Methods and compositions for targeting gene imbalances in cells and tissues

Abstract

Disclosed herein are methods and compositions for improving competitiveness of a host cell, tissue or organ against a metastatic, invading, or misplaced cell. Also provided are gene therapy compositions for expressing a serine protease inhibitor in a target cell.

Description

TITLEMETHODS AND COMPOSITIONS FOR TARGETING GENE IMBALANCES IN CELLS AND TISSUESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 678,411 , filed August 1 , 2024, and U.S. Provisional Application No. 63 / 724,179, filed November 22, 2024, the disclosures of each are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety for all purposes. The XML copy, created on July 30, 2025, is referred to as UTSD.P4282WO_SequenceListing.xml and is 29,819 bytes in size.BACKGROUND1. Field

[0003] The present disclosure is generally directed to methods and compositions for improving competitiveness of a host cell, tissue or organ against a metastatic, invading, or misplaced cell or improving competitiveness of a cell of a developing organ into a body region.2. Description of Related Art

[0004] Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. Given the particularly poor prognosis of BCLM (patients have a 5-year survival rate of 8%), there is an urgent need for novel therapeutics to target liver-specific treatment for BCLM.SUMMARY

[0005] In various aspects, the present disclosure is directed to a method of treating a metastatic tumor in a subject by administering a therapeutic agent that modulates the activity of one or more cellular factors in one or more host cells and / or one or more metastasizing cancer cells, wherein the one or more host cells are located in an organ or tissue that comprises a site of metastasis and / or the one or more metastasizing cancer cells, and the one or more cellular factors are selected from a serine protease inhibitor, C-X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20,solute carrier family 4 member 7, phosphoserine aminotransferase 1 , sterol O-acyltransferase 1 , protein phosphatase 1 regulatory subunit 18, Matrix Gia protein, Parathyroid hormone- related protein, Pleiotrophin, Midkine, Secreted frizzled-related protein 1 , Aldehyde dehydrogenase family 1 member A3, Nucleolin, Sclerostin domain-containing protein 1 , Histone-lysine N-methyltransferase EZH2, Insulin-like growth factor-binding protein 2, Transient receptor potential cation channel subfamily M member 2, Galectin-7, or any combination thereof.

[0006] In some aspects, the one or more cellular factors comprise the serine protease inhibitor (Serpin), Matrix Gia protein, Parathyroid hormone-related protein, C-X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20, solute carrier family 4 member 7, phosphoserine aminotransferase 1 , or any combination thereof. In some aspects, the one or more cellular factors comprise at least one serine protease inhibitor (Serpin) selected from SerpinE2, SerpinEI , SerpinE3, SerpinAI , SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAI 0, SerpinA11 , SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinBH , SerpinB12, SerpinB13, SerpinCI , SerpinDI , SerpinFI , SerpinF2, SerpinGI , SerpinHI , Serpinll , and Serpinl2. In some aspects, the one or more cellular factors comprise SerpinE2.

[0007] In some aspects, the therapeutic agent increases activity and / or expression levels of the cellular factor in the one or more host cells. In some aspects, the therapeutic agent comprises a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a gene therapy vector, a nanoparticle, a liposome, a polysaccharide, or any combination thereof. In some aspects, the therapeutic agent comprises a gene therapy vector. In some aspects, the gene therapy vector comprises an expression construct encoding the one or more cellular factors. In some aspects, the gene therapy comprises an expression construct as described herein and / or a vector as described herein.

[0008] In some aspects, the therapeutic agent decreases activity and / or expression levels of the cellular factor in the one or more metastatic cancer cells. In some aspects, the therapeutic agent comprises a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a gene therapy vector, a nanoparticle, a liposome, a polysaccharide, or any combination thereof. In some aspects, the therapeutic agent comprises a small molecule inhibitor of the cellular factor or an interfering nucleic acid that reduces expression of the cellular factor. In some aspects, the therapeutic agent comprises an interfering nucleic acid that specifically targets a 10-50 nucleotide sequence region on a sequence as set forth in SEQ ID NO: 2, or a sequence at least about 60% identical thereto.

[0009] In some aspects, the metastatic tumor comprises a breast tumor, a liver tumor, a pancreatic tumor, squamous cell tumor, prostate tumor, melanoma, skin tumor, urothelial tumor, kidney tumor, colon tumor, gastric tumor, gastrointestinal tumor, a lung tumor, melanoma, a sarcoma, an adnexal tumor, or a tumor of a peritoneum. In some aspects, the metastatic tumor comprises a breast cancer liver metastasis (BCLM). In some aspects, the organ or tissue comprising the site of metastasis is selected from liver, bone, lungs, brain, and adrenal glands. In some aspects, the one or more metastatic cancer cells comprise metastatic breast cancer cells. In some aspects, the one or more host cells comprise hepatocytes. In some aspects, the method further comprises reducing or preventing metastasis in the subject. In some aspects, the subject is human.

[0010] In various aspects, the present disclosure is directed to an expression construct comprising a nucleic acid, the nucleic acid comprising a nucleic acid sequence encoding for a serine protease inhibitor operably linked to a promoter sequence. In some aspects, the serine protease inhibitor comprises SerpinE2, SerpinEI , SerpinE3, SerpinAI , SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAI 0, SerpinA11 , SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinBH , SerpinB12, SerpinB13, SerpinCI , SerpinDI , SerpinFI , SerpinF2, SerpinGI , SerpinHI , Serpinll , or Serpinl2. In some aspects, the serine protease inhibitor comprises SerpinE2. In some aspects, the nucleic acid sequence encoding for the serine protease inhibitor has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

[0011] In some aspects, the promoter is a constitutive promoter or an inducible promoter. In some aspects, the promoter is a tissue specific promoter. In some aspects, the tissue specific promoter is a liver specific promoter. In some aspects, the liver specific promoter is a TBG promoter or an hAAT promoter. In some aspects, the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence to any one of SEQ ID NOs: 3 to 6. In some aspects, the expression construct is optimized for expression in mammalian cells. In some aspects, the expression construct is optimized for expression in human cells, mouse cells, rat cells, canine cells, non-human primate cells, or bovine cells.

[0012] In various aspects, the present disclosure is directed to a vector comprising the expression construct as described herein, wherein the vector is formulated for delivery to a cell, optionally wherein the promoter is heterologous to the cell. In some aspects, the vector comprises a viral vector. In some aspects, the viral vector comprises a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a poxviral vector, or a herpes viralvector. In some aspects, the viral vector comprises an adeno-associated viral (AAV) vector, wherein the AAV vector is serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), or any combination thereof. In some aspects, the AAV vector is an organ specific AAV vector. In some aspects, the vector is a recombinant adeno-associated viral (AAV) vector comprising a first AAV ITR sequence, the promoter sequence, a transgene nucleic acid molecule comprising the nucleic acid sequence encoding the serine protease inhibitor, a polyA sequence, and a second AAV ITR sequence. In some aspects, the AAV vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7 or 8. In some aspects, the vector comprises a non-viral vector. In some aspects, the non-viral vector comprises a lipid nanoparticle (LNP) or an antibody-oligo conjugate.

[0013] In various aspects, the present disclosure is directed to a cell comprising the expression construct as described herein or the vector as described herein. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a human cell, a mouse cell, a rat cell, a canine cell, a non-human primate cell, or a bovine cell. In some aspects, the cell is a hepatocyte or other host organ-specific non-cancerous cell. In some aspects, the cell is an induced pluripotent stem cell (iPSC).

[0014] In various aspects, the present disclosure is directed to a composition comprising (a) the expression construct as described herein, a vector as described herein, or a cell as described herein, and (b) at least one pharmaceutically appropriate carrier or excipient.

[0015] In various aspects, the present disclosure is directed to a kit comprising (a) an expression construct as described herein, a vector as described herein, a cell as described herein, a composition as described herein, or any combination thereof, and (b) at least one container.

[0016] In various aspects, the present disclosure is directed to a method of identifying one or more cellular factors that increase competitiveness of a metastasizing tumor cell against host cells in an organ or tissue comprising a site of metastasis. The method comprises obtaining or having obtained: (i) a first gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing tumor cell and having an elevated organ-specific metastatic potential relative to other tumor cells of the same tumor type; (ii) a second gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing cancer cell; (iii) a third gene expression dataset of one or more developing cells (e.g., embryonic cells) in an organ of origination of the metastasizing tumor cell; (iv) a fourth gene expression dataset of the metastasizing tumor cell or one or more metastasizing tumor cells of the same tumortype as the metastasizing cancer cell that have metastasized to the same target organ; and (v) a fifth gene expression dataset of one or more host cells in the organ or tissue comprising the site of metastasis. The method further comprises identifying one or more genes that have elevated expression in the first expression dataset, have elevated expression in both the second gene expression dataset and third gene expression dataset, and have higher expression in the fourth gene expression dataset relative to the fifth gene expression dataset. The method further comprises identifying one or more protein products encoded by the one or more genes as the one or more cellular factors that increase competitiveness of the metastasizing tumor cells.

[0017] In various aspects, the present disclosure is directed to a method of treating a cancer metastasis in a subject in need thereof, the method comprising identifying at least one cellular factor that increases competitiveness of metastasizing cells in the cancer metastasis according to the method described herein and administering at least one therapeutic agent that modulates activity of the cellular factor in a cell to the subject. In some aspects, the therapeutic agent decreases activity of the cellular factor in at least one metastatic cancer cell in the subject. In some aspects, the therapeutic agent that decreases activity of the cellular factor comprises a gene therapy vector, a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a nanoparticle, a liposome, a polysaccharide, or any combination thereof. In some aspects, the therapeutic agent that decreases activity of the cellular factor comprises a small molecule inhibitor of the cellular factor or an interfering nucleic acid that reduces expression of the cellular factor. In some aspects, the therapeutic agent increases activity and / or expression levels of the cellular factor in one or more host cells in the organ or tissue comprising the site of metastasis. In some aspects, the therapeutic agent that increases activity and / or expression levels of the cellular factor comprises a gene therapy vector, a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a nanoparticle, a liposome, a polysaccharide, or any combination thereof. In some aspects, the therapeutic agent that increases activity and / or expression levels of the cellular factor comprises a gene therapy vector encoding for the cellular factor.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. Aspects of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein:

[0019] FIG. 1 is a schematic of a current hypothesis that metastatic cancer cells (MCC) use cell competition to kill hepatocytes (HEP) to expand and that therapeutics aimed at restoring hepatocyte fitness may be effective anti-cancer therapies.

[0020] FIG. 2 is a schematic of an in vitro experiment for tracking growth of cancer cells (4T 1 cells, in red) and hepatocytes (H2.35 cells, in green) in co-culture.

[0021] FIG. 3 shows representative immunofluorescent images of 1:1 co-cultures of WT metastatic cancer cells (MCC, in red) and hepatocytes (in green) one (Day 2) and two days (Day 3) after seeding in a 1 :1 ratio (at day 1).

[0022] FIGS. 4A-4B are representative plots showing hepatocyte and WT cancer cell growth as measured by area (FIG. 4A) or total cell count (FIG. 4B) when cells were cultured in isolation.

[0023] FIG. 5 shows a process for quantifying apoptosis events among hepatocyte or cancer cell neighbors.

[0024] FIG. 6 is a representative image of cancer cells (red) and hepatocytes (green) in coculture showing caspase-3 activation in hepatocytes (pale green).

[0025] FIGS. 7A-7C are representative fluorescent image (FIG. 7A) and quantifications (FIG. 7B-7C) of caspase levels in co-cultures (FIG. 7A, 7B) or monocultures (FIG. 7C) of cancer cells (4T1) and / or hepatocytes (H2.35).

[0026] FIG. 8 is a bar graph of %caspase positive cancer cells (EMT6) and liver cells (H2.35) in co-cultures.

[0027] FIGS. 9A-9B show a representative schematic (FIG. 9A) of an in vivo cancer metastasis model and a resulting image (FIG. 9B) showing hepatocytes (green), metastatic cancer cells (MCC, red) and caspase (white).

[0028] FIGS. 10A-10B show a representative image (FIG. 10A) and plot (FIG. 10B) showing acidophil bodies in a human breast to liver metastasis.

[0029] FIG. 11 is a representative image of a human breast to liver metastasis showing ductal formation of tumor cells (outlines) in the liver tissue (labeled “HEP”).

[0030] FIGS. 12A-12D show a representative image of a mouse liver metastasis labeled for DAPI (FIG. 12A), hepatocytes (FIG. 12B), cancer cells (FIG. 12C) or merged (FIG. 12D) showing that metastatic cancer cells retain ductal architecture.

[0031] FIGS. 13A-13C show schematics showing the formation of a mammary bud (FIG. 13A), its invasion into a fat pad (FIG. 13B) and branching into the fat pad (FIG. 13C).

[0032] FIG. 14 shows results from a bioinformatic and biological screen for targets that contribute to cell competition.

[0033] FIGS. 15A-15C show representative plots of cell confluence in vitro (FIG. 15A), cell count in vitro (FIG. 15B) and growth in vivo (FIG. 15C) of WT, SerpinE2 KO, PTHLH KO, or MGP KO 4T1 cells.

[0034] FIGS. 16A-16B show representative plots showing cell survival of WT, SerpinE2 KO, PTHLH KO, or MGP KO 4T 1 cells in the presence of increasing concentrations of Doxorubicin (FIG. 16A) or Paclitaxel (FIG. 16B).

[0035] FIGS. 17A-17B show a representative plot (FIG. 17A) and image (FIG. 17B) of caspase positive cells in co-cultures of WT, SerpinE2 KO, PTHLH KO, or MGP KO 4T1 cells and hepatocytes showing levels of caspase positive tumor cells are increased in knock out cells compared to WT.

[0036] FIG. 18 shows representative images of co-cultures comprised of WT 4T1 -cancer cells (red) and hepatocytes (green) in left column and SerpinE2 KO 4T 1 cancer cells (red) and hepatocytes (green) in right column over three days of culture. All cells were seeded at a 1 :1 ratio at Day 0.

[0037] FIGS. 19A-19B are representative plots of caspase positive cancer cells (FIG. 19A) and hepatocytes (FIG. 19B) in co-cultures of hepatocytes with WT cancer cells, SerpinE2 KO cancer cells, or SerpinE2 KO cancer cells over expressing SerpinE2 (“Rescue CO”).

[0038] FIGS. 20A-20B are a representative schematic (FIG. 20A) and a plot of caspase positive cells (FIG. 20B) in co-cultures of WT or SerpinKO cancer cells seeded into an established hepatocyte culture.

[0039] FIG. 21 is a plot showing that SerpinE2 predicts poor outcomes in breast cancer.

[0040] FIGS. 22A-22B are representative images (FIG. 22A) and quantification (FIG. 22B) of liver metastasis of WT or SerpinE2KO 4T1 cells in a mouse model.

[0041] FIGS. 23A-23B depict a representative image (FIG. 23A) of 4T1-SerpinE2 KO cancer cells (red), hepatocytes (green), and caspase (white) in a mouse metastatic cancer model and quantification (FIG. 23B) of cancer lesions bordering caspase+ hepatocytes in animals injected with WT or SerpinE2 KO cancer cells.

[0042] FIGS. 24A-24B depict representative schematics of AAV8 vectors delivering GFP (FIG. 24A) or SerpinE2 (FIG. 24B) under control of a liver specific promoter (TBG).

[0043] FIG. 25 is a schematic of an AAV. TBG. SerpinE2 vector (SEQ ID NO: 7).

[0044] FIG. 26 is a schematic of an AAV. ApoE / hAAT. SerpinE2 vector (SEQ ID NO: 8).

[0045] FIGS. 27A-27C are representative fluorescent images showing AAV expression (green) in liver (FIG. 27A), but not brain (FIG. 27B) or lung (FIG. 27C) of infected animals.

[0046] FIGS. 28A-28B are representative plot (FIG. 28A) and image (FIG. 28B) showing caspase activity and caspase positive hepatocyte or cancer cells in co-cultures of WT or SerpinE2 overexpressing hepatocytes and WT cancer cells (4T-1).

[0047] FIGS. 29A-29K show that SerpinE2 is the top gene candidate affecting metastatic competition during BCLM. FIG. 29A shows an overview of bioinformatics strategy to identify human BCLM competition targets (BC cells: breast cancer cells; E16 and E18: embryonic day 16 and day 18 mammary epithelial cells). FIG. 29B is a Venn diagram showing common genes identified in the three-part screen. FIG. 29C is a heatmap for the 8 common genes, showing log2FC values for each gene candidate per individual dataset and mean log2FC for each gene candidate across all three datasets. SerpinE2, outlined in red has the highest mean log2FC. FIG. 29D depicts the quantification of liver metastases in mice injected with 4T-1KOcompared to 4T-1WTcells (n = 5 per group). FIG. 29E depicts a representative hematoxylin and eosin (H&E) stained liver section of mouse injected with 4T-1WT(top) and 4T-1KO(bottom), (scale bar = 375 pm). FIG. 29F is a graphical representation of tumor volume over time in 4T-1KOand 4T-1WT(n = 5 per group). FIG. 29G shows representative images of Transwell invasion assay (scale bar = 500 pm). FIG. 29H depicts the quantification of Transwell invasion assay (n = 3 per group). FIG. 29I shows representative images of colony formation assay (scale bar = 2 cm). FIG. 29J depicts the quantification of colony formation assay (n = 3 per group). FIG. 29K depicts the quantification of proliferation of 4T-1wrand 4T-1KOcells (n = 4 per group). ns=p>0.05, *=p<0.05, **=p<0.01 , ***=p<0.001 by Welch’s t test. Data is represented as mean ± SD. See also FIGS. 33A-33O, FIGS. 34A-34K.

[0048] FIGS. 30A-30K show that SerpinE2 mediates metastatic competition between breast cancer cells and hepatocytes. FIG. 30A is a schema of metastatic competition. FIG. 30B shows micrographs and a graph depicting the quantification of cell populations remaining in co-cultures of 4T-1WTand H2.35wrin co-culture over 72 hours (n = 4, scale bar = 100 pm, p<0.05 at 24hrs, p<0.001 at 48 and 72hrs). FIG. 30C shows micrographs of co-cultures of 4T- 1mand H2.35wrcells, depicting the localization of CC3 to hepatocytes (scale bar = 25 pm). FIG. 30D depicts the quantification of percent of CC3+ neighboring 4T-1WTand H2.35WTin cocultures (n = 4). FIG. 30E depicts the quantification of cell populations remaining in co-cultures of 4T-1KOand H2.35WTduring metastatic-competition over 72 hours (n = 4, scale bar = 100 pm, p<0.05 at 24hrs, p<0.001 at 48 and 72hrs). FIG. 30F shows micrographs of co-cultured 4T-1KOand H2.35WTcells, depicting the localization of CC3 to cancer cells (scale bar = 25 pm). FIG. 30G depicts the quantification of percent of CC3+ neighboring 4T-1KOand H2.35WTin cocultures (n = 4). FIG. 30H depicts the quantification of percent of CC3+ cells in monoculturesof 4T-1KOand H2.35WTcells (n = 4). FIG. 30I depicts the quantification of percent of CC3+ neighboring 4T-1Resand H2.35wrin co-cultures (n = 4). FIG. 30J depicts the quantification of secreted SerpinE2 concentration by human breast cancer lines HCC1954, MDA-MB-231 , MCF7, and MDA-MB-436 (n = 4 per group, ns between MCF7 and MDA-MB-436, all other groups were p<0.05). FIG. 30K depicts the quantification of cell populations remaining in cocultures of human breast cancer cells and THLE2 by 48 hours (n = 4 per group). ns=p>0.05, *=p<0.05, **=p<0.01 , ***=p<0.001 by Welch’s t test (FIGS. 30B, 30D, 30E, 30G-30I, 30K) and Mann-Whitney II test (FIG. 30J). Data is represented as mean ± SD. See also FIGS. 35A- 35G.

