pro-drug conversion genes, Thymidine Kinase and cytosine deaminase. While employment strategies can result in successful cellular removal through expression of these genes, these systems are dependent upon signaling cascades to drive cell death through the classical apoptotic pathway, a known inducer of immunologicaltolerance. The present disclosure provides an engineered genetically encoded cell death molecule that results in direct, efficient, and immunogenic tumor cell death.[000121] For the past decade and a half, programmed necrosis has been recognized as form of immunogenic cell death in mammals. Elegant functional, pharmaceutical and crystallographic studies have uncovered many of the pathways and protein effector molecules responsible for the characteristic swelling and rupture of cellular membranes under the influence of necrosis inducing stimuli. One of the most well described pathways is known as necroptosis whose terminal effector activity involves the confirmational rearrangement of the constitutively expressed molecule; mixed lineage kinase domain like pseudokinase (MLKL). Phosphorylation events in the pseudokinase domain region results in a structural rearrangement, hinging open the four helical bundle (4HB) domain and brace region (MLKL (amino acids 1-210)) from the pseudokinase domain. This open conformation permits functionality to the pore-forming effector domain, the 4HB. The 4HB must traffic to the plasma membrane where it self-oligomerizes to form the actual cationic pore. While additional scaffolding amino acids in the wildtype MLKL can assist in trafficking to the membrane, overexpression of the 4HB domain alone (or in conjunction with the brace region) has been shown capable of forming pores in both cells and acellular lipid membranes. In the present disclosure, to facilitate plasma membrane association and enable additional conjugation of genetic cargo without conformational hinderance to the 4HB domain, the MLKL (amino acids 1-210) has been fused with the cytoplasmic and transmembrane domain (amino acids 3-118) of the human transferrin receptor (TFR1, TFRC, gene). Thus, the present disclosure shows wildtype native human MLKL, 4HB and bridge domains of MLKL (amino acids 1-210) and MLKL-human transferrin receptor (TfR, amino acids 3-118) chimeric protein.[000122] The transferrin receptor, a type II transmembrane receptor, has membrane association and recycling properties and may be used as a fusion partner for visualization of membrane dynamics as well as exploring the immunogenicity of membrane associated proteins. Amino acids 68-88 encode for the type II transmembrane domain signal sequence, whereby amino acid residues occurring N-terminal to this sequence are expressed by the endoplasmic reticulum in the intracellular space, while those occurring C terminal are expressed extracellularly. Importantly, this means the transmembrane region not only endows membrane association but allows for the plasma membrane itself to act as an insulator, limiting interaction between the N terminal and C terminal entities during protein synthesis and expression. Thus, it is intended for the selected regions of the transferrin receptor (TfR) to endow not only potent membrane association of MLKL 4HB and brace regions but also provide a scaffold for encoding other genetic cargo, that would reliably not impair MLKL functionality. Lastly, native transferrin receptor is known tohomodimerize. This property of self-association is mediated through C terminal (extracellular) disulfide bonds formed by Cys residues at position 89 and 98. By including these and additional extracellular amino acids of the wildtype transferrin receptor, it was ensured that it simultaneously provides a scaffold for extracellular genetic cargo and further assistance in MLKL selfoligomerization by retaining TfR homodimerization domains. With the goal of employing this genetic construct in human cancer gene therapy, the functionality of this novel fusion protein gene construct was first tested in the human leukemic cell line K562 (Figs. 1A-1D).[000123] To test the functionality of the MLKL-TfR construct in the K562 human cell line, a commercial lentiviral transduction approach was utilized which tightly restricts the expression of gene constructs to conditions in which doxycycline (Doxy) is present. Generation of the doxycycline / tetracycline responsive transcriptional element itself is controlled by the constitutive promoter for glycolytic pathway enzyme phosphoglycerate kinase (PGK). For Lentiviral transduction of K562, two different Lentiviral constructs were utilized, one encoding MLKL amino acids 1-210 (4HB and brace region, hereafter MLKL) and one encoding the novel fusion construct with TfR (MLKL-TfR). After puromycin selection and subcloning of construct containing cell lines, a flow cytometric assay for determining apoptosis from necrosis and establishing the ability of selected cell lines to undergo both classical apoptosis and necrosis was validated. The most conventional assay for measuring apoptosis vs necrosis is the Annexin V, propidium iodide (PI) assay. In this assay, fluor-conjugated Annexin V binds to the phosphatidyl serine (PS) on the extracellular surface of the plasma membrane. In healthy cells, PS is chronically being flipped intracellularly. In cells undergoing classical apoptosis, active Caspase 3 targets and cleaves the enzymes responsible for chronically flipping these lipids, resulting in greater PS being exposed and maintained on the extracellular surface. PI is a cell membrane impermeant DNA intercalating dye whose emission is significantly enhanced when binding to DNA. In classical apoptosis, highly orchestrated processes of plasma and nuclear membrane breakdown (blebbing) and DNA digestion results in membrane enveloped portions of DNA, unreactive to PI. In necrosis, however, plasma membrane permeability and nuclear permeability rapidly occur and thus permit PI access to cellular DNA resulting in a bright signal. Alternatively, membrane permeability can be determined through employment of “Live-Dead” vital dyes. These dyes are membrane impermeant, broadly amine reactive dyes. For a given length of time, the amount of amine reactive proteins both inside and outside of a cell vastly exceed those expressed on the plasma membrane only. Thus, Live-Dead staining allows investigation of plasma membrane permeability, without requiring nuclear accessibility and is both compatible with fixation and available in a spectrum of colors including Near Infrared which minimizes concerns of heightened autofluorescence fromdying cells. First, the Annexin V / Live-Dead assay was employed on the transduced K562 cells in the presence of the alkylating chemotherapeutic agent Mitomycin C (Mme), historically established to drive cell death through apoptosis. Examination of flow plots obtained eight hours post Mme treatment (Fig. 1A, upper middle) cells clearly demonstrated an enhanced frequency of Annexin only (upper left quadrant) staining cells compared to vehicle (Veh) treated cells (Fig.1 A, left), that dramatically expands after 24 hours of Mme treatment (Fig. 1A, lower middle). At 24 hours of Mme treatments a small population of cells is seen occurring that stains brightly for both Live-Dead and Annexin V (upper right quadrant). This mirrors results seen with the classical Annexin V / PI assays in which cell death progresses from classical apoptosis to secondary necrosis in the absence of professional apoptotic phagocytes consuming the apoptotic material. Consequently, the membrane blebs induced by apoptosis breakdown, resulting in enhanced membrane permeability and associated increased staining of either Live-Dead or PI. To further validate reagents, we preincubated and continuously incubated the cells with the potent pan-Capsase inhibitor peptide, ZVAD, which correspondingly reduced the appearance of Annexin V single positive and Annexin V single positive, live-dead single positive cells at both 8 and 24 hours post Mme treatment (Fig. 1 A, right).[000124] Next, the functionality and cell death phenotype induced by the expression of the MLKL-TfR construct was assessed through doxycycline (Doxy) administration (Fig. 1B). Within 8 hours of Doxy treatment a large fraction of cells progressed to the Annexin V / Live Dead double positive state (Fig. 1B, middle upper panel, upper right quadrant) without seeing an initial accumulation in the Annexin V single positive state. This observation was amplified over the following 4 hours. In addition, pre and co-incubation with the caspase inhibitor ZVAD (Fig. 1B, lower panels) did not significantly suppress the double positive signal. Importantly, once membrane permeability occurs Annexin V can bind PS on the interior and exterior of the cell. Quantifying the effects across experimental replicates and comparing to the classical apoptosis induced by Mme and the staining seen by treatment with the membrane permeabilizing agent Tween 20 (Twn20) (Fig. 1 C), it was concluded that the MLKL-TfR expression induces caspase-independent cell death that is necrotic in nature (Fig. 1C, AV+ LD+, lower panel) rather than apoptotic (Fig. 1C, AV+ LD-, upper panel). As described above, constructs in K562 cells that express MLKL alone was also generated. These cell lines were treated likewise and then the degree and kinetics of Apoptotic vs Necrotic cell populations were compared over a 24-hour time course study upon doxycycline addition (Fig. 1D). It was observed that expression of MLKL alone was sufficient to induce necrosis like cell death. Over the course of the first initial 8 hours of doxycycline treatment, it was observed that the MLKL-TfR construct induced a statistically significantly elevated fraction ofNecrotic like cells compared to MLKL alone construct containing cells (Fig. 1 D, right panel). Collectively these data depict that the MLKL-TfR construct design is functional, inducing necrotic cell death in human leukemic cell lines and that the addition of TfR does not hinder MLKL function, nor alter mechanism of cell death.[000125] Example 3: MLKL-TfR platform rapidly induces cell death and nuclear permeability in phenotypically normal murine mammary epithelial cells.[000126] Much can be learned about mechanism of cell death by studying the kinetics and morphology of cells undergoing cell death. While the K562 cells generally confirmed functionality of the constructs, K562 cells are relatively small, round and featureless compared to epithelial cells. In addition, as a cancer cell line, there is the inherent possibility that cell death pathways are perturbed altering kinetics and mechanism of cell death induced by the construct. To validate and further the functionality and phenotype of the MLKL-TfR construct ROSA26-guide RNA paired with recombinant CAS9 proteins was utilized and a ROSA26 targeting vector delivered by lipofection to introduce doxycycline inducible MLKL-TfR constructs into the ROSA26 “safe-harbor” locus of the immortalized but phenotypically normal (non-cancerous, organoid and secretory capable) mouse mammary epithelial cell line EPH4 was used. An incubator dwelling live cell phase and fluorescence capable imaging microscope (Incucyte, SX5) was used to compare the kinetics, cell morphology and nuclear permeability of cells induced to express MLKL-TfR to those subjected to classical apoptotic (Staurosporine, Stauro) and necrotic (Nigericin, Ngrcn) cell death inducers. To visualize nuclear permeability, a cell impermeant nucleic acid binding dye, Nuc Green Dead (NGD) was utilized, that similar to PI described above, exhibits bright fluorescence when bound to DNA but with enhanced imaging properties that permit its addition to live cell imaging.