[0049] FIGS. 31A-31I show that SerpinE2 loss disrupts focal adhesions in breast cancer cells leading to extrusion during metastatic competition. FIG. 31A is an orthogonal xz projection of time-lapse imaging tracking a hepatocyte extrusion event (blue arrows) in co-cultures of 4T- 1wr(red) and H2.35WT(green) cells (scale bar = 25 pm). FIG. 31 B is an orthogonal xz projection of time-lapse imaging tracking a cancer cell extrusion event (yellow arrows) in cocultures of 4T-1KO(red) and H2.35wr(green) cells (scale bar = 25 pm). FIG. 31 C depicts the quantification of cancer cell extrusion of 4T-1WTand 4T-1KOafter co-culture with H2.35wrcells (n = 4 per group). FIG. 31 D depicts cell adhesion assay results with 4T-1WTcells and 4T-1KOcells (n = 10 per group). FIG. 31 E is a PCA plot of all expressed genes in the 4T-1WTversus 4T-1KORNA-seq experiment (PC1 : Principal component 1 , PC2: Principal component 2). FIG. 31 F is a volcano plot of DEGs from the comparison of 4T-1wrto 4T-1KOsamples. Top 5 upregulated and downregulated DEGs are labeled. FIG. 31 G depicts GSEA of upregulated DEGs in 4T-1wrversus 4T-1KO. Waterfall plot of significantly enriched KEGG gene sets (p < 0.05). FIG. 31 H is a heatmap of expression (normalized counts) of selected focal adhesion related genes. FIG. 311 shows immunofluorescence images of focal adhesion proteins Paxillin (top), Vinculin (middle), and Tensin2 (bottom) in 4T-1WT(left) and 4T-1KO(right) (scale bar = 100 pm). *=p<0.05, **=p<0.01 , ***=p<0.001 by Welch’s t test. Data is represented as mean ± SD. See also FIGS. 35A-35G.

[0050] FIGS. 32A-32H show that SerpinE2 overexpression in hepatocytes reduces BCLM. FIG. 32A shows micrographs of co-cultures with 4T-1wr(red) and H2.35OE(green) cells, depicting the localization of CC3 (white). FIG. 32B depicts the quantification of percent of CC3+ 4T-1WTcells in co-cultures with H2.35WTor H2.35OEcells (n = 4 per group). FIG. 32C depicts the quantification of percent of CC3+ H2.35 cells in co-cultures with H2.35WTor H2.35OEcells (n = 4 per group). FIG. 32D is a schema for AAV8-SerpinE2 administration to treat liver metastases experiment. FIG. 32E shows representative whole body BLI images of mice administered with AAV8-Ctrl (top) or AAV8-SerpinE2 (bottom) at Day 1 and Day 7. FIG. 32F is a graphical representation of tumor growth progression in mice after administration withAAV8-Ctrl or AAV-SerpinE2 (n = 10, 11 per group), as tracked by whole body BLI for 7 days. Data is presented as mean ± SEM. FIG. 32G shows representative ex-vivo liver BLI images at Day 7 of mice administered with AAV-Ctrl (top) or AAV8-SerpinE2 (bottom). FIG. 32H depicts liver metastasis burden in mice after administration with AAV8-Ctrl or AAV8-SerpinE2, as measured by BLI of ex-vivo liver (n = 10, 11 per group). Data is presented as mean ± SEM. *=p<0.05, **=p<0.01 , ***=p<0.001 by Welch’s t test (B and C), and Mann-Whitney U test (F and H). Data is represented as mean ± SD (B and C) and mean ± SEM (F and H). See also FIGS. 36A-36L.

[0051] FIGS. 33A-33O shows a strategy to identify gene candidates involved in metastatic competition. FIG. 33A is a representative image of hematoxylin and eosin (H&E) stained BCLM patient sample showing acidophilic bodies at the tumor-liver interface (scale bar = 5 inches). FIG. 33B depicts the quantification of acidophilic bodies in BCLM patient samples (n = 10 per group). FIG. 33C is a representative image of H&E stained BCLM patient sample showing tubular structure (arrows) of early metastasis (scale bar = 187.5 pm). FIG. 33D depicts violin plots showing pre-QC count distributions of detected genes, detected UMIs, and mitochondrial gene percentage per cell, and UMAP visualization of all cells labeled by sample post-QC filtering, for the mouse embryonic mammary dataset (Giraddi et al.). FIG. 33E shows violin plots showing pre-QC count distributions of detected genes, detected UMIs, and mitochondrial gene percentage per cell, and UMAP visualization of all cells labeled by sample post-QC filtering, for the mouse breast tumor model dataset (Yeo et al.). FIG. 33F is an UMAP visualizations of all cells labeled by original source dataset for the combined dataset (preintegration) and the integrated dataset. FIG. 33G is an UMAP visualizations of all cells in the integrated dataset with original sample labels and unsupervised clustering labels. Unsupervised clustering reveals one mixed cluster (c2) which consists of both E16 / E18 and breast cancer cells. FIG. 33H is a volcano plot of DEGs from the comparison of c2 (mixed) to all remaining clusters (predominantly breast cancer cells). FIG. 33I is a PCA plot showing human breast cancer cell lines labeled by liver metastatic potential, for the liver colonization dataset (Jin et al., MetMap). FIG. 33J is a heatmap of Euclidean distance and clustering dendrograms of human breast cancer cell lines in the liver colonization dataset. FIG. 33K is a volcano plot of DEGs from the comparison of human breast cancer cell lines with high versus low liver metastatic potential. FIG. 33L shows violin plots for the human BCLM dataset (Wang et al.), showing pre-QC count distributions of detected genes, detected UMIs, and mitochondrial gene percentage, and a scatterplot showing cancer versus non-cancer annotation of epithelial cells based on inferCNV analysis. CNV signal reflects the extent of CNVs, while CNV correlation reflects the similarity between the cellular CNV pattern and that of other cells from the same tumor. Thresholds are shown in red dashed lines. FIG. 33M showsLIMAP visualizations of all cells post-QC filtering labeled by tissue (BCLM versus adjacent normal liver) and sample. FIG. 33N is an LIMAP visualization showing final cell type annotation of all cells in the human BCLM dataset. FIG. 330 is a volcano plot of DEGs from the comparison of breast cancer epithelial cells in BCLM to hepatocytes in adjacent normal liver. *=p<0.05, **=p<0.01 , ***=p<0.001 by Mann-Whitney U test. Data is represented as mean ± SD.

[0052] FIGS. 34A-34K shows validation of SerpinE2 as a top gene candidate. FIG. 34A shows Kaplan-Meier plots depicting the effect of SERPINE2 expression on survival outcomes in METABRIC breast cancer patients. High SERPINE2 expression (z-score >2) is associated with worse 10-year OS and RFS (log-rank test, p-values < 0.05). FIG. 34B shows Kaplan- Meier plots depicting the association between CXCL2 expression and survival outcomes in METABRIC breast cancer patients. CXCL2 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34C shows Kaplan-Meier plots depicting the association between FSCN1 expression and survival outcomes in METABRIC breast cancer patients. FSCN1 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34D shows Kaplan-Meier plots depicting the association between MAP3K20 expression and survival probability in METABRIC breast cancer patients. MAP3K20 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (logrank test, ns). FIG. 34E shows Kaplan-Meier plots depicting the association between PPP1 R18 expression and survival probability in METABRIC breast cancer patients. PPP1 R18 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34F shows Kaplan-Meier plots depicting the association between PSAT1 expression and survival probability in METABRIC breast cancer patients. PSAT1 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34G shows Kaplan-Meier plots depicting the association between SLC4A7 expression and survival probability in METABRIC breast cancer patients. SLC4A7 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34H shows Kaplan-Meier plots depicting the association between SOAT1 expression and survival probability in METABRIC breast cancer patients. SOAT1 expression is not associated with a difference in 10-year OS or RFS in METABRIC breast cancer patients (log-rank test, ns). FIG. 34I shows pie charts of patients with high SERPINE2 expression and low SERPINE2 expression by tumor stage for the METABRIC breast cancer cohort. SERPINE2 expression is not significantly associated with tumor stage (Chi-squared = 0.54, p-value = 0.91). FIG. 34J depicts SerpinE2 mRNA expression fold change of 4T-1KOrelative to 4T-1wrcells (n = 4 pergroup). FIG. 34K depicts the quantification of secreted SerpinE2 concentration in 4T-1KOcells relative to 4T-1wrcells (n = 4 per group). ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001 by Welch’s t test. Data is represented as mean ± SD.

[0053] FIGS. 35A-35H show that SerpinE2-mediated metastatic competition is specific to breast cancer cells and hepatocytes and requires direct interactions. FIG. 35A depicts the quantification of proliferation of 4T-1WTand H2.35wrcells (n = 4 per group). FIG. 35B depicts the quantification of area confluency over time of 4T-1WTand H2.35wrcells (n = 4 per group). FIG. 35C depicts the quantification of percent of CC3+ cells in monocultures of 4T-1wrand H2.35WTcells (n = 4 per group) FIG. 35D depicts the quantification of percent of CC3+ cells in co-culture of 1 :2 (left) and 1:5 (right) ratio of 4T-1WTto H2.35wrcells (n = 4 per group). FIG. 35E depicts the quantification of percent of CC3+ cells in monocultures of 4T-1wrand H2.35wrcells after treatment with conditioned media from the other cell type (n = 4 per group) FIG. 35F depicts sensitivity to Paclitaxel (left) and Doxorubicin (right) of 4T-1WTand 4T-1KOcells. FIG. 35G depicts the quantification of percent of CC3+ neighboring 4T-1WTand 603B cells in cocultures (n = 4 per group). FIG. 35H depicts the quantification of secreted SerpinE2 concentration in 603B, 4T-1wrand H2.35WTcells (n = 4 per group). ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001 by Welch’s t test (FIGS. 35A-35E) and Mann-Whitney U test (FIGS. 35H). Data is represented as mean ± SD.

[0054] FIGS. 36A-36L show the efficacy and validation of SerpinE2 overexpression in hepatocytes in vitro and in vivo models. FIG. 36A depicts SerpinE2 mRNA expression fold change of H2.35WTand H2.35OEcells relative to 4T-1wrcells (n = 4 per group). FIG. 36B depicts the quantification of secreted SerpinE2 concentration in H2.35WT, H2.35OE, and 4T-1wrcells (n = 4 per group). FIG. 36C depicts the quantification of percent of CC3+ cells in monocultures of H2.35wrand H2.35OEcells (n = 4 per group). FIG. 36D depicts a plasmid map of AAV8-TBG-GFP. FIG. 36E show fluorescent micrographs of mouse tissues after AAV8- TBG-GFP delivery. Efficiency of AAV8-TBG-GFP delivery in mouse liver (top) with no off- target delivery in lungs (middle) and brain (bottom), (scale bar = 100 pm). FIG. 36F depicts a plasmid map of empty vector AAV8-TBG-Ctrl. FIG. 36G depicts a plasmid map of AAV8-TBG- SerpinE2. FIG. 36H depicts the quantification of SerpinE2 mRNA livers of mice after injection with AAV8-SerpinE2 relative to mice after injection with AAV8-Ctrl (n = 4 per group). FIG. 36I depicts the quantification of serum SerpinE2 levels in mice injected with AAV8-SerpinE2 relative to AAV8-Ctrl injected mice (n = 6 per group). FIG. 36J depicts AST level at Day 7 in mice after delivery with AAV8-Ctrl or AAV8-SerpinE2 relative to baseline levels before AAV administration (n = 10, 11 per group). FIG. 36K depicts ALT level at Day 7 in mice after delivery with AAV8-Ctrl or AAV8-SerpinE2 relative to baseline levels before AAV administration (n = 10, 11 per group). FIG. 36L depicts body weight of mice after AAV administration in AAV8-Ctrl and AAV8-SerpinE2 injected mice over 7 days (n = 10, 11 per group). ns=p>0.05, *=p<0.05, **=p<0.01 , ***=p<0.001 by Welch’s t tests (FIGS. 36A-36C, 36H, 36I, 36K, 36L) and Mann-Whitney II test (FIG. 36J). Data is represented as mean ± SD.

[0055] FIGS. 37A-37C show that SerpinE2 overexpression in hepatocytes reduces BCLM in animals with established cancer. FIG. 37A is an experimental schema for AAV8-SerpinE2 administration to treat liver metastases in mice with established cancer. FIG. 37B is a graphical representation of tumor growth progression in mice after administration with AAV8- Ctrl or AAV-SerpinE2 (n = 3 per group), as tracked by whole body BLI for 11 days. Data is presented as mean ± SEM. FIG. 37C depicts liver metastasis burden in mice after administration with AAV8-Ctrl or AAV8-SerpinE2, as measured by BLI of ex-vivo liver.DETAILED DESCRIPTION

[0056] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0057] Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. While this can occur normally, the present disclosure is based, in part, on the surprising discovery that this process also occurs in pathological circumstances and that targeted therapies can modulate the competitiveness of desired cell type, thereby improving outcomes in many conditions. Accordingly, provided herein are methods of improving competitiveness of one or more cells against a separate cell type. In an aspect, the method may improve competitiveness of one or more host cells against a foreign cell, as described further below. Also provided are specific therapies, especially gene therapies, aimed at modulating levels or activity of key proteins that improve competitiveness of a desired cell in these contexts. Also provided are methods of identifying targets for therapy in a pathologic condition (e.g., metastatic cancer such as breast cancer liver metastasis (BCLM)).I. Terminology

[0058] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as,but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0059] Further, as the present inventive concept is susceptible to embodiments of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific embodiments shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “embodiment,” “embodiments,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “embodiment,” “embodiments,” and / or the like in the description do not necessarily refer to the same embodiment and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one embodiment may also be included in other embodiments but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and / or integrations of the embodiments described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

[0060] As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1 %.

[0061] The terms “comprising,” “including,” “encompassing” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including,” “encompassing” and “having” mean to include, but not necessarily be limited to the things so described.

[0062] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0063] As used herein, the terms “treat”, “treating”, “treatment” and the like, unless otherwise indicated, can refer to reversing, alleviating, inhibiting the process of, or preventing the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein, to prevent the onset of the symptoms or the complications, or alleviating the symptoms or the complications, or eliminating the condition, or disorder.

[0064] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disease, disorder or condition (e.g., cancer or a metastasis) in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. In other words, in an aspect, preventing metastasis or and / or cancer is intended. The words “prevent” and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having metastasis but having cancer from progressing to that complication.

[0065] As used herein, the term “administering” an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0066] “Contacting” as used herein, e.g., as in “contacting a sample” refers to contacting a sample directly or indirectly in vitro, ex vivo, or in vivo (i.e. , within a subject as defined herein). Contacting a sample can include addition of a compound (e.g., a nucleic acid and / or vector as provided herein) to a sample, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture.

[0067] As used herein, the term “therapeutic agent” means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a subject, such as cancer, cancer metastasis or limb deformity.

[0068] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0069] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0070] As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e., living organism, such as a patient).

[0071] As used herein, the term “sequence identity” refers to the number of identical or similar residues (i.e., nucleotide bases or amino acid) on a comparison between a test and referencenucleotide or amino acid sequence. Sequence identity can be determined by sequence alignment of nucleic acid to identify regions of similarity or identity. As described herein, sequence identity is generally determined by alignment to identify identical residues. Matches, mismatches, and gaps can be identified between compared sequences. Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequence x 100. In one non-limiting embodiment, the term “at least 90% sequence identity to” refers to percent identities from 90 to 100%, relative to the reference nucleotide or amino acid sequence. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplary purposes a test and reference oligonucleotide or length of 100 nucleotides are compared, no more than 10% (i.e. , 10 out of 100) of the nucleotides in the test oligonucleotide differ from those of the reference oligonucleotide. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions.

[0072] As used herein, “operably linked” means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0073] As used herein, a “regulatory element” can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-ll sequences). Regulatory elements are discussed infra and can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0074] As used herein, “recombinant” is used herein to refer to new combinations of genetic material as a result of genetic engineering. For instance, a recombinant organism (e.g., bacteria) can be an organism that contains different genetic material from either of its parents as a result of genetic modification, recombinant DNA can be a form of artificial DNA, a recombinant protein or enzyme can be an artificially produced and purified form of the protein or enzyme, and a recombinant virus can be a virus formed by recombining genetic material.

[0075] As used herein, the term “open reading frame (ORF)” refers to the parts of a reading frame that has the ability to be translated. An ORF can be a continuous chain of codons that begins with a start codon (e.g., ATG) and ends at a stop codon (e.g., TAA, TAG, TGA). A reading frame is a sequence of nucleotides that are read as codons specifying amino acids.As used herein, the term “endogenous promoter / enhancer” refers to a disclosed promoter or disclosed promoter / enhancer that is naturally linked with its gene. In an aspect, a disclosed endogenous promoter can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene (such as, for example, a disclosed phosphorylase kinase, phosphorylase, or some other enzyme involved in the glycogen metabolic pathway). In an aspect, a disclosed endogenous promoter can be used for constitutive and efficient expression of a disclosed transgene (e.g., a nucleic acid sequence encoding a polypeptide capable of preventing glycogen accumulation and / or degrading accumulated glycogen). In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer.

[0076] As used herein, the term “exogenous promoter” or “heterologous promoter” refers to a disclosed promoter or a disclosed promoter / enhancer that can be placed in juxtaposition to a gene by means of molecular biology techniques such that the transcription of that gene can be directed by the linked promoter or linked promoter / enhancer.

[0077] As used herein, the term “host cell” refers primarily to a normal (non-pathological) cell native to the environment where cell competition is taking place. For instance, in the context of a metastatic cancer, a “host cell” is a normal cell in an organ where metastasis is occurring. As an example, in the context of a breast cancer metastasizing to the liver, a “host cell” would be a normal hepatocyte in the liver, wherein the liver has a “site of metastasis.” In the context of a benign or pre-cancerous tumor, the “host cell” would be nearby cells in an organ where the benign or pre-cancerous tumor is developing. For instance, in a benign breast tumor, the “host cell” would be the cells in the breast that are not a part of the tumor.

[0078] As used herein, the term “foreign cell” refers to any cell that is undesired or not-native in a space, and which is engaged in cell competition with nearby cells. For instance, in the case of a metastasis, a “foreign cell” would be a metastatic cancer cell (i.e., a metastatic breast cancer cell in the liver). In the instance of a benign tumor or pre-cancerous lesion, a “foreign cell” would be a part of the benign tumor or pre-cancerous lesion. That is, it would be “undesired” in the space. In any aspect herein, the foreign cell may be a metastatic cancerous cell, a benign tumor cell or any other migrating or invading cell.

[0079] As used herein, the term “site of metastasis” refers to an area of the body (e.g., in an organ or a tissue) that comprises one or more metastatic cancer cells.