[000127] Over 24 hours, when EPH4 cells containing doxycycline inducible expression of MLKL-TfR in their ROSA26 locus are treated with the pan-kinase inhibitor Staurosporine (Fig. 2A, top row) the cells can be observed to shrink, creating the characteristic small blebs of apoptotic bodies without permitting interaction of DNA with extracellular NGD dye. Shrinking a blebbing exaggerates over the course of 24 hours with evidence of secondary necrosis appearing at 24 hours as the blebs breakdown and NGD interacts with the DNA. In contrast, treatment of the cells with Nigericin (Fig. 2A, middle row), a cationic ionophore, results in cell swelling and membrane permeability observable at 4 hours and exaggerating through 24 hours. As a classical necroptosis inducer, Nigericin’s ability to alter cationic balance results in osmotic imbalance that causes cell swelling and eventually rupture. Upon rupture the intracellular constituents are released into theextracellular space and the residual cell mass shrinks with the remaining nuclear content staining brightly green by NGD. Approximately 4 hours after doxycycline (Doxy, Fig. 2A, lower row) treatment induces genetic expression of MLKL-TfR, the cells demonstrate a hyperdense (small dark, round) morphology and clear plasma and nuclear membrane permeability with vibrant NGD signal observed. This phenotype is amplified over time with the most dramatic changes happening within the first 12 hours of doxycycline administration.[000128] By quantifying the cellular confluence over observable phase microscopy area, the impact of treatments on cell growth kinetics as a surrogate for cell survival / cell death was assessed (Fig. 2B, left panel). As anticipated, significant rapid and durable impairment of cell growth over 48 hours of treatment was observed and the observation was for all treatment groups. Next to quantify necrosis / cell permeability, the signal from the NGD stain was quantified by dividing the NGD positive area by the area occupied by cells (phase + area, confluence). Reflecting the observations in the images above, rapid induction of NGD signal can be observed with doxycycline administration (black filled circles) and Nigericin (grey open down triangles) treatment, while Staurosporine (black filled up triangles) induced apoptosis rapidly kills cells (Fig.2B, growth) but does not produce early NGD signals (Fig. 2B, right panel). This demonstrates that NGD signal over a short time course is an excellent means of detecting necrotic cell death. Importantly and consistent with the results from the K562 assay, the highly significant decreases in cell growth and increases in normalized NGD signal (necrotic cell death) are not significantly impaired by pre and continuous treatment with pan caspase inhibitor ZVAD (grey open circles). As described earlier, one rationale behind the employment of the TfR for the fusion protein was to serve as membrane associating scaffold that could separate distinct N terminal and C terminal moieties preventing unintended inhibition of the 4HB pore forming activity. To test the success of this endeavor, ROSA26 targeting constructs that included GFP on the C terminal side of the transmembrane domain of the TfR (MLKL-TfR-eGFP) was generated. Subjecting EPH4 cell lines containing this construct to the same microcopy assays (Fig. 20), statistically indistinguishable results were observed, consistent with success in the presently disclosed approach and lending feasibility to TfR serving as a platform for co-expression of additional genetically encoded agents.[000129] Example 4: Expression of MLKL-TfR results in DAMP release.[000130] Having firmly established the MLKL-TfR induces rapid, robust cell permeant (necrosis like) cell death, it was sought to further characterize the features of this cell death. As described above, in necrosis, intracellular constituents are released into the extracellular space. When occurring in vivo, these molecules serve as damage associated molecular patterns (DAMPs) thatengage with receptors on immune cells and other cells in the body to alert the body to perturbations from homeostasis, often resulting into immune cell recruitment to the site of cellular damage. Extensive studies of immunogenic cell death cascades have identified several common and important DAMPs. To assess the ability of LKL-TfR expression to elicit DAMP responses, ROSA26, doxycycline inducible EPH4 cell lines were utilized in established commercially available assays comparing the performance of the MLKL-TfR constructs with those elicited by Staurosporine and Nigericin. First, the extracellular release of ATP was assessed (exATP, Fig.3A). Through a real-time luciferase mediated reporter assay, an early and highly significant presence of exATP with doxycycline induction was observed in both constructs (black filled circles, left and right plots). By comparison, induction of exATP was delayed, or delayed and reduced over the time span with Nigericin (gray open down triangles) or Staurosporine (black filled up triangles) respectively despite the robust induction of cell death previously demonstrated (Figs. 2A-2C). Next, the extracellular release of the histone associated nuclear proteins HMGB1 was characterized (Fig. 3B). For this assay, supernatants were collected 4, 8, and 24 hours post treatment and subjected to HMGB1 ELISA assay. Expression of MLKL-TfR (Fig. 3B, filled grey) results in rapid and statistically significant extra-cellular release of HMGB1, on par with those observed for the classical necroptosis inducer Nigericin (filled black) and in contrast with the low levels observed for either DMSO / vehicle treated cells (open black) or those treated with Staurosporine (patterned grey).[000131] A subset of necrotic cell death inducers results in a specific form of immunogenic cell death known as “pyrogenic” cell death. This form of cell death is so named because signaling cascades induce the activation of Caspase 1 which is necessary to cleave the pyrogenic cytokine IL-1 from its pro-form to its active form. IL-1 is a potent instigator of immune cell activation, tissue inflammation and inducer of systemic fever. To evaluate the ability of MLKL-TfR expression to induce activation of Caspase 1, a commercial reagent which contains a cell permeant fluorescent probe linked to a fluorescence quenching agent by a peptide specifically recognized by Caspase 1 was utilized. When Caspase 1 is active, it cleaves this peptide linker releasing the fluor from the quencher enabling signal detection. Using phase and fluorescence imaging at discrete time points Caspase 1 activity in response to MLKL-TfR expression was quantified (Fig. 3C, inset) as well as Staurosporine and Nigericin treatments were quantified (Fig. 3C). It was observed that Nigericin induced a rapid and statistically significant expression of active Caspase- 1 compared to vehicle (DMSO) treated cells. While construct expression significantly elevated active Caspase 1 levels above DMSO controls, they were delayed and subdued compared to Nigericin, while Staurosporine treatments never reached significance over the time course above vehicletreatment alone. Another common hallmark of immunogenic cell death is the translocation of the endoplasmic reticulum protein Calreticulin (CALR) to either the cytoplasm or plasma membrane. When this occurs, staining for CALR goes from discrete bright nuclear adjacent puncta (Fig. 3D, in-set right, Stauro) to cytoplasmic / plasma membrane associated staining (Fig. 3D, in-set left, Ngrcn). To robustly assess the CALR expression pattern upon treatment, cells were treated with inducers and during the final 30 minutes of incubation cells were incubated with anti-Calreticulin antibody. Excess antibody was washed off, cells were fixed and counterstained for nuclei. The amount of cytoplasmic and plasma membrane distribution was inferred from normalizing the CALR area to the nuclear area. In support of this approach, it was observed that Nigericin treatment (Fig. 3D, black filled circles) induced significant increases in CALR expression while Staurosporine (Fig. 3D, open grey up triangles) did not. Induction of MLKL-TfR expression likewise induced significant increases in normalized CALR staining, although again muted to what was observed for Nigericin.[000132] Example 5: Expression of MLKL-TfR results in immunogenic cell death in vivo.[000133] While the results of the DAMP assays provided strong evidence that construct expression induces features consistent with immunogenic cell death, it was next tested whether construct expression would be beneficial in reducing tumor burden in vivo in the context of an intact or impaired adaptive immune system. Once again employing the ROSA26-CRISPR-CAS9 targeting strategy, MC38 mouse colorectal tumor cell lines were transduced with gene constructs encoding doxcycyline inducible expression of MLKL-TfR-eGFP. After selection and subcloning, morphology (Fig. 4A), cell growth (Fig. 4B) and necrotic cell death (Fig. 4C, nuc green dead staining) were evaluated by live cell, in vitro phase and fluorescence microscopy (Fig. 4A). Analogous to what was demonstrated before in K562 and EPH4 cells, rapid (Fig. 4B), cell permeant (NGD+, Fig. 4C), necrosis like cell death was observed upon doxycycline administration (grey filled circles, grey box). Next, one million MC38 tumors were implanted unilaterally in the flanks of immune-deficient (NOD. SCI D Common Gamma Deficient, NCG) mice and were permitted to grow till the average size per randomized group was 35mm2. Mice then received either doxycycline (Doxy) or vehicle by i.p. injection every other day while tumor growth and overall survival was assessed (Fig. 4D). Across two experimental replicates, mice were treated with doxycycline and therefore tumors were forced to express MLKL-TfR-eGFP. A significant decrease in overall tumor size compared to vehicle treated animals was observed up through day 10, the day the first mouse was euthanized due to terminal tumor size (Fig. 4D). The reduction in tumor growth was most pronounced and significant 2- and 4-days post treatment. After this timethe tumor growth continued (Fig. 4E), ultimately resulting in a modest delay in overall survival (Fig. 4F). The lack of complete tumor eradication upon doxycycline treatment could potentially be attributed to tumor clonal selection, where during initial outgrowth and after doxycycline induction, subsets of non-construct containing tumor cells outgrow and fill in the space of construct eradicated cells. Regardless, these results established a benchmark for construct efficacy in an immunologically deficient animal and importantly provided a realistic model for outcome given it is certainly unlikely that future nanoparticle experiments would be able to target every tumor cell.