[0080] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.II. Methods

[0081] Provided herein are methods of treating a condition in a subject in need thereof, the method comprising administering a therapeutic agent that improves competitiveness of one or more host cells in an organ or tissue against one or more foreign cells and / or decreases the competitiveness of the one or more foreign cells against the host cells. Also provided are methods for treating a metastatic cancer in a subject, the method comprising administering a therapeutic agent that improves competitiveness of one or more host cells in an organ or tissue comprising a site of metastasis against one or more metastasizing cells and / or decreases competitiveness of the one or more metastasizing cells against the host cells. Methods for improving or decreasing competitiveness in desired cell populations are described further below as well as methods for identifying cellular factors that improve cell competitiveness.Methods for Treating Various Conditions by Increasing Cell Competitiveness

[0082] In various aspects, methods are provided for treating various conditions characterized by growth or spread of one or more undesired cells in a tissue or organ. These conditions can include metastatic cancer - including the initial seeding of one or more metastatic cells in a distal location from the organ of origin of the metastatic cells (e.g., the establishment of the metastasis) and / or growth and invasion of the metastasis in the target organ or tissue, (e.g., growth of the metastasis). In other aspects, the conditions may include growth of an undesired, but still benign, tumor into neighboring tissues. For example, a condition may comprise a benign tumor or a pre-cancerous lesion. In any of these methods, the methods comprise administering a therapeutic agent that improves competitiveness in a desired cell and / or reduces competitiveness of an undesired cell. In various aspects, the disclosed methods improve competitiveness of a host cell in an organ or tissue comprising a site of metastasis against one or more metastasizing cancer cells.

[0083] In an aspect, the therapeutic agent modulates activity of a gene product encoded by the one or more differently expressed genes in at least one cell (e.g., at least one host cell, at least one foreign cell (e.g., metastatic cancer cell)). As used herein, the term “differently expressed genes” is used in reference to the two or more different cell types engaged in cell competition (i.e. , a “desired” and an “undesired” cell). It has been surprisingly discovered that cells engaged in abnormal cell competition typically have dysregulated genes that are either over or under expressed in one or more of the two cell types relative to a normal matched cell. Accordingly, the therapeutic agent may modulate activity or levels of a gene product encoded by a gene that is under-expressed in a “host cell” or a “foreign cell” (e.g., a metastatic cancer cell), or any combination thereof. Likewise, the therapeutic agent may modulate activity or levels of a gene product encoded by a gene that is over-expressed in a “host cell” or a “foreigncell” (e.g., a metastatic cancer cell) or any combination thereof. In some aspects, the gene product corresponds to a cellular factor associated with increased cell competitiveness, as identified by the methods described further below.

[0084] In any of the methods provided herein, the condition to be treated may be a malignant tumor, a benign growth, or a pre-cancerous lesion in the subject. Example malignant tumors may include, but are not limited to, a liver tumor, a pancreatic tumor, squamous cell tumor, prostate tumor, melanoma, skin tumor, urothelial tumor, kidney tumor, colon tumor, gastric tumor, gastrointestinal tumor, a breast tumor, a lung tumor, melanoma, a sarcoma, an adnexal tumor, or a tumor of a peritoneum.

[0085] In an aspect, the therapeutic agent may reduce activity or levels of a gene product (or cellular factor) in at least one host cell or at least one foreign cell (e.g., metastatic cancer cell). In an aspect, the therapeutic agent may increase activity or levels of a gene product (or cellular factor) in at least one host cell or at least one foreign cell (e.g., metastatic cancer cell). Suitable therapeutic agents for increasing or decreasing activities and / or levels of a gene product (or cellular factor) are described further below, particularly in Section III.Methods of Identifying Cellular Factors Associated with Cell Competitiveness

[0086] In various aspects, methods are provided for identifying one or more cellular factors that increase competitiveness of a foreign cell against host cells in an organ or tissue. In an aspect, the methods may further comprise identifying one or more differently expressed gene in a host cell, or a foreign cell. In an aspect, these methods comprise obtaining or having obtained an expression profile of any of these cells. An “expression profile” as used herein refers to a data set that provides information about the level of expression of genes and / or their protein levels in the cell. An expression profile may in an aspect be a transcriptome or a set of data reflecting all transcribed genes in a cell (i.e. , mRNA, or RNA-seq).

[0087] In an aspect, the methods may comprise identifying a gene target in a metastasis, where the metastasis is from an originating organ. The method can comprise obtaining an expression profile of one or more metastatic cells in the metastasis and comparing the expression profile to an expression profile of a cell from the originating organ, either during development or after development of the originating organ. That is, for example, in the case of a breast cancer metastasis to the liver, the expression profile of a metastatic breast cancer cell can be compared to an expression profile of a developing breast cell. Likewise, the expression profile of a metastatic breast cancer cell can be compared to an expression profile of a developed (normal) breast cell. In both aspects, genes that are differently (e.g., over-) expressed in the metastatic cancer cell as compared to the cell from the originating organ can be identified.

[0088] In an aspect, genes that are over-expressed or highly expressed in the metastatic cancer cell as compared to the cell from the originating organ can be identified. These genes can be identified as “dysregulated” or “differently expressed” and targeted for modulation according to methods herein. For instance, in an aspect, identification of a gene that is highly expressed in metastatic cancer cells relative to normal cells in the originating organ, can lead to development of a therapeutic that decreases activity or expression levels of this gene or its gene product in cancer cells. Surprisingly, it was also discovered that identification of a gene that is highly expressed in metastatic cancer cells relative to normal cells in the originating organ can also lead to the development of a therapeutic that increases activity or expression levels of this gene in healthy cells at the site of metastasis. For instance, if a gene is identified that is highly expressed in a metastatic cell, this gene can be targeted for increased expression in a neighboring (healthy) cell - thereby restoring homeostasis of this gene expression profile across the tumor environment.

[0089] In another aspect, the methods may comprise identifying one or more cellular factors that increase competitiveness of a metastasizing tumor cell against host cells in a target organ (that is, the organ where metastasis is occurring or at risk of occurring). In various aspects, these methods may comprise:(a) obtaining or having obtained(i) a first gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing tumor cell and having an elevated organ-specific metastatic potential relative to other tumor cells of the same tumor type,(ii) a second gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing cancer cell;(iii) a third gene expression dataset of one or more developing or embryonic cells in an organ of origination of the tumor cell;(iv) fourth gene expression dataset of the metastasizing tumor cell or one or more metastasizing tumor cells of the same tumor type as the metastasizing cancer cell that have metastasized to the same target organ; and(v) a fifth gene expression dataset of one or more host cells in the target organ; and(b) identifying one or more gene(s) that:(i) have elevated expression in the first expression dataset,(ii) have elevated expression in both the second gene expression dataset and third gene expression dataset; and(iii) have higher expression in the fourth gene expression dataset relative to the fifth gene expression dataset; and(c) identifying one or more protein product(s) encoded by the one or more gene(s) of (b) as the one or more cellular factors that increases competitiveness of the metastasizing tumor cells.

[0090] In some aspects, the first gene expression dataset of (i) comprises a transcriptome of a tumor cell which is the same type of tumor cell as the metastasizing tumor cell and also has an elevated organ-specific metastatic potential relative to other tumor cells of the same tumor type. Identifying a cell that has elevated organ-specific metastatic potential relative to other tumor cells of the same type can be performed by consulting a metastasis map of human cancer cell lines {e.g., MetMap which may be accessed at depmap.org / metmap / ). To derive such a metastasis map, a practitioner can use an in vivo barcoding approach as described in Jin, X., et al. (2020), A metastasis map of human cancer cell lines. Nature 588, 331-336. 10.1038 / s41586-020-2969-2, which is incorporated herein by reference in its entirety. This method classifies tumor cells on a Iog10 scale from -4 to +4, where a higher score correlates with higher metastatic potential. For example, in a non-limiting example, a cancer cell may be identified as having an elevated metastatic potential relative to other tumor cells of the same type if it has a score of +2.9 or greater (e.g., +2.9, +3.0, +3.1 , +3.2, +3.3, +3.4, +3.5, +3.6, +3.7, +3.8, +3.9, or +4.0) as assessed by the MetMap method. As used herein, the term “organ-specific” means that the metastatic potential is evaluated in the context of the organ targeted by the metastasis. For example, a cancer cell may be identified as having an elevated metastatic potential (e.g., have a score of +2.9 or greater as measured by MetMap) to metastasize to a specific organ (e.g., a liver) relative to other tumor cells of the same type. In all these instances, the comparative “tumor cells of the same type” may include cells having an elevated metastatic potential to metastasize to a different organ and / or cells that have a low metastic potential.

[0091] In some aspects, the second gene expression dataset of (ii) comprises a transcriptome of tumor cells of the same type as the metastasizing cancer cell. As used throughout, tumor cells “of the same type” as the metastasizing cancer cell are understood to encompass tumor cells that are derived from tumors that would be classified by a skilled practitioner as the same type as the tumor of origin of the metastasizing cancer. Databases of transcriptomes of different tumor cells are available and readily available to a skilled artisan. For example, when the metastasizing cancer cell is a metastasizing breast cancer cell, a transcriptome of breastepithelial cancer cells may be used to generate the second gene expression dataset. For the second gene expression dataset, the metastatic potential of the cancer cells is not considered (e.g., the transcriptome may be obtained from a cancer cell having a high or low metastatic potential as determined by the methods above).

[0092] In some aspects, the third gene expression dataset of (iii) comprises a transciptome of a developing cell in an organ of origin of the metastatic cancer cells. In some aspects, the developing cell may be an embryonic cell. In various aspects, the stage of development of the developing cell is chosen to align with the time when the developing organ invades surrounding tissue during embryonic development. In a specific example, when the organ of origin is breast (e.g., in the case of metastatic breast cancer), the developing cell may be a murine mammary epithelial cell of embryonic day 16, 17, or 18.

[0093] In various aspects, the fourth gene expression dataset of (iv) may comprise a transcriptome of the metastatic tumor cell or one or more metastasizing tumor cells of the same tumor type as the metastasizing cancer cell that have metastasized to the same target organ. There are published transcriptomes of various metastatic lesions for various tumor types. An illustrative dataset of liver metastatic lesions is GSE249361. In some aspects, the metastatic cells may be obtained and analyzed from surgical resection of metastatic lesions from a subject.

[0094] In various aspects, the fifth gene expresison dataset may comprise a transcriptome of one or more host cells located in an organ or tissue that contains a site of metastasis. These host cells may be obtained and analyzed from adjacent tissue obtained during surgical resection of metastastic lesions. In some instances, when organ or tissue that contains a site of metastasis is a liver, the host cells may comprise hepatocytes.

[0095] In further aspects, methods of applying the methods above to treat a cancer metastasis in a subject in need thereof are provided. For instance, in an aspect, a method of treating a cancer metastasis in a subject may comprise identifying at least one cellular factor that increases competitiveness of metastasizing cells in the cancer metastasis according to the methods herein and administering at least one therapeutic agent that modulates activity of the cellular factor to the subject. In some aspects, the therapeutic agent may decrease activity of the cellular actor in at least one cancer cell in the cancer metastasis. In some aspects, the therapeutic agent may increase activity of the cellular factor in at least one host cell of an organ where the cancer metastasis is located in the subject. Suitable therapeutic agents that can modulate activity of the cellular factor are described herein below.Methods of Treating Cancer (e.g., Metastatic Cancer)

[0096] In view of the foregoing, it is appreciated that a method of treating cancer (and in particular, metastatic cancer) is provided. Accordingly, in an aspect a method of treating cancer (i.e. , metastatic cancer) is provided comprising administering a therapeutic agent that improves competitiveness of one or more host cells in an organ or tissue against one or more cancer cells. In various aspects, the methods comprise administering a therapeutic agent that modulates activity of one or more cellular factors (e.g., cellular factors identified by the methods provided above) in one or more host cells and / or one or more metastasizing cancer cells.

[0097] In various aspects, the cellular factors are selected from a serine protease inhibitor, C- X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20, solute carrier family 4 member 7, phosphoserine aminotransferase 1 , sterol O-acyltransferase 1 , protein phosphatase 1 regulatory subunit 18, Matrix Gia protein, Parathyroid hormone-related protein, Pleiotrophin, Midkine, Secreted frizzled-related protein1 , Aldehyde dehydrogenase family 1 member A3, Nucleolin, Sclerostin domain-containing protein 1 , Histone-lysine N-methyltransferase EZH2, Insulin-like growth factor-binding protein2, Transient receptor potential cation channel subfamily M member 2, Galectin-7, or any combination thereof. I

[0098] In various aspects, the cellular factors comprise protein products encoded by one or more genes selected from: a gene encoding a serine protease inhibitor (e.g., SERPINE2 (GDN)), CXCL2, FSCN1, MAP3K20, SLC4A7, PSAT1, SOAT1, PPP1R18, MGP, PTHLH, PTN, MDK, SFRP1, ALDH1A3, NCL, SOSTDC1, EZH2, IGFBP2, TRPM2, and LGALS7.

[0099] In some aspects, the cellular factor comprises a serine protease inhibitor or a protein product encoded by a serine protease inhibitor gene. Exemplary serine protease inhibitors include SerpinE2, SerpinEI , SerpinE3, SerpinAI , SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAIO, SerpinA11 , SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinB11 , SerpinB12, SerpinB13, SerpinCI , SerpinDI SerpinFI , SerpinF2, SerpinGI , SerpinHI , Serpinll , Serpinl2, which are encoded, respectively, by the following serine protease inhibitor genes: SERPINE2, SERPINE1, SERPINE3, SERPINA1, SERPINA2, SERPINA3, SERPINA4, SERPINA5, SERPINA6, SERPINA7, SERPINA8, SERPINA9, SERPINA10, SERPINA11, SERPINA 12, SERPINB1, SERPINB2, SERPINB3, SERPINB4, SERPINB5, SERPINB6, SERPINB7, SERPINB8, SERPINB9, SERPINB10, SERPINB11, SERPINB12, SERPINB13, SERPINC1, SERPIND1 SERPINF1, SERPINF2, SERPING1, SERPINH1, SERPINI1, SERPINI2.

[0100] Accordingly, in some aspects, the cellular factor comprises SerpinE2 and / or a protein product encoded by a SERPINE2 gene. In some aspects, the cellular factor comprises SerpinEI and / or a protein product encoded by a SERPINE1 gene. In some aspects, the cellular factor comprises SerpinE3 and / or a protein product encoded by a SERPINE3 gene. In some aspects, the cellular factor comprises SerpinAI and / or a protein product encoded by a SERPINAI gene. In some aspects, the cellular factor comprises SerpinA2 and / or a protein product encoded by a SERPINA2 gene. In some aspects, the cellular factor comprises SerpinA3 and / or a protein product encoded by a SERPINA3 gene. In some aspects, the cellular factor comprises SerpinA4 and / or a protein product encoded by a SERPINA4 gene. In some aspects, the cellular factor comprises SerpinA5 and / or a protein product encoded by a SERPINA5 gene. In some aspects, the cellular factor comprises SerpinA6 and / or a protein product encoded by a SERPINA6 gene. In some aspects, the cellular factor comprises SerpinA7 and / or a protein product encoded by a SERPINA7 gene. In some aspects, the cellular factor comprises SerpinA8 and / or a protein product encoded by a SERPINA8 gene. In some aspects, the cellular factor comprises SerpinA9 and / or a protein product encoded by a SERPINA9 gene. In some aspects, the cellular factor comprises SerpinAI 0 and / or a protein product encoded by a SERPINA10 gene. In some aspects, the cellular factor comprises SerpinA11 and / or a protein product encoded by a SERPINA11 gene. In some aspects, the cellular factor comprises SerpinA12 and / or a protein product encoded by a SERPINA 12 gene. In some aspects, the cellular factor comprises SerpinBI and / or a protein product encoded by a SERPINBI gene. In some aspects, the cellular factor comprises SerpinB2 and / or a protein product encoded by a SERPINB2 gene. In some aspects, the cellular factor comprises SerpinB3 and / or a protein product encoded by a SERPINB3 gene. In some aspects, the cellular factor comprises SerpinB4 and / or a protein product encoded by a SERPINB4 gene. In some aspects, the cellular factor comprises SerpinB5 and / or a protein product encoded by a SERPINB5 gene. In some aspects, the cellular factor comprises SerpinB6 and / or a protein product encoded by a SERPINB6 gene. In some aspects, the cellular factor comprises SerpinB7 and / or a protein product encoded by a SERPINB7 gene. In some aspects, the cellular factor comprises SerpinB8 and / or a protein product encoded by a SERPINB8 gene. In some aspects, the cellular factor comprises SerpinB9 and / or a protein product encoded by a SERPINB9 gene. In some aspects, the cellular factor comprises SerpinBI 0 and / or a protein product encoded by a SERPINB10 gene. In some aspects, the cellular factor comprises SerpinB11 and / or a protein product encoded by a SERPINB11 gene. In some aspects, the cellular factor comprises SerpinB12 and / or a protein product encoded by a SERPINB12 gene. In some aspects, the cellular factor comprises SerpinB13 and / or a protein product encoded by a SERPINB13 gene. In some aspects, the cellular factor comprises SerpinCI and / or a protein product encoded by a SERPINC1 gene. In some aspects, the cellular factor comprisesSerpinDI and / or a protein product encoded by a SERPIND1 gene. In some aspects, the cellular factor comprises SerpinFI and / or a protein product encoded by a SERPINF1 gene. In some aspects, the cellular factor comprises SerpinF2 and / or a protein product encoded by a SERPINF2 gene. In some aspects, the cellular factor comprises SerpinGI and / or a protein product encoded by a SERPING1 gene. In some aspects, the cellular factor comprises SerpinHI and / or a protein product encoded by a SERPINH1 gene. In some aspects, the cellular factor comprises Serpinll and / or a protein product encoded by a SERPINI1 gene. In some aspects, the cellular factor comprises Serpinl2 and / or a protein product encoded by a SERPINI2 gene.

[0101] In some aspects, the cellular factor comprises C-X-C motif chemokine ligand 2 and / or a protein product encoded by a CXCL2 gene. In some aspects, the cellular factor comprises fascin actin-bundling protein 1 and / or a protein product encoded by a FSCN1 gene. In some aspects, the cellular factor comprises mitogen-activated protein kinase kinase kinase 20 and / or a protein product encoded by a MAP3K20 gene. In some aspects, the cellular factor comprises solute carrier family 4 member 7 and / or a protein product encoded by a SLC4A7 gene. In some aspects, the cellular factor comprises phosphoserine aminotransferase 1 and / or a protein product encoded by a PSAT1 gene. In some aspects, the cellular factor comprises sterol O-acyltransferase 1 and / or a protein product encoded by a SOAT1 gene. In some aspects, the cellular factor comprises protein phosphatase 1 regulatory subunit 18 and / or a protein product encoded by a PPP1R18 gene. In some aspects, the cellular factor comprises Matrix Gia protein and / or a protein product encoded by a / WGP gene. In some aspects, the cellular factor comprises Parathyroid hormone-related protein and / or a protein product encoded by a PTHLH gene. In some aspects, the cellular factor comprises Pleiotrophin and / or a protein product encoded by a PTN gene. In some aspects, the cellular factor comprises Midkine and / or a protein product encoded by a MDK gene. In some aspects, the cellular factor comprises Secreted frizzled-related protein 1 and / or a protein product encoded by a SFRP1 gene. In some aspects, the cellular factor comprises Aldehyde dehydrogenase family 1 member A3 and / or a protein product encoded by a ALDH1A3 gene. In some aspects, the cellular factor comprises Nucleolin and / or a protein product encoded by a NCL gene. In some aspects, the cellular factor comprises Sclerostin domain-containing protein 1 and / or a protein product encoded by a SOSTDC1 gene. In some aspects, the cellular factor comprises Histone-lysine N-methyltransferase EZH2 and / or a protein product encoded by a EZH2 gene. In some aspects, the cellular factor comprises Insulin-like growth factor-binding protein 2 and / or a protein product encoded by a IGFBP2 gene. In some aspects, the cellular factor comprises Transient receptor potential cation channel subfamily M member 2 and / or a protein product encodedby a TRPM2 gene. In some aspects, the cellular factor comprises Galectin-7 and / or a protein product encoded by a LGALS7 gene.