[000134] Given the objective was to generate immunogenic tumor cell death, next the construct efficacy in providing tumor control was evaluated in syngeneic (C57BL6, B6), fully immunocompetent hosts using the exact same ROSA26 locus doxycycline inducible MLKL-TfR-EGFP construct containing MC38 tumor cell line used in the NCG experiments. Recent work has highlighted that one complication for implantable tumor models has been the eliciting of an adaptive immune response during the initial seeding of tumors. Since the interest is in evaluating the newly elicited cytotoxic (CD8) T cell immune response upon construct expression, one day prior to tumor implantation the animals were pre-treated by administering a single 200 pg dose of a CDS depleting antibody. Studies have demonstrated that such a dose is sufficient to deplete CD8 T cell populations for 4 days. Therefore, when tumors reached the established treatments size of 35mm2a week and a half after implantation, the re-populated CD8 T cells present in the animal could only be educated by the established tumor microenvironment or the induction of construct expression and not a “pseudo-vaccination” response to the initial tumor implantation (Figs. 4G-4J). Once again administering doxycycline or vehicle i.p. to the fully immunocompetent, syngeneic host with established 35mm2, a highly significant reductions in tumor size was observed in doxycycline treated animals (Fig. 4G, grey filled circles). Unlike experiments in the immune-compromised NCG mice, this reduction of tumor size persisted throughout the 10-day duration (Fig. 4G) till the first animal reach terminal tumor size (Fig. 4H). In addition, tumor growth retardation was observed in several mice 14-30 days post doxycycline administration (Fig. 4H), which was interpreted as evidence of immune mediated control, since this was not observed in the NCG mice. Correspondingly, this resulted in a highly significant extension of overall mouse survival (Fig. 4I) in construct expressing mice (doxycycline administered) compared to mice receiving vehicle only. To quantify a potential immune mediated control effect, the number of days elapsed for each mouse from when their tumor reached 100mm2to achieving terminal tumor size of 200mm2was plotted (Fig. 4J). Rationally by the time mouse tumors reach 100mm2, there should be no impact of doxycycline administration, as all construct expressing tumors should be deleted, as observed in NCG experiments. Therefore, the prolonged duration of time to progressfrom 100mm2to 200mm2can be attributed to immune mediated control. Aligned with this rationale, it was observed that vehicle and doxycycline administered NCG animals progress from 100mm2to 200mm2at the same time as vehicle treated wildtype B6 animals and each other. While the doxycycline treated animals in the fully immune competent background demonstrated highly significant delay in tumor growth, consistent with construct expression and consequent tumor cell death imparting immune mediated tumor control (Fig. 4J).[000135] Example 6: PTTG1 promoter element enables nanoparticle delivered immunogenic cell death cancer gene therapy[000136] To this point, utilizing genetic constructs stably expressed by either lentivirus or ROSA26 directed recombination in 3 different mouse and human cell lines has allowed us to robustly validate MLKL-TfR efficacy as well as characterize the mechanism and immunogenic “flavor” of construct induce cell death without confounding variables that could be attributed to delivery vehicles. However, to succeed in the intended efforts to generate a novel and translationally relevant genetic construct therapy for inducing immunogenic cancer cell death, an effective nanoparticle delivery platform was employed as well as a means for tumor selective expression of MLKL-TfR in vivo was engineered. Given the established nature of plasmid DNA (pDNA) for conditional construct expression in nanoparticle gene therapy, a promoter element was sought to be identified with activity that would be enriched in tumor cells over normal tissue. Specifically, promoters of transcription factors whose expression levels differed greatly in tumor vs normal human tissues were targeted. The level and activity of PTTG1 in tumors indicate that the PTTG1 promoter may be a viable candidate for controlling tumor specific / enriched construct expression. To further PTTGI’s candidacy, the overall RNA expression levels (FPKM, Upper Quartile normalized, UQ) of PTTG1 in a broad selection of normal and cancerous tissues extracted from the TOGA public data set were examined (Fig. 5A). Analysis of this data independently validated the observations that PTTG1 is overexpressed or highly active in wide range of cancer tissues. Next, whether or not PTTG1 expression was associated with poor prognosis was considered. While using a promoter from a gene with high tumor expression might seem sufficient for therapeutic employment, if higher gene expression is associated with good outcomes, it could be that within the heterogeneity of tumor mass, the tumors that overexpress that given gene, are those that are already favorably responsive to traditional therapy. In which case, augmentation with cancer gene therapy would confer a more limited benefit. Fortunately for PTTG1, when patient outcomes for a given tumor type were divided into PTTG1 high (black line)and PTTG1 low (grey line) cohorts, PTTG1 expression significantly associated with poorer overall survival 5 years post initial diagnosis (Fig. 5B).[000137] Next, the proximal regions in the human PTTG1 promoter for recognized regulatory elements and levels of evolutionary conservation were examined. From this analysis, a 561 base pair fragment of the promoter was identified as a putative gene expression control element. To determine which processes and transcription factors might be associated with this region, JASPAR promoter motif analysis was first performed. Given the first in vivo validation of these experiments would be in mice, the mouse transcription factor annotations were utilized. The predicted active transcription factors were then taken from this analysis and STRING database analyses was performed to see if specific cellular activities were associated with the predicted transcription factors. Interestingly and reassuringly, the specific activities of greatest significance were the KEGG annotated “Pathways in Cancer" and Wikipathways “EGFR1”. Thus, STRING database analysis of JASPAR predicted transcription factors associated with the selected PTTG1 promoter sequence showed statistically significant enrichment for pathways upregulated in cancer and EGFR1 signaling. To test the biological activity of this promoter in tumors, the PTTG1 promoter element was cloned synthesized and cloned into a commercially available, well-established vaccine / gene therapy plasmid backbone NTC-7482, where it was positioned to drive GFP expression. Utilizing commercial lipid-based transfection reagent 4K (Roche), a multitude of tumor and phenotypically normal cell lines were lipo-transfected. Using phase and fluorescence microscopy, it was observed that this promoter element was sufficient to drive gene expression in some but not all tumor cells and cell lines. From these experiments, the CT26 Balb / c derived colorectal tumor cell lines were selected as being a reasonable candidate for elevated PTTG1 activity (Fig. 5C).[000138] For in vivo delivery of the pDNA containing the PTTG1 promoter element driving expression of MLKL-TfR-eGFP, PEG-PPS nanoparticles were employed. PEG-PPS based nanoparticles have been shown to be effective for in vitro transfection of both DNA and RNA in myeloid immune cells. PEG-PPS based nanoparticles have also demonstrated high in vivo safety, tolerability and efficacy for targeted delivery of drugs to tumors. In the present disclosure, the use of the lipid co-block-polymer PEG-PPS polymer system (PPDP2) was explored. The PPDP2 system is composed of polymeric (17mer) PEG module linked to polymeric PPS (80mer) module connected to a di-peptide (LHRK, LHR) branched network or double cationic peptide scaffold. The di-peptide branched network contains positively charged amine groups or protonated primary amines that are intended to interact with the negatively charged phosphate (P -) backbone of the double stranded DNA. The PPS module is responsible for orchestrating nanoparticle self-assembly, while the PEG module enhances biocompatibility and stability by shielding charges conferring aqueous solubility. The modules are linked together through sulfur bonding which is susceptible to reduction in vivo by actions of reduced pH (as occurs in the endosome) and interaction with reactive oxygen species, thus the polymeric nanoparticle is intrinsically biodegradable. Once the polymer is degraded, protonated amines of the peptide backbone not bound to DNA are intended to facilitate endosomal escape. Earlier studies of PEG-PPS nanoparticles, report highly reproducible spherical morphology of self-assembled nanoparticles. Results from dynamic light scattering (DLS, Fig. 5D) of multiple separate batches of self-assembled nanoparticles with and without plasmid corroborated this regularity with PPDP2 alone creating nanoparticles ~ 24nm in size while inclusion of the DNA resulted in particles ~167nm in size.[000139] To test the potential in vivo translational relevance of this genetic construct -nanoparticle system, 4 x 105CT26 tumor cells were implanted unilaterally, in the flanks of syngeneic, wildtype Balb / c mice. When the randomized tumor cohorts reached 35mm2average tumor size, mice received either 15 pg pDNA encoding CMV-GFP on the NTC-7482 backbone encapsulated by PPDP2 (Fig. 5E, black open circles), 15 pg pDNA encoding PTTG1-MLKL-TfR-eGFP on the NTC-7482 backbone encapsulated by PPDP2 (Fig. 5E, grey filled circles), an equivalent dose of PPDP2 polymeric nanoparticle alone (Fig. 5E grey open circles) or equivalent volume of vehicle alone (grey open squares) through intra-tumoral injection. Importantly, mice received repeat intra-tumoral injections every 4 days. Across two separate experimental replicates, it was observed that administration of PTTG1-MLKL-TfR-eGFP significantly impaired tumor growth compared to all other treatment groups through the first 8 days of treatment, the extent of time prior to the first control mouse being euthanized due to tumor size, (Figs. 5E and 5F). Examination of individual tumor / mouse growth plots over 4 weeks (Fig. 5F) showed significantly reduced tumor growth which translated to significantly extended overall survival (Fig.5G) in the PTTG1-MLKL-TfR-eGFP (dark grey line) compared to PPDP2 administration of the irrelevant plasmid CMV-GFP (black line). In addition, it was noted that CMV-GFP containing PPDP2, as well as PPDP2 only nanoparticles overlapped with regards to tumor growth and survival with those of vehicle treated mice, meaning despite repeated administration neither plasmid DNA backbone, nor the polymeric nanoparticle initiated biological responses (i.e. inflammation) that significantly hindered tumor growth.[000140] Concluding Paragraphs[000141] Each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.[000142] Unless otherwise indicated, all numbers expressing quantities of ingredients, agent / drug / inhibitor concentrations, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.[000143] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.[000144] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.[000145] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.[000146] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.[000147] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.[000148] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.[000149] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.[000150] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006).