[0102] In particular aspects, the methods comprise modulating activity of SerpinE2 or a protein product encoded by a SERPINE2 gene.

[0103] In any of the foregoing or related aspects, the therapeutic agent may comprise a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a gene therapy vector, a nanoparticle, a liposome, a polysaccharide or any combination thereof. For example, in some aspects, the therapeutic agent may comprise a nucleic acid (e.g., DNA or RNA), a protein (e.g., an antibody), a small molecule therapeutic (e.g., a small molecule activator or inhibitor of a cellular factor), or any combination thereof. In various aspects, the therapeutic agent may comprise a gene therapy. In various aspects, the therapeutic agent comprises an mRNA (e.g., an mRNA encoding for a therapeutic protein encapsulated in a liposome), an interfering RNA targeting a gene or RNA transcript of a cellular factor (e.g., a gene or gene transcript encoding SerpinE2), or any combination thereof. In various aspects, the therapeutic agent comprises DNA (e.g., a DNA expression construct, such as a viral vector). In various aspects, the therapeutic agent may comprise an antibody or antigen binding fragment thereof.

[0104] In various aspects, the therapeutic agent increases levels and / or activity of the cellular factor in a host cell (e.g., a host cell located in an organ or tissue that comprises a site of metastasis). Accordingly, in various aspects, the therapeutic agent may comprise an agent that directly or indirectly increases activity of the cellular factor and / or an agent that increases expression levels of the cellular factor in the host cell. In various aspects, the therapeutic agent may be formulated for delivery to the host cell in vivo, according to standard methods in the art, including but not limited to those described herein. In some aspects, the therapeutic agent may comprise any of the Activating Agents described herein below in Section III.

[0105] In various aspects, the therapeutic agent decreases levels and / or activity of the cellular factor in a metastatic cell. Accordingly, in various aspects, the therapeutic agent may comprise an agent that directly or indirectly decreases activity of the cellular factor and / or an agent that decreases expression levels of the cellular factor in the metastatic cell. In various aspects, the therapeutic agent may be formulated for delivery to the metastatic cell in vivo, according to standard methods in the art, including but not limited to those described herein. In some aspects, the therapeutic agent may comprise any of the Inhibiting Agents described herein below in Section III.

[0106] In any of these methods, the metastatic cancer may be derived from the metastatic tumor that may comprise a breast tumor, a liver tumor, a pancreatic tumor, squamous cell tumor, prostate tumor, melanoma, skin tumor, urothelial tumor, kidney tumor, colon tumor,gastric tumor, gastrointestinal tumor, a lung tumor, melanoma, a sarcoma, an adnexal tumor, or a tumor of a peritoneum. That is, the one or more metastatic cancer cells may comprise metastatic cancer cells from any of these tumors. In some aspects, the one or more metastatic cancer cells may comprise breast cancer cells.

[0107] In further aspects, the organ or tissue comprising the site of metastasis may be selected from liver, bone, lungs, brain, and adrenal glands or any other common site of metastases. That is, the one or more host cells may comprise liver cells (e.g., hepatocytes, Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, or cholangiocytes), bone cells (e.g., osteoblasts, osteocytes, osteoclasts, and / or osteogenic or osteoprogenitor cells), lung cells (e.g., alveolar cells, lung epithelial cells, or basal cells), brain cells (e.g., neurons, astrocytes, glia, or microglial), or adrenal cells (e.g., adrenal cortex cells or adrenal medulla cells). In any of the methods herein, the one or more host cells comprise hepatocytes.Additional Anti-Cancer Therapies

[0108] The methods may comprise or further comprise administration of an additional anticancer therapy or agent. The compositions may comprise or further comprise an additional anti-cancer therapy or agent. The methods and / or compositions may exclude an additional anti-cancer therapy.Immunostimulators

[0109] The anti-cancer therapy or agent may be an immunostimulator. The term “immunostimulator” as used herein refers to a compound that can stimulate an immune response in a subject, and may include an adjuvant. In some embodiments, an immunostimulator is an agent that does not constitute a specific antigen, but can boost the strength and longevity of an immune response to an antigen. Such immunostimulators may include, but are not limited to stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such as alum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri or specifically with MPL (ASO4), MPL A of above-mentioned bacteria separately, saponins, such as QS-21 , Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomal formulations such as AS01 , synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic block co-polymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial toxoids or toxin fragments.

[0110] The additional anti-cancer therapy may comprise an agonist for pattern recognition receptors (PRR), including, but not limited to Toll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof. The additional anti-cancer therapy may comprise agonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists for Toll-Like Receptor 9; preferably the recited immunostimulators comprise imidazoquinolines; such as R848; adenine derivatives, such as those disclosed in U.S. Pat. No. 6,329,381 , U.S. Published Patent Application 2010 / 0075995, or WO 2010 / 018132, which are each incorporated herein by reference in their entirety; immunostimulatory DNA; or immunostimulatory RNA. In some embodiments, the additional anti-cancer therapies also may comprise immunostimulatory RNA molecules, such as but not limited to dsRNA, poly l:C or poly I: poly C12U (available as Ampligen.RTM., both poly l:C and poly I: polyC12U being known as TLR3 stimulants), and / or those disclosed in F. Heil et al., “Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8” Science 303(5663), 1526- 1529 (2004); J. Vollmer et al., “Immune modulation by chemically modified ribonucleosides and oligoribonucleotides”, WO 2008033432 A2; A. Forsbach et al., “Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway” WO 2007062107 A2; E. Uhlmann et al., “Modified oligoribonucleotide analogs with enhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US 2006241076; G. Lipford et al., “Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections” WO 2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods”, WO 2003086280 A2 which are each incorporated herein by reference in their entirety. In some embodiments, an additional anti-cancer therapy may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB- 1. In some embodiments, additional therapies may comprise TLR-5 agonists, such as flagellin, or portions or derivatives thereof, including but not limited to those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725, which are incorporated by reference in their entirety.

[0111] In some embodiments, additional anti-cancer therapies may be proinflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, additional anti-cancer therapies may be activated components of the complement cascade (e.g., CD21 , CD35, etc.). In some embodiments, additional anti-cancer therapies may be activated components of immune complexes. Additional anti-cancer therapies also include complement receptor agonists, such as a molecule that binds to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some embodiments, immunostimulators are cytokines, which are small proteins or biological factors (in the range of 5 kD-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. Insome embodiments, the cytokine receptor agonist is a small molecule, antibody, fusion protein, or aptamer.Immunotherapies

[0112] In some embodiments, the additional anti-cancer therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.Inhibition of co-stimulatory molecules

[0113] In some embodiments, the immunotherapy comprises an inhibitor of a co-stimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1 BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.Dendritic cell therapy

[0114] The additional anti-cancer therapy may comprise dendritic cells. Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0115] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0116] Dendritic cells can also be activated in vivo by making tumor cells express GM-CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0117] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (i.e., a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0118] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.CAR-T cell therapy

[0119] The additional anti-cancer therapy may comprise a chimeric antigen receptor (CAR). CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

[0120] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.Cytokine therapy

[0121] The additional anti-cancer therapy may comprise cytokines. Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immuneresponses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0122] Interferons are produced by the immune system. They are usually involved in anti-viral response, but also have use for cancer. They fall in three groups: type I (IFNa and I FNp), type II (IFNy) and type III (IFNA).

[0123] Interleukins have an array of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.Adoptive T-cell therapy

[0124] The additional anti-cancer therapy may comprise adoptive T cell therapy. Adoptive T cell therapy is a form of passive immunization by the transfusion of T-cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically, they activate when the T-cell’s surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune- mediated tumor death.

[0125] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.Checkpoint Inhibitors and Combination Treatment

[0126] In some embodiments, the additional anti-cancer immunotherapy comprises immune checkpoint inhibitors. Certain embodiments are further described below.

[0127] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Additional anti-cancer therapies of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0128] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1 , B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1 , PDL1 , and PDL2 are human PD-1 , PDL1 and PDL2.

[0129] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD- 1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1 . In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021 , and US2011 / 0008369, all incorporated herein by reference.

[0130] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011 , hBAT, or hBAT-1 , is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-FC fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional anticancer PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0131] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHlgM12B7.

[0132] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1 , CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1 , CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1 , PDL1 , or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0133] Another immune checkpoint that can be targeted in the methods provided herein as an additional anti-cancer therapy is the cytotoxic T-lymphocyte-associated protein 4 (CTLA- 4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell costimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1 , and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA- 4 and B7-2 interaction.

[0134] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0135] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA- 4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent ApplicationNo. WO2001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0136] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1 , MDX- 010, MDX- 101 , and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424).

[0137] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1 , CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1 , CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD- 1 , B7-1 , or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.Oncolytic virus

[0138] In some embodiments, the additional anti-cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for longterm immunotherapy.Polysaccharides

[0139] In some embodiments, the additional anti-cancer therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.Neoantigens

[0140] In some embodiments, the additional anti-cancer therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.Chemotherapies

[0141] In some embodiments, the additional anti-cancer therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0142] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg / m2 to about 20 mg / m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and / or subject in conjunction with the construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.

[0143] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (TAXOL®) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and / or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0144] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg / m2to about 75 mg / m2at about 21 -day intervals or about 25 mg / m2to about 30 mg / m2on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.

[0145] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®), available from Mead Johnson™, and NEOSTAR®, available from Adria™, is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0146] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-Fll) and floxuridine (fluorodeoxyuridine; FudR). 5-Fll may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-Fll dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0147] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co.™, “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.

[0148] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, thechemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.Targeted Therapies

[0149] In some embodiments, the additional anti-cancer therapy comprises a targeted therapy. Targeted therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules (“molecular targets”) that are involved in the growth, progression, and / or spread of cancer. Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names. Non-limiting examples of targeted therapies include hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, toxin delivery molecules, and the like. In particular embodiments, the targeted therapy may be a poly ADP ribose polymerase (PARP) inhibitor (e.g., niraparib). PARP (e.g., PARP-1 and / or PARP-2) inhibitors are well known in the art (e.g., Olaparib, ABT- 888, BSI-201 , BGP-15, INO-1001 , PJ34, 3-aminobenzamide, 4-amino-1 ,8- naphthalimide, 6(5H)-phenanthridinone, benzamide, NLI1025).Hormone Therapy

[0150] In some embodiments, the additional anti-cancer therapy comprises hormone therapy. In some embodiments, one or more anticancer therapies may be hormonal therapy, Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LLIPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).Radiotherapy

[0151] In some embodiments, the additional anti-cancer therapy comprises radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactionsto produce ionization (gain or loss of electrons). An exemplary and preferred ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0152] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 18, 19, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1 , 4, 8, 12, or 24 hours or 1 , 2, 3, 4, 5, 6, 7, or 8 days or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0153] In some embodiments, the amount of IR may be presented as a total dose of IR, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21 , 22, 23, 24, 25, 26, 27,28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52,53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77,78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 ,102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1 , 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7,8, 9, 10, 11 , or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.Surgery

[0154] The additional anti-cancer therapy may comprise surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0155] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1 , 2, 3, 4, 5, 6, or 7 days, or every 1 , 2, 3, 4, and 5 weeks or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months. These treatments may be of varying dosages as well.Outcomes

[0156] In certain embodiments, compositions disclosed herein can treat and / or prevent cancer in a subject in need. In some embodiments, compositions disclosed herein can impair tumor growth compared to tumor growth in an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be stopped following treatment with compositions disclosed herein. In other embodiments, tumor growth can be impaired at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In other words, tumors in subject treated using a composition of the disclosure have tumors that grow at least 5% less (or more as described above) when compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subjectwith identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated subject with identical disease condition and predicted outcome.

[0157] In some embodiments, treatment of tumors with compositions disclosed herein can result in a shrinking of a tumor in comparison to the starting size of the tumor. In some embodiments, tumor shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the tumor.

[0158] In some embodiments, treatment of tumors with compositions disclosed herein can result in a reduction in metastasis in comparison to the level of metastasis that occurs, or would be expected to occur, in an untreated patient. In some embodiments, the reduction in metastasis is measured as metastasis frequency over a period of time, which quantifies the number of new metastases detected over the period of time. In some embodiments, thenumber of new metastases detected over a period of time is reduced by at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that there are no new metastases detected after treatment), compared to the number of new metastases that are detected during an equivalent time period for an untreated patient.

[0159] In some embodiments, the reduction in metastasis is measured as a shrinking of an already established metastasis in comparison to the starting size of the metastasis. In some embodiments, metastasis shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the metastasis.

[0160] In certain embodiments, compositions disclosed herein can improve patient life expectancy compared to the cancer life expectancy of an untreated subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is definedas the time at which 50 percent of subjects are alive and 50 percent have passed away. In some embodiments, patient life expectancy can be indefinite following treatment with a composition disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted ou tcome. In some embodiments, patient life expectancy can be increased at least about 5% > or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease < condition and predicted outcome. In some embodiments, patient life expectancy can be inc reased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.

[0161] In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and / or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject. In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in a subject before and afteradministration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a cancer in a subject allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, methods disclosed herein may include administration of the compositions herein to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1 %, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject’s tumor volume, size, load or burden prior to treatment. In other embodiments, methods disclosed herein may include administration of the compositions herein to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.

[0162] In some embodiments, a subject to be treated by any of the methods and / or compositions herein can present with one or more cancerous solid tumors, metastatic nodes, or any combination thereof. In some embodiments, a subject herein may have a cancerous tumor cell source that can be less than about 0.2 cm3to at least about 20 cm3or greater, at least about 2 cm3to at least about 18 cm3or greater, at least about 3 cm3to at least about 15 cm3or greater, at least about 4 cm3to at least about 12 cm3or greater, at least about 5 cm3to at least about 10 cm3or greater, or at least about 6 cm3to at least about 8 cm3or greater.

[0163] In certain embodiments, the compositions disclosed herein can be effective for treating at least one tumor cell in a solid tumor from a subject in need. In some embodiments, the amount of viable tumor cells may be reduced by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about25% or greater, at least about 30% or greater, at least about 35% or greater, at least about40% or greater, at least about 45% or greater, at least about 50% or greater, at least about55% or greater, at least about 60% or greater, at least about 65% or greater, at least about70% or greater, at least about 75% or greater, at least about 80% or greater, at least about85% or greater, at least about 90% or greater, at least about 95% or greater, at least about100% compared to an untreated subject with identical disease condition and predicted outcome.

[0164] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the compositions disclosed herein to a subject, depending upon the type of disease to be treated or the site of the disease. In some embodiments, compositions herein can be administered to a subject by intravenous infusion, by subcutaneous administration, by inhalation, by intranasal administration or other mode of administration. In some embodiments, compositions herein can be administered to a subject orally.

[0165] In some embodiments, any of the methods disclosed herein can further include monitoring occurrence of one or more adverse effects in the subject. Exemplary adverse effects include, but are not limited to, hepatic impairment, hematologic toxicity, neurologic toxicity, cutaneous toxicity, gastrointestinal toxicity, or any combination thereof. When one or more adverse effects are observed, the method disclosed herein can further include reducing or increasing the dose of one or more of the disclosed active agents (e.g., a gene therapy) depending on the adverse effect or effects in the subject. For example, when a moderate to severe hepatic impairment is observed in a subject after treatment, one or more compositions can be reduced in concentration, frequency of dosing, or a combination thereof.III. Compositions

[0166] The present disclosure provides various therapeutic agents for increasing or decreasing activity and / or levels of a cellular factor associated with cellular competition in a target cell. In various aspects, therapeutic agents for increasing activity and / or levels of a cellular factor are referred to herein as “Activating Agents” and therapeutic agents for decreasing activity and / or levels of a cellular factor are referred to herein as “Inhibiting Agents.”Activating Agents

[0167] The present disclosure provides activating agents that increase activity of one or more cellular factors in a target cell. These activating agents may include small molecule activators, antibody activators, polynucleotide activators, or any combination thereof. In particular aspects, the activating agents comprise polynucleotide activators, particularly nucleic acids, expression constructs, and vectors to deliver a therapeutic protein into a cell of interest. These nucleic acids, expression constructs and / or vectors may be used to express a protein product, either for subsequent purification and delivery to a cell / subject, or for use directly in a geneticbased delivery approach. As an exemplary aspect, the activating agent may comprise an expression construct containing a nucleic acid coding for a cellular factor, as described further herein below.Inhibiting Agents

[0168] The present disclosure also provides inhibiting agents that decrease activity of one or more cellular factors in a target cell. These inhibiting agents may include small molecule activators, antibody activators, polynucleotide inhibitors, or any combination thereof. In particular aspects, the inhibiting agents comprise polynucleotide inhibitors, particularly interfering nucleic acids, expression constructs, and vectors to deliver the interfering nucleic acid into a cell of interest to disrupt expression of the cellular factor. These polynucleotide inhibitors can be in some aspects an interfering nucleic acid that specifically targets a 10-50nucleotide sequence region on a nucleic acid that encodes for a cellular factor associated with cell competitiveness. In particular aspects, the polynucleotide inhibitor can comprise an interfering nucleic acid that specifically targets a 10-50 nucleotide sequence region on a sequence as set forth in SEQ ID NO: 2 (encoding SerpinE2 as described below) or a sequence at least about 60% identical thereto.Expression Constructs

[0169] Throughout this application, the term “expression construct” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e. , is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “operably linked” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression constructs comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0170] Expression constructs provided herein may express a protein that is under-expressed in a cell population, where the under-expression of the protein directly leads to a loss of competitive advantage against another cell population. For instance, a protein underexpressed by normal cells at or around a metastatic site in a body may lead to a competitive advantage of the metastasizing cells. In general, the inventors have found that cell competition occurs in many circumstances not previously appreciated - such as in cancer cell metastasis. In each of these situations, defined by a “host” population (native to a region) and a “foreign” cell (defined as a cell that is invading (i.e., a metastatic cell)) , certain genes were found to be dysregulated (i.e., over or under-expressed). In certain aspects, the proteins encoded by these dysregulated genes (i.e., genes having increased or decreased expression relative to a baseline) in certain cell populations may be selected for inclusion in these expression constructs.

[0171] For instance, in some aspects, the protein encoded by an expression construct provided herein may comprise a serine protease inhibitor, C-X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20, solute carrier family 4 member 7, phosphoserine aminotransferase 1 , sterol O-acyltransferase 1 , protein phosphatase 1 regulatory subunit 18, Matrix Gia protein, Parathyroid hormone-related protein, Pleiotrophin, Midkine, Secreted frizzled-related protein 1 , Aldehyde dehydrogenasefamily 1 member A3, Nucleolin, Sclerostin domain-containing protein 1 , Histone-lysine N- methyltransferase EZH2, Insulin-like growth factor-binding protein 2, Transient receptor potential cation channel subfamily M member 2, Galectin-7 or any combination thereof.

[0172] In some aspects, the protein encoded by an expression construct provided herein may comprise a serine protease inhibitor (Serpin). Serine protease inhibitors have unique structural properties, allowing them to trap and inactivate serine proteases. Serine proteases are involved in a wide number of biological functions, including liver regeneration, coagulation, and immunologic response and are tightly regulated by serpins. Disruption of serpin / serine protease homeostasis results in apoptosis and it has been surprisingly discovered that this contributes to the establishment and growth of cancer metastases. In an aspect, the serpin encoded by an expression construct herein may comprise SerpinE2, SerpinEI , SerpinE3, SerpinAI , SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAIO, SerpinA11 , SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinB11 , SerpinB12, SerpinB13, SerpinCI , SerpinDI SerpinFI , SerpinF2, SerpinGI , SerpinHI , Serpinll , Serpinl2, or any combination thereof. For example, in some aspects the serine protease inhibitor can comprise SerpinE2. For ease of reference, the amino acid sequence and nucleic acid sequences of SerpinE2 are provided herein below as SEQ ID Nos: 1 and 2, respectively. In an aspect, an expression construct provided herein may encode a therapeutic protein or peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1. In an aspect, an expression construct provided herein may encode for a protein or polypeptide comprising SEQ ID NO: 1. In an aspect, an expression construct provided herein may comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2. In an aspect, an expression construct provided herein may comprise a nucleic acid sequence of SEQ ID NO: 2.Table 1 - Illustrative Serpin SequencesNucleic Acids Encoding System Components

[0173] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a protein or polypeptide, such as a therapeutic protein described above. The term “nucleic acid sequence,” “nucleic acid molecule,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid molecules can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments generated, for example, by a polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any one or more of ligation, scission, endonuclease action, or exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally-occurring nucleotides), or a combination thereof. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, morpholino, or the like. Nucleic acid molecules can be either single stranded or double stranded (e.g., ssDNA, dsDNA, ssRNA, or dsRNA).

[0174] The term “nucleotide” refers to sequences with conventional nucleotide bases, sugar residues and internucleotide phosphate linkages, but also to those that contain modifications of any or all of these moieties. The term “nucleotide” as used herein includes those moieties that contain not only the natively found purine and pyrimidine bases adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (II), but also modified or analogous forms thereof. Polynucleotides include RNA and DNA sequences of more than one nucleotide in a single chain. Modified RNA or modified DNA, as used herein, refers to a nucleic acid molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature.

[0175] As used herein, the term “isolated” nucleic acid molecule (e.g., an isolated DNA, isolated cDNA, or an isolated vector genome) means a nucleic acid molecule separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid.

[0176] Likewise, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0177] In any of the aspects of the present disclosure, the nucleic acid encoding a protein (e.g., a Serpin) can comprise a nucleic acid expression cassette. As used herein, the term“nucleic acid expression cassette” refers to an isolated nucleic acid molecule that includes one or more transcriptional control elements (e.g., promoters, enhancers, and / or regulatory elements, polyadenylation sequences, and introns) that are operably linked to and direct gene expression in one or more desired cell types, tissues or organs. A nucleic acid expression cassette can contain a transgene, although it is also envisaged that a nucleic acid expression cassette directs expression of an endogenous gene in a cell into which the nucleic acid sequence is inserted.

[0178] The term “operably linked” means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term “regulatory sequence” is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology, 1990, 185, Academic Press, San Diego, CA. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the nucleic acid expression cassette can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0179] In some examples, a nucleic acid expression cassette provided herein can comprise one or more transcription and / or translation control elements. Depending on the host and system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. The transcription and translation control element can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. The transcription and translation control element can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0180] Suitable transcription and translation control elements include promoters, enhancers, and / or transcriptional termination signals.

[0181] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor- regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter, C AG promoter). In some cases, the promoter can be a spatially restrictedand / or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).

[0182] The promoter can be chosen so that it will function in the target cell(s) of interest. Tissue-specific promoters refer to promoters that have activity in only certain cell types. The use of a tissue-specific promoter in a nucleic acid expression cassette can restrict unwanted transgene expression in the unaffected tissues as well as facilitate persistent transgene expression by escaping from transgene induced host immune responses. Tissue specific promoters include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters. Examples of liver-specific promoters include, but are not limited to, the alpha-1 - microglobulin / bikunin enhancer / thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the alpha-1 - antitrypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human alpha 1 antitrypsin (hAAT) promoter, the ApoEhAAT promoter composed of the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter consisting of the hAAT promoter and the alpha 1-microglobulin enhancer, the DC190 promoter containing the human albumin promoter and the prothrombin enhancer, the TBG promoter, and other natural and synthetic liver-specific promoters. In one embodiment, the promoter comprises a human TBG promoter provided herein as SEQ ID NO: 3 or a minimal functional portion thereof. In another embodiment, the promoter comprises a hAAT promoter provided herein as SEQ ID NO: 4. In another embodiment, the promoter comprises the ApoE enhancer provided herein as SEQ ID NO: 5. In another embodiment, the promoter comprises the ApoEhAAT promoter (comprising hAAT promoter and ApoE enhancer) provided herein as SEQ ID NO: 6.

[0183] For ease of reference, illustrative promoters and / or enhancers that may be used in the expression constructs of the present disclosure are provided in Table 2 below. In an aspect, the expression constructs provided herein may comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence to any one of SEQ ID Nos: 3 to 5. In an aspect, the expression constructs provided herein may comprise a nucleic acid sequence of any one of SEQ ID NOs: 3 to 5.Table 2 - Illustrative Promoters and Enhancers

[0184] In other aspects, the promoter can be a constitutive promoter. Constitutive promoters refer to promoters that allow for continual transcription of its associated gene. Constitutive promoters are always active and can be used to express genes in a wide range of cells and tissues, including, but not limited to, the liver, kidney, skeletal muscle, cardiac muscle, smooth muscle, diaphragm muscle, brain, spinal cord, endothelial cells, intestinal cells, pulmonary cells (e.g., smooth muscle or epithelium), peritoneal epithelial cells and fibroblasts. Examples of constitutive promoters include, but are not limited to, a CMV major immediate-early enhancer / chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFI -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (llbc) promoter, aMFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a Il- kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous promoters. In some embodiments, the constitutively active promoter is selected from the group consisting of human P-actin, human elongation factor-la, chicken P-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, or herpes simplex virus thymidine kinase.

[0185] Inducible promoters refer to promoters that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0186] The tissue-specific promoters can be operably linked to one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) enhancer elements (e.g., a neuron-specific promoter fused to a cytomegalovirus enhancer) or combined to form a tandem promoter (e.g., neuron-specific / constitutive tandem promoter). When two or more tissue-specific promoters are present, the isolated nucleic acid can be targeted to two or more different tissues at the same time.

[0187] As discussed above, a disclosed promoter can be an endogenous promoter. Endogenous refers to a disclosed promoter or disclosed promoter / enhancer that is naturally linked with its gene. In an aspect, a disclosed endogenous promoter can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene (such as, for example, a disclosed phosphorylase kinase, phosphorylase, or some other enzyme involved in the glycogen metabolic pathway). In an aspect, a disclosed endogenous promoter can be used for constitutive and efficient expression of a disclosed transgene (e.g., a nucleic acid sequence encoding a polypeptide capable of preventing glycogen accumulation and / or degrading accumulated glycogen). In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer.

[0188] As discussed above, a disclosed promoter can be an exogenous promoter. Exogenous (or heterologous) refers to a disclosed promoter or a disclosed promoter / enhancer that can be placed in juxtaposition to a gene by means of molecular biology techniques such that the transcription of that gene can be directed by the linked promoter or linked promoter / enhancer. An enhancer element is a nucleic acid sequence that functions to enhance transcription. As used herein, the terms “enhance” and “enhancement” with respect to nucleic acid expression or polypeptide production, refers to an increase and / or prolongation of steady-state levels of the indicated nucleic acid or polypeptide, e.g., by at least about 2%, 5%, 10%, 15%, 20%,25%, 40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50- fold, 100-fold or more. As used herein, the term “intron” refers to nucleic acid sequences that can enhance transgene expression. An intron can also be a part of the nucleic acid expression cassette or positioned downstream or upstream of the expression cassette in the expression vector. Introns can include, but are not limited to, the SV40 intron, elongation factor 1 alpha (EF-1 alpha) first intron, or the MVM intron. In some embodiments, the nucleic acid expression cassettes do not contain an intron. Representative enhancer elements that can be used herein include any enhancer elements normally associated with a Serpin gene.

[0189] In other aspects, the nucleic acid expression cassettes according to the present disclosure can further comprise a transcriptional termination signal. A transcriptional termination signal is a nucleic acid sequence that marks the end of a gene during transcription. Examples of a transcriptional termination signal include, but are not limited to, bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (Sv40 Poly A), and a synthetic polyadenylation signal. A polyadenylation sequence can comprise the nucleic acid sequence AATAAA. In some embodiments, the nucleic acid encoding the therapeutic protein (e.g., the nucleic acid encoding a serine protease inhibitor) comprises a FLAG tag at the C-terminus.

[0190] In any of the foregoing or related aspects, the nucleic acids disclosed herein may be “codon optimized” to ensure expression in a target cell or organism. As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es / OPTIMIZER / ).

[0191] In any of the aspects of the present disclosure, the nucleic acid expression constructs as provided herein may be packaged or formulated for delivery to the target cell(s) or subject of interest. Therefore, in various aspects, a vector (e.g., delivery vector) is provided that comprises any of the nucleic acid expression constructs disclosed herein. The choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro vs. in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or enzyme production), the target cell or organ, route of delivery, size of the isolated nucleic acid, safety concerns, and the like.

[0192] The expression constructs can be viral or non-viral as described further below. In some aspects, a viral or non-viral expression construct are also called “vectors”. Suitable vectors that are known in the art and that can be used to deliver, and optionally, express the isolated nucleic acids of the disclosure (e.g., viral and non-viral vectors), including, virus vectors (e.g., retrovirus, adenovirus, AAV, lentiviruses, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as a plasmid, and the like. In some embodiments, the non-viral vector can be a polymer-based vector (e.g., poly ethyleimine (PEI), chitosan, poly (DL-Lactide) (PLA), or poly (DL-lactic-co- glycolic acid) (PLGA), dendrimers, polymethacrylate) a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid based vector.

[0193] Other types of vectors include “plasmids”, which are circular double-stranded DNA loops into which additional nucleic acid segments can be ligated and viral vectors wherein additional nucleic acid segments can be ligated into the viral genome and which comprises the vector genome (e.g., viral DNA) packaged within a virion. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In some examples, the vectors, like the nucleic acid expression cassettes above, can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors”, or more simply “expression vectors”, which serve equivalent functions.

[0194] In some embodiments, the nucleic acid expression cassettes and / or transgenes (e.g., Serpin-E2 and variants thereof) can be incorporated into a recombinant viral vector. As used herein, the term “viral vector” refers to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA) packaged within a virion. Alternatively, in some contexts, the term “vector” is used to refer to the vector genome / viral DNA alone.

[0195] Expression vectors contemplated include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTI, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia™).

[0196] In some aspects, the vector is a recombinant viral vector suitable for gene therapy. Examples of such viral vectors include, but are not limited to vectors derived from: Ade novi ridae', Bimaviridae', Bunyavi ridae', Caliciviridae, Capillovirus group; Carlavirus group; Carmovirus virus group; Group Caulimovirus', Closterovirus Group; Commelina yellow mottle virus group; Comovirus virus group; Coronavi ridae’ PM2 phage group; Corcicoviridae', Group Cryptic virus; group Cryptovirus; Cucumovirus virus group family ([PHgr]6 phage group); Cysioviridae', Group Carnation ringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae; Flavivi ridae', Furovirus group; Group Germinivirus', Group Giardiavirus; Hepadnavi ridae', Herpesvi ridae', Hordeivirus virus group; lllarvirus virus group; I novi ridae', Iridoviridae', Leviviridae Lipothrixviridae', Luteovirus group; Marafivirus virus group; Maize chlorotic dwarf virus group; icroviridae', Myovi ridae', Necrovirus group; Nepovirus virus group; Nodaviridae', Orthomyxovi ridae', Papovaviridae', Pa ramyxovi ridae-, Parsnip yellow fleck virus group; Pa rtitivi ridae, Parvoviridae', Pea enation mosaic virus group; Phycodnavi ridae', Picornavi ridae', Plasmaviridae', Prodovi ridae Polydnaviridae', Potexvirus group; Potyvirus: Poxyiridae: Reovi ridae: Retroviridae: Rhabdoviridae: Group Rhizidiovirus: Siphoviridae: Sobemovirus group; SSV 1-Type Phages; Tectiviridae: Ten ui virus', Tetraviridae: Group Tobamovirus', Group Tobra virus: Togaviridae: Group Tombusvirus: Group Toroviruis; Totivi ridae: Group Tymovirus: and plant virus satellites.

[0197] In some embodiments, the recombinant viral vector is selected from the group consisting of adenoviruses, Adeno-associated viruses (AAV) (e.g., AAV serotypes and genetically modified AAV variants), a herpes simplex viruses (e.g., e.g., HSV-1 , HSV), a retrovirus vector (e.g., MMSV, MSCV), a lentivirus vector (HIV-1 , HIV-2), and alphavirus vector (e.g., SFV, SIN, VEE, Ml), a flavivirus vector (e.g., Kunjin, West Nile, Dengue virus), a rhabdovirus vector (e.g., Rabies, VSV), a measles virus vector (e.g., MV-Edm), a Newcastle disease virus vector, a poxvirus vector (VV), or a picomavirus vector (e.g., Coxsackievirus). The recombinant viral vector of the present disclosure includes any type of viral vector that is capable of packaging and delivering the G6PC transgene or viral vectors that can be designed engineered and generated by methods known in the art.

[0198] In some embodiments, the delivery vector is an adenovirus vector. The term “adenovirus” as used herein encompasses all adenoviruses, including the Mastadenovirus and Aviadenovirus genera.

[0199] The various regions of the adenovirus genome have been mapped and are understood by those skilled in the art. The genomic sequences of the various Ad serotypes, as well as the nucleotide sequence of the particular coding regions of the Ad genome, are known in the art and may be accessed from GenBank and NCBI (see, e.g., GenBank Accession Nos. J0917,M73260, X73487, AF108105, L19443, NC 003266 and NCBI Accession Nos. NC 001405, NC 001460, NC 002067, NC 00454).

[0200] A recombinant adenovirus (rAd) vector genome can comprise the adenovirus terminal repeat sequences and packaging signal. An “adenovirus particle” or “recombinant adenovirus particle” comprises an adenovirus vector genome or recombinant adenovirus vector genome, respectively, packaged within an adenovirus capsid. Generally, the adenovirus vector genome is most stable at sizes of about 28 kb to 38 kb (approximately 75% to 105% of the native genome size). In the case of an adenovirus vector containing large deletions and a relatively small transgene, “stutter DNA” can be used to maintain the total size of the vector within the desired range by methods known in the art.

[0201] The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 (Ad5) or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. In some embodiments, the viral vector comprises a recombinant Adeno-Associated Viruses (AAV). AAV are parvoviruses and have small icosahedral virions and can contain a single stranded DNA molecule about 4.7 kb (e.g., about 4.5 kb, 4.6 kb, 4.8 kb, 4.9 kb, or 5.0 kb) or less in size. The viruses contain either the sense or antisense strand of the DNA molecule and either strand is incorporated into the virion. Two open reading frames encode a series of Rep and Cap polypeptides. Rep polypeptides (e.g., Rep50, Rep52, Rep68 and Rep78) are involved in replication, rescue and integration of the AAV genome, although significant activity may be observed in the absence of all four Rep polypeptides. The Cap proteins (e.g., VP1 , VP2, VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5’ and 3’ ends of the genome are inverted terminal repeats (ITRs). Typically, in recombinant AAV (rAAV) vectors, the entire rep and cap coding regions are excised and replaced with a transgene of interest. Recombinant AAV vectors generally require only the inverted terminal repeat(s) (ITR(s)) in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans. Typically, the rAAV vector genome will only retain the one or more ITR sequence so as to maximize the size of the transgene that can be efficiently packaged by the vector. The structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell). In embodiments of the present disclosure, the rAAV vector genome comprises at least one terminal repeat (ITR) sequence (e.g., AAV TR sequence), optionally two ITRs (e.g., two AAV ITRs), which typically will be at the 5’ and 3’ ends of the vector genome and flank the heterologous nucleic acid sequence, but need not be contiguous thereto. The ITRs can be the same or different from each other.

[0202] The term “inverted terminal repeat” or “ITR” is used equivalently herein with the term “terminal repeat” or “TR” and includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and / or provirus rescue, and the like). The ITR can be an AAV ITR or a non-AAV ITR. For example, a non-AAV ITR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as a ITR, which can further be modified by truncation, substitution, deletion, insertion and / or addition. Further, the ITR can be partially or completely synthetic, such as the “double-D sequence.”

[0203] An “AAV inverted terminal repeat” or “AAV ITR” may be from any AAV, including but not limited to serotypes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered. An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV ITR sequence may be altered by insertion, deletion, truncation and / or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and / or provirus rescue, and the like. In some embodiments, the vector comprises flanking ITRs derived from the AAV2 genome.

[0204] Wild-type AAV can integrate their DNA into non-dividing cells and exhibit a high frequency of stable integration into human chromosome 19. A rAAV vector genome will typically comprise the AAV terminal repeat sequences and packaging signal.

[0205] An “AAV particle” or “rAAV particle” comprises an AAV vector genome or rAAV vector genome, respectively, packaged within an AAV capsid. The AAV rep / cap genes can be expressed on a single plasmid. The AAV rep and / or cap sequences may be provided by any viral or non-viral vector. For example, the rep / cap sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the Ela or E3 regions of a deleted adenovirus vector). EBV vectors may also be employed to express the AAV cap and rep genes. One advantage of this method is that EBV vectors are episomal, yet will maintain a high copy number throughout successive cell divisions (i.e., are stably integrated into the cell as extrachromosomal elements, designated as an “EBV based nuclear episome,” see Margolski (1992) Curr. Top. Microbiol. Immun. 158:67). The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs.

[0206] However, the rAAV vector itself need not contain AAV genes encoding the capsid (cap) and Rep proteins. In particular embodiments of the disclosure, the rep and / or cap genes aredeleted from the AAV genome. In a representative embodiment, the rAAV vector retains only the terminal AAV sequences (ITRs) necessary for integration, excision, and replication.

[0207] Sources for the AAV capsid genes can include naturally isolated serotypes, including but not limited to, AAV1 , AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7, as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In particular embodiments, the AAV capsids are chimeras either created by capsid evolution or by rational capsid engineering from the naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and host immune response escape, including but not limited to AAV- DJ, AAV-HAE1 , AAV-HAE2, AAVM41 , AAV-1829, AAV2 Y / F, AAV2 TA / , AAV2i8, AAV2.5, AAV9.45, AAV9.61 , AAV-B1 , AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS),AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS., AAV-PHP. B, AAV-PHP. eB, and AAV-PHP.S. In these and other aspects, the AAV vectors may be optimized for targeted delivery to an organ (i.e. , “organ specific AAV vector”). Exemplary AAV vectors designed for targeted delivery to the nervous system, for instance, include but are not limited to those described in Huang Q et al., BioRxiv [Preprint]. 2023 Dec 22:2023.12.20.572615; and Krolak T. et al., Nat Cardiovasc Res. 2022 Aprl; 1 (4):389-400 which are both incorporated herein by reference in their entirety.