Claims

CLAIMS1. A composition comprising a nucleic acid encoding an isolated mixed lineage kinase domain-like (MLKL) protein for use in inhibition of a tumor in a subject harboring the tumor.

2. The composition of claim 1, wherein the nucleic acid encoding the isolated MLKL protein further comprises a sequence encoding transferrin receptor.

3. The composition of any of claims 1 and 2, wherein the isolated MLKL protein induces necroptotic-like death of the tumor in the subject.

4. The composition of any of claims 1-3, wherein the isolated MLKL protein enhances an immune response to the tumor in the subject.

5. The composition of any of claims 1-4, wherein the tumor is selected from the group of leukemia, colorectal cancer, breast cancer, and ovarian cancer.

6. The composition of any of claims 1-5, wherein the nucleic acid encoding the isolated MLKL protein comprises a sequence coding for a portion of a native LKL protein.

7. The composition of claim 6, wherein the portion comprises a four helical bundle (4HB) domain and a brace region of the native LKL protein.

8. The composition of any of claims 6 and 7, wherein the portion is devoid of a pseudokinase domain of the native MLKL protein.

9. The composition of any of claims 2-8, wherein the sequence encoding transferrin receptor comprises sequences coding for a cytoplasmic domain and a transmembrane domain of the transferrin receptor.

10. The composition of any of claims 2-9, wherein the isolated MLKL protein is configured to be fused with the transferrin receptor to form a fusion construct.

11. The composition of any of claims 1-10, wherein the nucleic acid comprises a DNA sequence at least 95% identical to SEQ ID NO: 1.

12. The composition of claim 11, wherein the nucleic acid comprises the DNA sequence of SEQ ID NO: 1.

13. The composition of any of claims 11 and 12, wherein the nucleic acid is plasmid DNA, linear double strand DNA, DNA included in a viral vector, or complexed DNA.

14. The composition of any of claims 1-13, wherein the nucleic acid is delivered using a nanoparticle system.

15. The composition of any of claims 1-14, wherein the composition is administered to the subject by intra-tumoral delivery.

16. The composition of any of claims 2-15, wherein an expression of the isolated MLKL protein fused with the transferrin receptor in the tumor is transient or inducible.

17. A fusion protein comprising the isolated MLKL protein and the transferrin receptor encoded by the nucleic acid of any of claims 1-16.

18. The fusion protein of claim 17, wherein the fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

19. The fusion protein of claim 18, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

20. The fusion protein of any of claims 17-19, wherein the transferrin receptor is included on a C terminal side of the isolated MLKL protein.

21. A composition comprising a nucleic acid molecule encoding a chimeric protein for use in treatment of a tumor, cancer, or neoplasm in a subject in need thereof, the chimeric protein comprising a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor.

22. The composition of claim 21, wherein the tumor, cancer, or neoplasm is selected from the group of leukemia, colorectal cancer, breast cancer, and ovarian cancer.

23. The composition of any of claims 21 and 22, wherein the nucleic acid molecule comprises a sequence at least 95% identical to SEQ ID NO: 1.

24. The composition of claim 23, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

25. The composition of any of claims 21-24, wherein the chimeric protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

26. The composition of claim 25, wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO: 2.

27. The composition of any of claims 21-26, wherein the isolated MLKL protein of the chimeric protein comprises a four helical bundle (4HB) domain and a brace region of a native MLKL protein.

28. The composition of claim 27, wherein the isolated MLKL protein of the chimeric protein is devoid of a pseudokinase domain of the native MLKL protein.

29. The composition of any of claims 21-28, wherein the isolated transferrin receptor of the chimeric protein comprises a cytoplasmic domain and a transmembrane domain of a native human transferrin receptor.