[0208] Accordingly, when referring herein to a specific AAV capsid protein (e.g., an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 or AAV12 capsid protein) it is intended to encompass the native capsid protein as well as capsid proteins that have alterations other than the modifications of the invention. Such alterations include substitutions, insertions and / or deletions.

[0209] In some embodiments, the recombinant AAV vectors are selected from the group consisting of AAV8, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV12 . In certain embodiments, the recombinant AAV vector comprises AAV8.

[0210] In some embodiments, the recombinant viral vectors (e.g., rAAV) according to the present disclosure generally comprise, consist of, or consist essentially of one or more of the following elements: (1) an Inverted Terminal Repeat sequence (ITR); (2) a promoter (e.g., a liver-specific promoter); (3) a transgene (e.g., a nucleic acid sequence encoding Serpin-E2, a fragment thereof, an isoform thereof, or a homologue thereof); (4) a transcription terminator (e.g., a polyadenylation signal); and (5) a flanking Inverted Terminal Repeat sequence (ITR).

[0211] In some embodiments, the recombinant viral vectors can comprise a linker sequence. The term “linker sequence” as used herein refers to a nucleic acid sequence that encodes a short polypeptide sequence. A linker sequence can comprise at least 6 nucleotide sequences, at least 15 nucleotides, 27 nucleotides, or at least 30 nucleotides. In some embodiments, the linker sequence has 6 to 27 nucleotides. In other embodiments, the linker sequence has 6 nucleotides, 15 nucleotides, and / or 27 nucleotides. A linker sequence can be used to connect various encoded elements in the vector constructs. For example, a transgene and Myc tag can be operably linked via a linker, or a Myc tag and FLAG can be operably linked via a linker or a FLAG tag and mCherry tag can be operably linked via a linker. Alternatively, the vector elements can be directly linked (e.g., not via a linker).

[0212] In accord with any of the foregoing, an expression construct provided herein may comprise a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NOs: 7 or 8, which are provided herein below for reference. These AAV8 vectors comprise a coding region for Serpin-E2 (i.e., SEQ ID NO: 2) operably linked to either a TPG promoter (in SEQ ID NO: 7) or an hAAT / ApoE promoter / enhancer (in SEQ ID NO: 8). For ease of reference, SEQ ID NOs 7 and 8 are provided in Table 3 below.Table 3 - Illustrative expression constructs / vectors

[0213] In some embodiments, the AAV vectors are pseudotyped, which refers to the practice of creating hybrids of certain AAV strains to be able to refine the interaction with desired target cells. The hybrid AAV can be created by taking a capsid from one strain and the genome from another strain. For example, AAV2 / 5, a hybrid with the genome of AAV2 and the capsid of AAV5, can be used to achieve more accuracy and range in brain cells than AAV2 would be able to achieve unhybridized. Production of pseudotyped rAAV is disclosed in, for example, WOOI / 83692.

[0214] Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014), incorporated herein by reference in its entirety. It is understood that the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0215] Examples of recombinant AAV that can be constructed to comprise the nucleic acid molecules of the disclosure are set out in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352) incorporated by reference herein in its entirety.

[0216] Any suitable method known in the art can be used to produce AAV vectors. In one particular method, AAV stocks can be produced by co-transfection of a rep / cap vector plasmid encoding AAV packaging functions and the vector plasmid containing the recombinant AAV genome into human cells infected with the helper adenovirus. General principles of recombinant AAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, (1992) Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. Nos. 5,173,414; 5,658,776; WO 95 / 13392; WO 96 / 17947; WO 97 / 09441 ; WO 97 / 08298; WO 97 / 21825; WO 97 / 06243; WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3: 1124-1132; U.S. Pat. Nos. 5,786,211 ; 5,871 ,982; and 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to recombinant AAV production.

[0217] The recombinant viral vectors (e.g., rAAV) may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying recombinant viral vectors from helper virus are known in the art.

[0218] The nucleic acid encoding Serpin-E2 (or any other therapeutic protein) can be provided to the cell using any method known in the art. For example, the template can be supplied by a non-viral (e.g., plasmid) or viral vector.

[0219] The AAV rep and / or cap genes can alternatively be provided by a packaging cell that stably expresses the genes. A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for viral (e.g., AAV) particle production. For example, in one embodiment, a plasmid (or multiple plasmids) comprising a viral rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077- 2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells. In one embodiment, packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), Wl- 38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0220] In still further embodiments, the delivery vectors are a hybrid Ad- AAV delivery vector. Briefly, the hybrid Ad-AAV vector comprises an adenovirus vector genome comprising adenovirus (i) 5’ and 3’ cis-elements for viral replication and encapsidation and, further, (ii) a recombinant AAV vector genome comprising the AAV 5’ and 3’ inverted terminal repeats (ITRs), an AAV packaging sequence, and a heterologous sequence(s) flanked by the AAV ITRs, where the recombinant AAV vector genome is flanked by the adenovirus 5’ and 3’ cis- elements. The adenovirus vector genome can further be deleted, as described above.

[0221] Another vector for use in the present disclosure comprises Herpes Simplex Virus (HSV). HSV can be modified for the delivery of transgenes to cells by producing a vector thatexhibits only the latent function for long-term gene maintenance. HSV vectors are useful for nucleic acid delivery because they allow for a large DNA insert of up to or greater than 20 kilobases; they can be produced with extremely high titers; and they have been shown to express transgenes for a long period of time in the central nervous system as long as the lytic cycle does not occur.

[0222] Herpes virus may also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate scalable AAV vector production schemes. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al. (1999) Gene Therapy 6:986 and WO 00 / 17377).

[0223] In other embodiments of the present disclosure, the delivery vector of interest is a retrovirus. Retroviruses normally bind to a species-specific cell surface receptor, e.g., CD4 (for HIV); CAT (for MLV-E; ecotropic Murine leukemic virus E); RAM1 / GLVR2 (for murine leukemic virus- A; MLV-A); GLVR1 (for Gibbon Ape leukemia virus (GALV) and Feline leukemia virus B (FeLV-B)). The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes. A replication-defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.

[0224] Yet another suitable vector is a lentiviral vector. Lentiviruses are a subtype of retroviruses but they have the unique ability to infect non-dividing cells, and therefore can have a ride range of potential applications.

[0225] Yet another suitable vector is a poxvirus vector. These viruses contain more than 100 proteins. Extracellular forms of the virus have two membranes while intracellular particles only have an inner membrane. The outer surface of the virus is made up of lipids and proteins that surround the biconcave core. Poxviruses are very complex antigenically, inducing both specific and cross-reacting antibodies after infection. Poxvirus can infect a wide range of cells. Poxvirus gene expression is well studied due to the interest in using vaccinia virus as a vector for expression of transgenes.

[0226] In another representative embodiment, the nucleic acid sequence encoding a serine protease inhibitor (i.e., Serpin-E2) is provided by a replicating rAAV virus. In still other embodiments, an AAV provirus comprising the nucleic acid sequence encoding a serine protease inhibitor (i.e., Serpin-E2) can be stably integrated into the chromosome of the cell.

[0227] To enhance virus titers, helper virus functions (e.g., adenovirus or herpesvirus) that promote a productive AAV infection can be provided to the cell. Helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences will be provided by a helper adenovirus or herpesvirus vector. Alternatively, the adenovirus or herpesvirus sequences can be provided by another non -viral or viral vector, e.g., as a non-infectious adenovirus miniplasmid that carries all of the helper genes that promote efficient AAV production.

[0228] Further, the helper virus functions may be provided by a packaging cell with the helper sequences embedded in the chromosome or maintained as a stable extrachromosomal element. Generally, the helper virus sequences cannot be packaged into AAV virions, e.g., are not flanked by TRs.

[0229] In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed. Many non-viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In particular embodiments, non-viral delivery systems rely on endocytic pathways for the uptake of the nucleic acid molecule by the targeted cell. Exemplary nucleic acid delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. In particular embodiments, plasmid vectors are used in the practice of the present disclosure. Naked plasmids can be introduced into cells by injection into the tissue. Expression can extend over many months. Cationic lipids can aid in introduction of DNA into some cells in culture. Injection of cationic lipid plasmid DNA complexes into the circulation of mice can result in expression of the DNA in organs (e.g., the lung). One advantage of plasmid DNA is that it can be introduced into non-replicating cells.

[0230] In a representative embodiment, a nucleic acid molecule (e.g., a plasmid) can be entrapped in a lipid particle bearing positive changes on its surface and, optionally, tagged with antibodies against cell surface antigens of the target tissue.

[0231] Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for nucleic acid delivery in vitro and in vivo. The positively charged liposomes are believed to complex with negatively charged nucleic acids via electrostatic interactions to form lipidmucleic acid complexes (also known as lipoplexes). The lipidmucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipidmucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they can evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.

[0232] Amphiphilic cationic lipidmucleic acid complexes can be used for in vivo transfection both in animals and in humans and can be prepared to have a long shelf-life.

[0233] In addition, vectors according to the present disclosure can be used in diagnostic and screening methods, whereby a nucleic acid encoding a serine protease inhibitor (i.e. , Serpin- E2) is transiently or stably expressed in a cell culture system, or alternatively, a transgenic animal model screening method, whereby a nucleic acid of interest is transiently or stably expressed in a cell culture system, or alternatively, a transgenic animal model.

[0234] The vectors of the present disclosure can also be used for various non-therapeutic purposes, including but not limited to use in protocols to assess gene targeting, clearance, transcription, translation, etc., as would be apparent to one skilled in the art. The vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Such data, for example, are considered by the United States Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.Cells

[0235] Also contemplated herein are cells comprising an expression construct provided herein. Suitable cells may be in vitro or in vivo (i.e., in a subject). In an aspect, the cell is a mammalian cell. In an aspect, the cell is of a subject or a patient. In an aspect, the cell is a cancerous cell (e.g., a metastatic cancer cell). In an aspect, the cell may be a human cell, a mouse cell, a rat cell, a canine cell, a non-human primate cell, or a bovine cell. In an aspect, the cell may be a hepatocyte or other host organ-specific non-cancerous cell. In an aspect, the cell may be a pluripotent stem cell (iPSC).Pharmaceutical Compositions

[0236] Another aspect of the present disclosure provides a composition and / or pharmaceutical formulation comprising, consisting, or consisting essentially of a nucleic acid, a nucleic acid expression construct, a vector, or a cell as provided herein.

[0237] In some embodiments, compositions of the present disclosure comprise, consist of, or consist essentially of a recombinant viral vector (e.g., rAAV) and / or a pharmaceutically acceptable carrier and / or excipient, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form.

[0238] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with theisolated nucleic acid or vector without causing any undesirable biological effects such as toxicity. Thus, such a pharmaceutical composition can be used, for example, in transfection of a cell ex vivo or in administering an isolated nucleic acid or vector directly to a subject.

[0239] The compositions can also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and can include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

[0240] The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0241] In some embodiments, sterile injectable solutions are prepared by incorporating the recombinant viral vector (e.g., rAAV) in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0242] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of recombinant viral vector (e.g., rAAV) as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of recombinant viral vector (e.g., rAAV) can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0243] Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the subject by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The recombinant viral vector can be used with any pharmaceutically acceptable carrier and / or excipient for ease of administration and handling.Dosage formulations

[0244] In certain embodiments, the present disclosure provides compositions formulated for one or more routes of administration. Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal, and / or parenteral delivery. In some embodiments, compositions herein formulated can be formulated for parenteral delivery. In some embodiments, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, and / or intranasal injections.

[0245] In certain embodiments, one may administer a composition herein in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or any combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to subject as disclosed herein. In some embodiments, a pharmaceutical composition disclosed herein can administered to human patient. In some embodiments, a pharmaceutical composition disclosed herein can administered to a humanpatient via at least two administration routes. In some embodiments, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and / or intra-parenchymal injection and intravenous injection.

[0246] In certain embodiments, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0247] In certain embodiments, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, physiological salt buffer, or any combination thereof.

[0248] In certain embodiments, pharmaceutical compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection herein may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions herein may be suspensions, solutions or emulsions in oily or aqueous vehicles, and / or may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0249] In certain embodiments, pharmaceutical compositions herein formulated for parenteral administration may include aqueous solutions of the active preparation in water-soluble form. In some embodiments, compositions herein comprising suspensions of the active preparation may be prepared as oily or water-based injection suspensions. Suitable lipophilic solvents and / or vehicles for use herein may include, but are not limited to, fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. In some embodiments, compositions herein comprising aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and / or dextran. In some embodiments, compositions herein comprising a suspension may also contain one or more suitable stabilizers and / or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0250] In some embodiments, compositions herein may comprise the active ingredient in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0251] Pharmaceutical compositions suitable for use in context of the present disclosure may include compositions wherein the active ingredients can be contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0252] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0253] In some embodiments, toxicity and therapeutic efficacy of the active ingredients disclosed herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In some embodiments, data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human subject. In some embodiments, a dosage for use herein may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1).

[0254] In certain embodiments, dosage amounts and / or dosing intervals may be adjusted individually to brain or blood levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). In some embodiments, the MEC for an active ingredient may vary for each preparation but can be estimated from in vitro data. In some embodiments, dosages necessary to achieve the MEC herein may depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0255] In certain embodiments, depending on the severity and responsiveness of the condition to be treated, dosing with compositions herein can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0256] In certain embodiments, amounts of a composition herein to be administered will be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like. In some embodiments, effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.IV. Kits

[0257] The present disclosure further provides kits comprising the compositions provided herein and for carrying out the subject methods as provided herein. For example, in one embodiment, a subject kit can comprise, consist of, or consist essentially of one or more of the following: (i) nucleic acid construct as provided herein; (ii) a vector(s) and / or vector systems as provided herein; (iii) delivery systems comprising a nucleic acid constructs and / or vector(s) and / or vector systems as provided herein; (iv) cells comprising a nucleic acid construct(s) and / or vector(s), and / or vector systems and / or delivery system comprising a nucleic acid construct and / or vector(s), vector systems, compositions as provided herein; and / or (v) pharmaceutical compositions as provided herein.

[0258] In other embodiments, a kit can further include other components. Such components can be provided individually or in combination and can provide in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, (i) one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more wash buffers; one or more storage buffers, one or more control reagents and the like, (ii) one or more control expression vectors or RNA polynucleotides; (iii) one or more reagents for in vitro production and / or maintenance of the of the molecules, cells, delivery systems etc. provided herein; and the like. Components (e.g., reagents) can also be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrated or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.

[0259] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. As such, the instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.EXAMPLES

[0260] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 : Summary of Examples

[0261] Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. The following examples describe data testing the hypothesis MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis (see FIG. 1).

[0262] New in-vitro and mouse lineage-traced models were developed to assay cell competition between MCC and hepatocytes. These models directly capture MCC and hepatocyte interactions and other modes of cell competition, including apoptosis, can be assessed through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, a bioinformatics screen was performed to identify potential signals that contribute to cell competition between MCC and hepatocytes. A new single-cell RNA-seq dataset was generated that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched inboth. Next, genes were validated by testing for their high expression in MCC when compared to hepatocytes. Then, the impact of target genes on cell competition was determined in these models.

[0263] These models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice with MCC also resulted in increased neighboring hepatocyte apoptosis. Using a bioinformatics screen, SerpinE2, a protease inhibitor, was identified as a target that could drive breast cancer metastasis and cell competition. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild-type (WT) conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM.Example 2: Materials and Methods

[0264] Hepatocyte and Cancer co-cultures'. GFP labeled H2.35 and mCherry labeled 4T1 , were plated together in 1 :1 ratio (50,000:50,000 cells) in each well of an 8 well chambered slide ( Nunc™ Lab-Tek™ II Chamber Slide™ System). Cells were plated using 14 - 14 media with equal ratios of DMEM 1X supplemented with 4% FBS, 200nM dexamethasone, and 1% PenStrep media and RPMI 1640 1X supplemented with 10% FBS, and 1 % PenStrep. Coculture slides were fixed after a 48-hour incubation time.

[0265] Hydrodynamic Tail Vein Injection: Mice were anesthetized with 2-3% isoflurane gas, 1 L / min 02. Mice were weighed both pre-and post-injection and kept on supplemental heat during the procedure. Hydrodynamic tail vein injections (HDTVIs) were accomplished by delivery of 700K mCherry labeled cancer cells in 1.5mL injectate warmed to 28-37 degrees C via tail vein injection over 5-8s. Anesthesia lines were immediately flushed of isoflurane and oxygen increased to 5L / min after successful injection, measured by visual assessment of tail vein clearance and no resistance to injection. Aggressive stimulation was provided to mice post-injection until they regained consciousness. Mice were kept on supplemental heat 1-3h post-injection and monitored daily according to IACUC protocols.

[0266] Tissue Processing: Mice were euthanized according to the UT Southwestern Institutional Animal Care and Use Committee (IACUC) guidelines. Animals were weighed andimaged post-euthanasia and their livers removed, weighed, and imaged. Transcardiac perfusion was performed with PBS until liver turned pale. Mice were then perfused with 4% PFA. All tissues collected were fixed in 4% PFA for 4-8hrs. Tissues were moved to 10, 20, and 30% sucrose consecutively for 12-24h each. Tissues were then OCT embedded and the interior 75% of the frozen tissue block was cryosectioned at 5 pm thickness. Two slides every ~50 pm were sampled for immunofluorescence, slide scanning, and quantification.

[0267] Immunofluorescence staining: Slides were fixed with 4% PFA and then washed twice with DPBS and permeabilized with 2% FBS, 0.5% Triton X-100 in DPBS for 10 minutes at room temperature. Slides were subsequently blocked using 10% FBS, 0.2% Triton X-100 in DPBS for 30 minutes at room temperature. Antibodies were diluted in blocking buffer. Primary antibodies were applied to the slide overnight at 4 °C. After primary antibody incubation, slides were washed 3 times for 15 minutes each in DPBS. Secondary antibodies and DAPI (1 :1000 dilution) were applied to the slide for 1 h at room temperature. After secondary antibody incubation, slides were washed 3 times for 15 minutes each in DPBS. Excess liquid was removed from the slide and mounted with Prolong® Gold Antifade Reagent. Slides were left at room temperature in the dark to cure for 24h, and then sealed with nail polish the next day.

[0268] Quantification of immunofluorescence staining: For in vitro slides whole slides were scanned used Zeiss Axioscan 7 and quantified using QuPath Software. Quantification was performed blinded to image and sample identities. Images were loaded onto a project file in QuPath 0.4.0. Appropriate ROIs were selected such that as many cells as possible were included per well, without included the well border. All image manipulation was carried out blinded to sample identities. The cell-detection protocol was run through QuPath using DAPI channel. All scans were visually verified for appropriate and reasonable detection of cell boundaries, and the protocol was rerun with appropriate changes based on the slide. Next, object classifier scripts were run with mCherry or GFP fluorescence such that each cell was classified as either cancer or hepatocyte based on their fluorescent intensity. For in vitro slides, a spatial analysis feature was used to detect centroid distance between cells. Finally, a threshold was set to detect Alexa Fluor 647 activity. Cells were then screened out by a distance threshold between classes and only those within 30 pm distance were classified as neighbors. Out of these, the number of cells positive for Alexa Fluor 647 were measured for both cancer cells and hepatocytes. In vivo slides were quantified manually by counting the number of micrometastasis in each liver section.

[0269] AAV Production and concentration: AAV8.TBG. GFP and AAV8.TBG. SerpinE2 were produced using HEK293T cells. 1 x 107cells were plated on a 15 cm plate one day before transfection so they could be around 80% confluent during transfection. Media was changed on cells the next day and 25mL of fresh media was added ~2-4 hrs before transfection. 10pgpAdDeltaF6 (Addgene #112867), 10ug pAAV 2 / 8 (Addgene #112864), and 10pg of pAAV.TBG.PI.eGFP.WPRE. bGH (Addgene #105535) or pAAV.TBG. SerpinE2 were mixed in 1.25 mL Opti-MEM™ and 18pL of PEI (5pg / mL) was added in 1.25 mL Opti-MEM™ in another tube. Both DNA and PEI solutions were then mixed and incubated at room temperature for ~20 minutes, and then added to cell culture. One day after transfection media was removed and DMEM 1X with 1% FBS and 1% PenStrep was added. AAV particles were purified and concentrated 72 hrs later using AAVpro® Cell & Sup. Purification Kit Max (Takara). AAV particles were quantified using AAVpro® Titration Kit (for Real Time PCR) Ver.2 (Takara).

[0270] AAV Injections: Mice were kept on supplemental heat during the procedure. Tail vein injections were performed by injecting ~ 2 .5 x 1010vector copies of AAV particles in 200pL of injectate. Mice were monitored daily to assess health status and body weights were recorded daily.Example 3: Development of new in vitro model for metastatic competition

[0271] An in vitro model for monitoring cell competition in a dish was developed. This lineage traced, in vitro metastasis competition assay is shown in FIG. 2 and involves co-culturing mCherry-labeled metastatic cancer cells (MCC) (as depicted, 4T 1 breast cancer cells) and GFP-labeled healthy cells (as depicted, H2.35 liver cells) and then using QuPath software to identify mCherry+ cancer cells and GFP+ hepatocytes that neighbor each other. Visually, it was apparent that cancer cells outcompete hepatocytes within 48-72 hours where WT MCC and HEP co-cultured initially at a 1 :1 ratio results in a culture that is 94.3% MCC cells and 5.7% HEP by day three (FIG. 3), despite having similar growth rates when cultured in isolation (FIGS. 4A-4B). To determine how the cancer cells outcompeted neighboring hepatocytes, fluorescently labeled cleaved caspase-3 that localized to either a metastatic MCC or HEP that neighbor each other was quantified as shown in FIG. 5. It was found that, indeed, metastatic MCC induced apoptosis of neighboring hepatocytes (FIG. 6 and FIGS. 7A-7C). Interestingly, monocultures of MCC or HEP (FIG. 7C) did not induce apoptosis in MCC or HEP cultures, nor did conditioned media experiments (data not shown), suggesting that direct cell-cell contact is required for competition. The same results were also seen with a different cancer cell line (EMT6) in co-cultures with normal hepatocytes (H2.35 cell line) (see FIG. 8).Example 4: Cell competition between hepatocytes and cancer cells exists in vivo

[0272] To determine whether the cell competition observed in vitro occurs in vivo, liver models of metastasis were generated. Specifically, mCherry+ transduced WT metastatic cancer cells (4-T1 cell line, “MCC”) were injected into NOD-scid gamma mice using hydrodynamic tail vein injection (HD-TVI) (FIG. 9A). FIG. 9B shows how neighboring hepatocytes (HEP, in green)have high caspase-3 expression (white) bordering MCC (red), demonstrating that cell competition between cancer cells and hepatocytes occurs in these in vivo models.

[0273] In another experiment, human liver metastases were biopsied and examined for evidence of apoptosis. It was found that acidophil bodies (a marker of apoptosis) were significantly increased in liver cells, and especially at the interface of liver cells and tumor cells in these patients (FIGS. 10A-10B). This demonstrates that cell competition is occurring in human metastases and identifies it as a valid therapeutic target.Example 5: Identification of key genes driving metastatic outgrowth of breast cancer in liver.

[0274] Histological analysis of breast-to-liver metastases suggests that initial metastatic formation largely retains the function and architecture of their cell-of-origin. That is, the metastatic cells still form ductal structures (FIG. 11 , FIGS. 12A-12D), just like the developing mammary gland (FIGS. 13A-13C). It was hypothesized that there are shared mechanisms between mammary gland development and breast cancer metastasis. Both embryonic mammary development and development of metastasis occur in well characterized steps that mirror one another. During embryogenesis, mammary gland development occurs with a mammary bud that seeds a fat pad on embryonic day 16 (E16) and then branches and grows on embryonic day 18 (E18). In metastasis, cancer cells similarly seed a distant organ and then branch and grow in that organ (see FIGS. 13A-13C, adapted from Cowin and Wyolmerski, CSH Perspectives in Biology, 2010, which is incorporated herein by reference in its entirety). Further, cell competition plays a major biological role in branching morphogenesis, the fundamental process underlying mammary gland development.

[0275] Therefore, the inventors sought to identify shared genes among developing mammary epithelial cells (at E16 and E18) and metastatic breast cancer, using publicly available single cell RNA-seq (scRNA-seq) datasets of mouse embryonic mammary gland and primary breast cancer as provided in Giraddi, R.R et al., (Cell Rep, 2018. 24(6): p. 1653-1666 e7) and Yeo, S.K., et al., (Elife, 2020. 9) which are both incorporated by reference herein in their entirety. These datasets were integrated using procedures developed by the inventors and described in Xu, L et al., (Cell Rep Med, 2024: p.101511), incorporated herein by reference in its entirety. This resulted in the identification of differential genes expressed between E16, E18, and primary cancer epithelial cells that cluster together when compared to the remaining epithelia. These results are shown in FIG. 14) and the list of genes is provided in Table 4, below.Table 4

[0276] From a literature search, 4 uniquely overexpressed genes were identified as possibly integral to seeding and metastasis. Of these 4, SerpinE2 had high expression levels in MCC compared to HEP.Example 6: Growth and proliferation of 4T1 knockout cell lines in isolation in vitro and in vivo.

[0277] SerpinE2 (GTN), PTHLH and MGP were each individually knocked out in 4T1 cells using lentivirally delivered CRISPR-Cas9. The growth of these knockout cells was then measured in vitro and in vivo. It was found that that 4T 1 KO cells have similar growth rates as 4T1 WT cells as measured both by confluence (FIG. 15A), cell count (FIG. 15B) in vitro, and tumor volume in vivo (FIG. 15C). At the same time, it was found that they do not have any altered chemosensitivity to either doxorubicin (FIG. 16A) or paclitaxel (FIG. 16B). Therefore, knocking out any of the identified genes did not affect cancerous properties of these cells in isolation.Example 7: Altered competition between knockout metastatic cancer cells (MCC) and normal hepatocytes (HEP) in vitro.

[0278] As discussed above, WT 4T-1 cells rapidly outcompete WT HEP cells in co-cultures. However, surprisingly, it was found that knocking out any of GTN (Serpin-E2), MGP or PTHLH completely reversed this phenomena. That is, WT HEP cells outcompeted KO 4T-1 cells in co-cultures. This is shown in FIGS. 17A-17B which quantify and visualize increased caspase levels in 4T-1 KO (but not WT HEP) cells. This suggests that KO of target genes in 4T 1 cells reverse competition with hepatocytes. Looking at Serpin-E2 specifically, co-culturingexperiments further show how WT MCC and HEP co-cultured initially at a 1 :1 ratio results in a culture that is 94.3% MCC cells and 5.7% HEP by day three (FIG. 18, left column) but coculturing KO SerpinE2 4T-1 cells with WT HEP cells, resulted in a culture with 29.5% MCC and 70.5% HEP by day 3 (FIG. 18, right column). Indeed, it was found that SerpinE2 KO MCC in co-culture with HEP results in a 9.05-fold increase in MCC apoptosis compared to WT MCC (FIG. 19A) and a 4.75-fold decrease in HEP apoptosis (FIG. 19B). This effect could be rescued by overexpressing SerpinE2 in the KO MCC cells resulting in restoration of MCC competitiveness (reduced MCC apoptosis by 4.1 -fold) (FIG. 19A) and increased neighboring HEP apoptosis (FIG. 19B) by 5.89-fold compared to SerpinE2 KO MCC in co-culture with HEP.Example 8: SerpinE2-mediated competition occurs in established hepatocyte cultures

[0279] To test whether SerpinE2 mediates competition even in established hepatocyte cultures, another culturing experiment was designed. As shown in FIG. 20A, hepatocytes (H2.35 cells) were cultured for a period of time before 4T-1 cancerous cells (in red) were added. After a period of time, the number of caspase positive 4T-1 or H2.35 cells were quantified. As shown in FIG 20B, SerpinE2 KO cells failed to induce apoptosis of neighboring cancer cells and were in fact killed by those cells. Therefore, SerpinE2-mediated competition can occur even in established cultures.Example 9: SerpineE2 mediated competition occurs in tumors

[0280] SerpinE2 belongs to a family of serpins which are serine protease inhibitors that have unique structural properties, allowing them to trap and inactivate serine proteases. Serine proteases are involved in a wide number of biological functions, including liver regeneration, coagulation, and immunologic response and are tightly regulated by serpins. Disruption of serpin / serine protease homeostasis results in apoptosis. Interestingly, SerpinE2 predicts poor outcomes in breast cancer (FIG. 21).

[0281] To test whether SerpinE2 might regulate invasiveness in vivo, mCherry+ transduced Serpin E2 KO or WT metastatic cancer cells (4-T1 cell line, “MCC”) were injected into NOD- scid gamma mice using hydrodynamic tail vein injection (HD-TVI). The colonization efficiency of the cancer cells were evaluated post transplantation by counting the number of immunofluorescent labeled MCC in liver sections. As shown in FIGS. 22A-22B, the engraftment of SerpinE2 KO MCC into the liver results in 10-fold decreased metastases vs. WT MCC. Further experiments will measure on days 5 and 21 post transplantation and will also test for decrease in apoptosis (measured by caspase-3) in HEP. To determine whether SerpinE2 mediated competition involves apoptosis of neighboring liver cells in vivo (as it doesin vitro), tumor sections were stained for caspase activity. FIGS. 23A-23B show how KO of SerpinE2 results in less hepatocyte apoptosis and more cancer cell apoptosis.Example 10: Preparing AAV vectors encoding SerpinE2

[0282] AAV vectors were generated to overexpress SerpinE2 in hepatocytes to determine whether doing so would have a similar effect to knocking it out in cancer cells. Two sets of vectors expressing SerpinE2 were prepared under the control of two different liver specific promoters: TBG (AAV.TBG. SerpinE2; SEQ ID NO: 7) or hAAT (AAV. ApoE / hAAT. SerpinE2; SEQ ID NO: 8). These vectors included a nucleic acid sequence (SEQ ID NO: 2) encoding SerpinE2 (SEQ ID NO: 1) under the control of TBG promoter (SEQ ID NO: 3) or an ApoEhAAT promoter / enhancer (SEQ ID NO: 6). FIGs. 24A-24B shows a schematic of the expression constructs for a control vector expressing GFP (FIG. 24A) or Serpin-E2 vector (FIG. 24B) containing the TBG promoter. FIG. 25 provides a labeled schematic of the AAV.TBG. SerpinE2 vector (SEQ ID NO: 7). FIG. 26 provides a labeled schematic of the AAV. ApoE / hAAT. SerpinE2 vector (SEQ ID NO: 8). For ease of reference, key features for these two vectors are described in Tables 5 and 6 below.Table 5: Features of AAV.TPG. SerpinE2 vector (SEQ ID NO: 7)Table 6: Features of AAV.ApoE.hAAT. SerpinE2 vector (SEQ ID NO: 8)

[0283] Tail vein injections were performed by injecting ~ 2 .5 x 1010vector copies of AAV particles in 200 pL of injectate into mice. Samples from the liver, brain and lungs were obtained and analyzed for expression in these organs. As expected, the liver specific AAV vectors showed robust expression in the liver (at 60-85% efficiency) (FIG. 27A), but not brain (FIG. 27B) or lungs (FIG. 27C).Example 11 : Overexpression of SerpinE2 in hepatocytes protects against cancer cell competition

[0284] The AAV vectors described in Example 10 were used to overexpress Serpin-E2 in hepatocytes in culture. These overexpressing (OE) hepatocytes were then co-cultured with WT cancer cells (4T-1) and levels of caspase were assessed. It was surprisingly found that overexpression of SerpinE2 in hepatocytes still protected against cancer cell competition and significantly increased death of 4T1 cells and survival of the OE hepatocyte cells (FIGS. 28A- 28B). Therefore, delivering a gene therapy vector to increase liver cell expression of Serpin- E2 may be a viable anti-cancer therapy.Example 12: Determining how MCC produced SerpinE2 can alter serine protease homeostasis in the liver

[0285] It was found that HEP secrete 8.46-fold increased tPA (a serine protease partner of SerpinE2) compared to WT MCC and that when tPA was introduced into the co-culturing system, SerpinE2 KO MCCs had a 2-fold increase in death compared to WT MCC. Based on these results, it was hypothesized that cancer produced SerpinE2 disrupts tPA homeostasis in the tumor microenvironment, resulting in cancer cells’ competitive advantage. To test this, site directed mutagenesis will be performed to alter a conserved tPA cleavage region on SerpinE2 at arginine residues 365-366. MCC will be transfected with this altered SerpinE2 construct and cell competition assays will be performed to determine whether it alters tumor growth and metastasis.Example 13: SerpinE2 mediated cell competition during breast cancer metastasis to the liver

[0286] Patients with breast cancer liver metastasis (BCLM) have a 5-year survival rate of 8%, highlighting an urgent need for novel therapeutics to target liver-specific treatment for BCLM. The liver is a cellularly dense organ, and it remains unclear how metastatic cancer cells (MCC) colonize and expand in the liver. Cell competition is a developmentally conserved process that explains how cells use fitness-sensing mechanisms leading to survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. This process is critical to organogenesis and tumorigenesis. It was previously shown that MCC are able to create space during metastatic colonization of the liver by causing apoptosis in neighboring hepatocytes (HEP), which make up 80% of liver mass. Through a biological and bioinformatic screen, it was found that MCC expresses SerpinE2, a serine protease inhibitor. SerpinE2 has been shown to correlate with increased metastatic potential but the mechanistic cause is still unknown. In this study, the following hypothesis was tested: MCC engage in SerpinE2-mediated cell competition with HEP to create space and expand within the liver by eliminating neighboring HEP through apoptosis resulting in tumor expansion.

[0287] New in-vitro and mouse lineage-traced models were developed to model cell competition between MCC and HEP using syngeneic mouse breast cancer cells and hepatocytes. These models directly capture cell-cell interaction between MCC and HEP so it was possible to assess for apoptotic and extrusion events, known modes of cell competition, through immunostaining and live imaging. Hydrodynamic tail vein injections (HDTVI) were used to engraft mice livers with MCC. Prior literature suggests that the developing mammary gland informs both cell competition and metastasis, so a bioinformatics screen was performed to identify signals contributing to cell competition between MCC and HEP. A new single-cell RNA-seq dataset incorporating information from the developing mammary gland and breast cancer cells was generated. Through this, a target gene, SerpinE2, was identified as being enriched in both breast cancer and the developing mammary gland. It was further found that SerpinE2 is more highly expressed in MCC compared to HEP. CRISPR-Cas9 technology was used to generate a SerpinE2 knock-out (KO) 4T 1 cell line and lentiviral gene editing to restore the full length SerpinE2 gene in SerpinE2 KO MCC to generating a genetic rescue line. Previously established in vitro and in vivo models were then used to evaluate the functional role of SerpinE2 in cell competition and metastatic colonization in the liver.

[0288] Using in vitro coculture models it was demonstrated that wild-type (WT) MCC induce apoptosis in neighboring HEP through cell competition. MCC-induced apoptosis in neighboring HEP is 7.62-fold higher compared to apoptosis in neighboring MCC. KO of SerpinE2 in MCC did not affect their viability or growth rates in monocultures. In coculture with HEP, SerpinE2 KO resulted in increased apoptosis in MCC by 9.04-fold compared to WT MCC that neighbor HEP. There was a 4.75-fold decrease in HEP apoptosis compared to HEP in coculture with WT MCC. SerpinE2 was then genetically rescued in MCC and the resulting cells cocultured with HEP. SerpinE2 KO MCC with SerpinE2 restored increased apoptosis in HEP by 5.89-fold while decreasing MCC apoptosis by 4.10-fold when compared to cocultures with SerpinE2 KO MCC, similar to WT MCC. HDTVI of SerpinE2 KO MCC in mice showed a 10-fold decrease in micrometastasis in livers compared to those injected with WT MCC. These data suggest SerpinE2 regulates competition between MCC and HEP. Future work involves understanding the mechanistic regulation of metastatic colonization by SerpinE2 in the liver. These findings may suggest a novel therapy for BCLM patients that reduces MCC fitness or restores HEP fitness during liver colonization by MCC.Example 14: Introduction to Examples 15-21.

[0289] While metastasis is the primary cause of mortality for patients with breast cancer, not all metastases have similar survival rates. For example, patients with bone metastases have a 50% five-year overall survival rate, while patients with liver metastases have an 8-10% five- year overall survival rate. In contrast to other cancers where localized therapies have shown efficacy at extending overall survival, multiple studies exploring surgical resection, radiation therapy, or systemic therapies to broadly curtail BCLM growth have not resulted in durable responses.

[0290] The lack of effective therapies against BCLM underscores a limited understanding of how breast cancer cells colonize the liver. While recent research has focused on cancer cell intrinsic features which drive liver metastases development, these have not yet translated into clinical therapies. The following examples explore the possibility that breast cancer cell interactions with hepatocytes, liver epithelial cells that form 80% of the liver’s mass, are also significant. Given that the liver is a cellularly dense organ predominated by hepatocytes, the inventors first asked how cancer cells first create space in the liver. Cells in tissues compete for space and survival through a conserved process called cell competition. Cell competition has been described between cells of similar lineages during a variety of developmental stages and primary tumorigenesis as a mechanism to create space. Cell competition is directed by specific gene and protein imbalances that lead to elimination of one cell type through programmed cell death and the expansion of the other neighboring cell type. In the following examples, experiments are described that tested whether the early colonizing epithelial cancer cells create space in the liver via metastatic competition with hepatocytes, leading to hepatocyte death and BCLM.Example 15: Materials and Methods for Examples 16 to 21

[0291] The following materials and methods were used to perform the experiments described in the following examples.Table 7: Reagents and ResourcesBacterial and virus strainsAll references cited in Table 7 are incorporated herein by reference in their entireties.Three-part comparative transcriptomics bioinformatics screenPart 1 - Integration and analysis of scRNA-seq mouse embryonic mammary and mouse breast cancer model datasets

[0292] The inventors obtained two scRNA-seq datasets: one consisting of embryonic mouse mammary epithelial cells at embryonic day 16 and embryonic day 18 from Giraddi et al. (GSE111113), and one consisting of mouse breast cancer epithelial cells from MMTV-Neu, MMTV-PyMT, and 4T1 transplant tumor models from Yeo et al. (GSE123366). The inventors refer to the original papers for the details of their approach to scRNA sequencing of mouse embryonic mammary tissue (Giraddi et al. 2018) and cancer epithelial cells from multiple breast cancer mouse models (Yeo et al. 2020).