30. The composition of any of claims 21-29, wherein the nucleic acid molecule encoding the chimeric protein is DNA or RNA.

31. The composition of claim 30, wherein the DNA is plasmid DNA, linear double strand DNA, DNA included in a viral vector, or complexed DNA.

32. The composition of claim 30, wherein the RNA is mRNA, RNA included in a viral vector, or complexed (m)RNA.

33. The composition of claim 32, wherein the mRNA further comprises a 5' cap and / or a 3' poly(A)tail and / or a 5' untranslated region and / or a 3' untranslated region.

34. The composition of any of claims 30-33, wherein an expression of the chimeric protein in the tumor, cancer, or neoplasm is transient or inducible.

35. The composition of claim 34, wherein the expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof.

36. The composition of any of claims 21-35, wherein the nucleic acid molecule encoding the chimeric protein is delivered to the tumor, cancer, or neoplasm of the subject in need thereof using a system selected from the group of lentivirus, adenovirus, polymeric nanoparticle, lipidpolymeric hybrid nanoparticle, liposome, lipid polyplex, polymersome, dendrimer, metal-based nanoparticle, mesosilicate nanoparticle, exosome, membrane-derived nanovesicle, virus like particle, protein nanocage, proteolipid vehicle, lipid nanoparticle, and stabilized plasmid-lipid particle.

37. The composition of claim 36, wherein the nucleic acid molecule encoding the chimeric protein is delivered to the tumor, cancer, or neoplasm of the subject using the lipid-polymeric hybrid nanoparticle system.

38. A method of treating a tumor, cancer, or neoplasm in a subject experiencing the tumor, cancer, or neoplasm, the method comprising administering to the subject in need thereof a therapeutically effective amount of the composition of claim 21.

39. The method of claim 38, wherein the composition further comprises a nanocarrier.

40. The method of claim 38, wherein expression of the chimeric protein in the tumor, cancer, or neoplasm causes an immunogenic cell death in the tumor, cancer, or neoplasm of the subject in need thereof.

41. The method of claim 39, wherein the nanocarrier comprises a system selected from the group of lentivirus, adenovirus, polymeric nanoparticle, lipid-polymeric hybrid nanoparticle, liposome, lipid polyplex, polymersome, dendrimer, metal-based nanoparticle, mesosilicate nanoparticle, exosome, membrane-derived nanovesicle, virus like particle, protein nanocage, proteolipid vehicle, lipid nanoparticle, and stabilized plasmid-lipid particle.

42. The composition of claim 29, wherein one or more amino acid residues of the transmembrane domain of the native human transferrin receptor are modified to alter membrane dynamics.

43. The composition of claim 29, wherein one or more amino acid residues of the transmembrane domain of the native human transferrin receptor are modified to attach an intracellular or extracellular cargo.

44. The composition of any of claims 21-37, wherein the nucleic acid molecule encoding the chimeric protein is operably linked to a tumor-specific promoter.

45. The composition of claim 44, wherein the tumor-specific promoter comprises PTTG1.

46. The composition of claim 45, wherein PTTG1 comprises a sequence at least 95% identical to SEQ ID NO: 5.

47. The composition of claim 45 or claim 46, wherein treatment of the tumor, cancer, or neoplasm further comprises PTTG1 being used for cell depletion therapy of the tumor, cancer, or neoplasm or tumor enhanced expression of other DNA encoded therapeutic agents selected from the group of antigens, antigenic peptides, adjuvants, antibodies, and cytokines.

48. A composition comprising a nucleic acid molecule encoding a chimeric protein for use in prophylactic vaccination or therapeutic treatment of an infectious disease in a subject in need thereof, the chimeric protein comprising a fusion of an isolated mixed lineage kinase domain-like (MLKL) protein and an isolated transferrin receptor.

49. The composition of claim 48, wherein the nucleic acid molecule comprises a sequence at least 95% identical to SEQ ID NO: 1.

50. The composition of claim 49, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

51. The composition of any of claims 48-50, wherein the infectious disease comprises a viral infection.

52. The composition of claim 51, wherein the viral infection further comprises Hepatitis B, Hepatitis C, or HIV.

53. The composition of any of claims 48-50, wherein the infectious disease comprises a bacterial infection.

54. The composition of any of claims 48-53, wherein expression of the chimeric protein leads to an elimination of cellular viral reservoirs by inducing a direct cell death.

55. A pharmaceutical composition comprising a pharmaceutically effective amount of the composition of any of claims 1 to 37 and a pharmaceutically acceptable carrier.

56. The pharmaceutical composition of claim 55 further comprising an adjuvant.

57. The use of the composition of any of claims 48 to 54, in the manufacture of a pharmaceutical composition.

58. A kit comprising the composition of any one of claims 1 to 37 or the pharmaceutical composition according to claim 55 or 56.

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