[0293] To integrate these datasets, the inventors used the previous integration pipeline for scRNA-seq data. The scRNA-seq data is found in Xu, L., et al. (2024), A comprehensive single-cell breast tumor atlas defines epithelial and immune heterogeneity and interactions predicting anti-PD-1 therapy response. Cell Rep Med 5, 101511 , which is incorporated herein by reference in its entirety. In brief, for the embryonic samples from Giraddi et al., cells were filtered by percent mitochondrial transcripts less than 5, and number of features greater than 200 and less than 3000. For breast tumor cell samples from Yeo et al., cells were filtered by percent mitochondrial transcripts less than 20, and number of features greater than 200 and less than 6000. The inventors used DoubletFinder (v2.0.3) to identify and remove doublets from the dataset. After removal of low-quality cells, gene expression matrices were normalized using sctransform (vO.3.5). The datasets were integrated using canonical correlation analysis (CCA) via the IntegrateLayers function in Seurat (v5.0.3). Louvain algorithm was applied to iteratively group proximal cells together by the FindClusters function with resolution of 0.2. Visualization was achieved using Uniform Manifold Approximation and Projection (UMAP). Success of integration and batch effect correction was determined by ensuring original source datasets were not driving clustering of the integrated dataset.

[0294] Using the FindMarkers method in Seurat (v5.0.3) and MAST (v1.20.0), DEG analysis was performed on raw counts between the mixed cluster containing both breast cancer cells and developing mammary epithelial cells (c2) and all remaining clusters. Benjamini-Hochberg adjusted p-value less than 0.05, pct.1 greater than 0.05, pct.2 greater than 0.007, and log2FC greater than 1 were used as thresholds for determining significant DEGs. This resulted in 1 ,543 significant DEGs. For the significant DEGs, mouse gene symbols were converted to human gene symbols using the Mouse Genome Informatics (MGI) mouse and human ortholog database (accessed from www.informatics.jax.org / downloads / reports / HOM_MouseHumanSequence.rpt), resulting in 1 ,397 unique human gene symbols. Volcano plots were generated using ggplot2 (v3.4.1) and gg repel (vO.9.1).Part 2 - Analysis of bulk RNA-seq human cell line and MetMap dataset

[0295] The inventors accessed bulk RNA-seq data from the Broad Cancer Cell Line Encyclopedia (CCLE) DepMap portal from depmap.org / portal using DepMap Public 24Q2. For human breast cancer cell lines, bulk RNA-seq data containing values of protein-coding genes were inferred using the RSEM tool and loaded into Seurat (v5.0.3) and normalized.

[0296] The metastasis map of human cancer cell lines (MetMap) was used to infer degree of liver colonization based on the liver metastatic potential of human breast cancer cell lines. Briefly, metastatic potential of human cancer cell lines in mice following injection were reported on a Iog10 scale (ranging from -4 to +4), as determined by the in vivo barcoding approach described in Jin, X., et al. (2020), A metastasis map of human cancer cell lines. Nature 588, 331-336. 10.1038 / s41586-020-2969-2, which is incorporated herein by reference in its entirety. The inventors refer to the original paper for details of the approach to calculate metastatic potential of human cancer cell lines in mouse xenograft models at scale. Human breast cancer cell lines were defined to have high versus low liver colonization using the following MetMap cohort-specific cutoffs for liver metastatic potential: +2.9 in the MetMap basal breast cancer cohort, and -3.2 in the MetMap500 cohort.

[0297] Using DESeq2 (v1.34.0), DEG analysis was performed on raw counts between cell lines with high versus liver metastatic potential. Benjamini-Hochberg adjusted p-value less than 0.05, and log2FC greater than 1 were used as the thresholds for determining significant DEGs. This resulted in 320 significant DEGs, with 309 unique human gene symbols. Volcano plots were generated using ggplot2 (v3.4.1) and ggrepel (vO.9.1).Part 3 - Analysis of human BCLM dataset

[0298] A published scRNA-seq dataset (Wang, X., et al., 2024) (GSE249361) consisting of samples of patient liver metastatic lesions and adjacent liver tissue following surgical resection was obtained. The inventors analyzed BCLM and adjacent normal liver samples from the 6 female breast cancer patients, with all subtypes represented (HR+ (n = 3), TNBC (n = 2), HER2+ (n = 1)). In accordance with the approach described by Wang et al., cells were filtered by number of features greater than 250 and number of UMIs greater than 500. After removal of low-quality cells, gene expression matrices were normalized using sctransform (vO.3.5). Removal of batch effects between samples was realized with integration in Seurat (v4.0.5), using sample site as the variable for integration. Louvain algorithm was applied to iteratively group proximal cells together by the FindClusters function with resolution of 0.5. Visualization was achieved using Uniform Manifold Approximation and Projection (UMAP). To identify major cell types, SingleR (v1.8.1) was applied, with the Human Primary Cell Atlas used as a reference. Specific marker genes for cell types were visualized to confirm major cell type labeling by SingleR (v1 .8.1 ). SingleR (v1.8.1 ) is described in Aran, D., et al. (2019), Referencebased analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage, Nature Immunology 20, 163-172. 10.1038 / s41590-018-0276-y, which is incorporated herein by reference in its entirety.

[0299] Copy number variant (CNV) profile analysis was used to determine cancer versus noncancer annotations of all epithelial cells in the dataset. To minimize batch effects between samples, inferCNV (v1.23.0) was run one tumor at a time, using the BCLM sample and adjacent normal liver tissue sample for each patient. For each tumor, CNV signal for individual epithelial cells was estimated with a 100-gene sliding window: genes with mean count less than 0.1 across all cells were filtered out, and the signal was denoised using a dynamic threshold of 1.3 SD from the mean. B cells, myeloid cells, and stromal cells from the adjacent normal liver sample were used for the reference cell profiles. Epithelial cells were classified as cancer (malignant) or non-cancer (non-malignant) using the method previously described by Neftel, C., et al. (2019), An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma. Cell 178, 835-849. e821 , which is incorporated herein by reference in its entirety. Briefly, inferred changes at each genomic locus were scaled (between -1 and +1) and the mean of the squares of these values was used to define CNV signal for each cell. For each tumor, an average CNV profile was created, and each epithelial cell within the tumor was then correlated to this profile to calculate the CNV correlation score. All epithelial cells were then classified as cancer versus non-cancer based on CNV signal and CNV correlation values, with thresholds of 0.02 for CNV signal and 0.4 for CNV correlation. This assigned 24,336 epithelial cells as cancer and 99 epithelial cells as non-cancer.

[0300] Using the FindMarkers method in Seurat (v5.0.3) and MAST (v1.20.0), DEG analysis was performed on raw counts between cancer epithelial cells in BCLM versus hepatocytes in adjacent normal liver. Benjamini-Hochberg adjusted p-value less than 0.05, pct.1 greater than 0.05, pct.2 greater than 0.007, and log2FC greater than 1 were used as the thresholds for determining significant DEGs. This resulted in 8,611 significant DEGs with unique human gene symbols. Volcano plots were generated using ggplot2 (v3.4.1) and ggrepel (vO.9.1).Overlap analysis of three-part comparative transcriptomics screen

[0301] Across the three-part screen, overlap was calculated with the VennDiagram (v1.7.3) package between the three significant DEG lists using unique human gene symbols. Results were visualized using ggVennDiagram (v1.5.3), with 8 common genes present across all DEG lists. For the 8 common genes, log2FC values for each dataset and mean log2FC value across all 3 datasets were plotted using ComplexHeatmap (v2.10.0). Methods using VennDiagram (v1 .7.3), ggVennDiagram (v1.5.3), and ComplexHeatmap (v2.10.0) were done as described in Chen, H., and Boutros, P.C. (2011), VennDiagram: a package for the generation of highly- customizable Venn and Euler diagrams in R. BMC Bioinformatics 12, 35. 10.1186 / 1471-2105- 12-35, Gao, C.H., et al., (2021), ggVennDiagram: An Intuitive, Easy-to-Use, and Highly Customizable R Package to Generate Venn Diagram. Front Genet 12, 706907. 10.3389 / fgene.2021.706907, and Gu, Z., et al., (2016), Complex heatmaps reveal patterns and correlations in multidimensional genomic data, Bioinformatics 32, 2847-2849. 10.1093 / bioinformatics / btw313, respectively.Survival analysis

[0302] The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) consists of clinically annotated primary fresh-frozen breast cancer specimens from patients diagnosed with non-metastatic breast cancer between 1977 and 2005 in the UK and Canada. Available gene expression data from microarrays for the subset of 1 ,980 patients (the METABRIC molecular dataset) and corresponding clinical data were accessed from www.cbioportal.org. To determine samples with high expression of a gene of interest, a z- score greater than +2 was set as threshold. To determine samples with low expression of a gene of interest, a z-score less than -2 was set as the threshold. For genes of interest with no samples with z-score less than -2 (i.e. , CXCL2, PSAT, SOAT1), samples with expression in the bottom 5th percentile were considered to have low expression of the gene of interest. Survival analysis and generation of Kaplan-Meier curve figures were done using the survival (v3.2.10) and survminer (vO.4.9) packages.In vitro and in vivo methodsCell lines

[0303] 4T-1 and H2.35 cell lines, both derived from a Balb / c background were purchased from ATCC (CRL-3539, CRL-1995). THLE2, MDA-MB-231 , MDA-MB-436 cell lines were also purchased from ATCC (CRL-2706, HTB-26, HTB-130). 603B cell line, derived from Balb / c background, was a gift from Dr. Anna Mae Diehl. MCF7 cell line was a gift from Dr. Carlos Arteaga. HCC1954 cell line was a gift from Dr. John Minna. 4T-1 , MDA-MB-436, and HCC1954 cell lines were maintained in RPMI media supplemented with 10% FBS and 1 % penicillin / streptomycin solution at 37°C, 5% CO2. MCF7 and MDA-MB-231 cell lines were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin solution at 37°C, 5% CO2. THLE2 cells were maintained with BEGM Bullet Kit (Lonza CC-1370) from which the inventors discarded Gentamycin / Amphotericin (GA) and Epinephrine and to which the inventors added extra 5 ng / mL EGF, 70 ng / mL Phosphoethanolamine, 10% FBS, and 1% penicillin / streptomycin solution at 37°C, 5% CO2. H2.35 cell line was maintained in DMEM supplemented with 4% FBS, 200nM Dexamethasone, and 1 % penicillin / streptomycin solution at 33°C, 10% CO2.Lentivirus generation

[0304] 4 million HEK293T cells were initially plated in a 10 cm tissue culture plate. Cells were transfected 24 hours later with 7.5ug lentiviral plasmid, 7.5ug helper plasmid (3:1 addgene #12260: #12259) and 45uL of the Mirus TransitLT reagent, in 1.5mL Opti-MEM™ (ThermoFisher #31985062). Media was replaced 24 hours after transfection by adding 10mL of fresh DMEM media supplemented with 10% FBS, 1 % PenStrep. Virus media was collected 48hrs later and stored at -80°C.Generation of lentiviral transduced cell lines

[0305] pLenti-PGK-mCherry and pLenti-PGK-eGFP were used to fluorescently label cell lines. pLenti-PGK-SerpinE2 was used to generate H235OEand 4T-1Rescell line. SerpinE2 sgRNA was cloned into a lentiCRISPRv2 backbone (Addgene, 52961) for generating SerpinE2 CRISPR knock-out cell line. Plasmid sequencing was confirmed through Plasmidsaurus. After lentivirus generation, 50% virus media diluted in complete media was added to cells with 8ug / mL polybrene. Media was replaced 24hrs after lentivirus transduction. Fluorescent lentiviral transduced cells were grown to at least 1x10A6 cells and FACS was used to sort cells into single cell clones after 2 weeks to generate stably transduced fluorescent cell lines. Cell lines were then grown to confluency from single cell clones. Non-fluorescent lentiviral transduced cells were selected using 5ug / mL puromycin for 48-72hrs and then grown toconfluency. Selected cells were then sorted into single cell clones and expanded. Knockout and overexpression clones were validated by qPCR and ELISA.RNA isolation, cDNA generation, and quantitative real-time PCR

[0306] For liver tissue samples mouse livers were flash frozen in liquid nitrogen homogenized using a tissue homogenizer / grinder. Homogenized tissue was then transferred into an Eppendorf tube containing 1mL TRIzol™ (Invitrogen 15596026). For RNA isolation from cells, approximately 1x106cells were pelleted and resuspended in 1 mL TRIzol. 10OuL of chloroform was added to the TRIzol suspension. Tubes were shaken vigorously to homogenize the samples and were centrifugated at 15000g for 15 mins, and the aqueous (clear) layer was extracted. Extracted samples were then processed for RNA isolation using the RNeasy Plus Mini Kit (Qiagen #74134). cDNA was generated using LunaScript RT Supermix (NEB # E3010S). SYBR Green Master Mix (Biorad #1725270) was used for the quantitative Real- Time (qPCR) reactions. qPCR was cycled and measured using the QuantStudio3 or QuantStudio6 (AppliedBiosystems) according to manufacturer recommendations using the primer sequences listed in T able 7. GAPDH was used as a housekeeping gene for all samples. Reactions were run in technical triplicate and analyzed using AACt method.SerpinE2 Enzyme-linked immunosorbent assay (ELISA)

[0307] 1x106cells were seeded on a 96 well plate in 100uL of complete growth media. Media was then collected 24 hours later. The inventors used a precoated mouse SerpinE2 ELISA kit for mouse cell lines (Mouse Protease Nexin 1 ELISA Kit, Innovative Research). For human cell lines the inventors used a precoated human SerpinE2 kit (Human Serpin E2 / PN1 ELISA Kit, Novus Biologicals).Animals

[0308] All mice were handled in accordance with the guidelines of the Institutional Animal Care and Use Committee at University of Texas Southwestern (UTSW). All experiments were done in an age-controlled fashion. NOD-scid Gamma (NSG) mice and Balb / c were obtained from The Jackson Laboratory.Hydrodynamic tail vein injection

[0309] 8-12-week-old mice were anesthetized with 2-3% isoflurane gas, 1 L / min O2. Mice were weighed both pre-and post-injection and kept on supplemental heat during the procedure. Hydrodynamic tail vein injections (HDTVIs) were accomplished by delivery of strict 8-10% volume of total mouse body weight in 5-8 secs. Anesthesia lines were immediately flushed of isoflurane and oxygen increased to 5L / min after successful injection, measured byvisual assessment of tail vein clearance and no resistance to injection. Aggressive stimulation was provided to mice post-injection until they regained consciousness. Mice were kept on supplemental heat 1-3hrs post-injection and monitored daily according to IACUC protocols.Orthotopic mammary fat pad transplant

[0310] 8-12-week-old mice were anesthetized with 2-3% isoflurane gas, 1 L / min O2. Mice were weighed both pre-and post-injection and kept on supplemental heat during the procedure. The abdominal area was shaved and cleaned 3x times using ethanol swabs and Betadine alternatively. Tweezer was used to pinch the area around the 4thnipple for easy access to mammary fat pad. 50uL of 1x107 / mL cell suspension was injected into the mammary fat pad. 1 mg / kg meloxicam was administered subcutaneously post injection. Mice were kept on supplemental heat until they regained consciousness.Tissue processing

[0311] Mice were euthanized according to the UT Southwestern Institutional Animal Care and Use Committee (IACUC) guidelines. All tissues collected were fixed in 4% PFA for 4-24 hrs. Tissues were moved to 10, 20, and 30% sucrose consecutively for 12-24hrs each. Tissues were then OCT embedded and the interior 50% of the frozen tissue block was sectioned at 10pm thickness. Two slides every -100 pm were sampled for immunofluorescence, slide scanning, and quantification.Transwell migration assay

[0312] Transwell migration assays were performed using 8.0 pm pore Polycarbonate Membrane inserts (Corning 3422). 1x104cells were seeded in the top chamber in serum-free media. The lower chamber was filled with RPMI containing 10% FBS. After incubation for 24hrs, migrated cells were fixed in 4% formaldehyde and stained with 0.1% crystal violet. Experiments were performed in triplicates, and the area of cell coverage was quantified using Imaged.Colony formation assay

[0313] 500 cells were plated in a 6 well plate in triplicates. After 72 hrs, cells were washed with PBS and stained with 6.0% glutaraldehyde and 0.5% crystal violet for 30 minutes. Plates were washed with tap water 3x times. Plates were then allowed to dry at room temperature overnight. Images were acquired using Leica DMi8 Inverted Phase Contrast Microscope and stitched. Image analysis of colony formation was done using Imaged.Proliferation assay

[0314] 5x104cells were plated in each well of Falcon® 8-well Culture Slide (Corning 354118).3 plates were used for a complete cohort to monitor proliferation over 72hrs. One plate was fixed every 24hrs and stained with DAPI. Slides were slide scanned and QuPath was used to quantify slides for further analysis.Metastatic competition assay

[0315] Breast cancer cells and hepatocyte were plated together in 1 :1 , 1 :2, or 1 :5 ratios in each well of a Falcon® 8-well Culture Slide. Cells were plated using 1 :1 ratio of complete media of breast cancer cells and hepatocyte media. For analysis of cell population, a cohort of slides were fixed every 24 hrs until 72 hrs. For end point analysis of cleaved caspase-3 (CC3) slides were fixed at 48 hrs and stained for CC3.Immunofluorescence staining

[0316] PFA fixed slides were washed twice with DPBS and permeabilized with 2% FBS, 0.5% Triton X-100 in DPBS for 10 mins at room temperature. Slides were subsequently blocked using 10% FBS, 0.2% Triton X-100 in DPBS for 30 mins at room temperature. Antibodies were diluted in 10% FBS, 0.2% Triton X-100 in DPBS. Primary antibodies were applied to the slide overnight at 4°C. After primary antibody incubation, slides were washed 3x times for 15 mins each in DPBS. Secondary antibodies and DAPI (1 :1000 dilution) were applied to the slide for 1 hr at room temperature. After secondary antibody incubation, slides were washed 3x times for 15 mins each in DPBS. Excess liquid was removed from the slide and mounted with Gold Antifade media (Cell Signaling, 9071 S). Slides were left at room temperature in the dark to cure for 24 hrs and then imaged. A Nikon SoRa Spinnng Disk Confocal microscope was used to take 100x images.Immunohistochemistry staining

[0317] OCT embedded tissue sections were given to the LITSW Tissue Management Shared Resource Core (TMSR) for hematoxylin and eosin (H&E) staining. All mouse or human H&E slides were then imaged and analyzed by a medical pathologist.Time-lapse imaging

[0318] Cells were plated in 1 :1 ratio on glass bottom dishes (MatTek) 24h prior to live imaging start time in 1 :1 4T-1 growth media and H2.35 growth media. Cells were placed in the Nikon Biostation CT incubation chamber, at 37 degrees C and 5% CO2. The Nikon A1 R confocal microscope was used to acquire GFP and mCherry channel images in parallel at 40x oil immersion continuously at every 15-30 min interval over one location for 36-48hrs of liveimaging. Imaged was used to generate a 2D time-lapse video by assembling images in TIFF format from the median z-plane and an xz projection of time-lapse imaging.Slide scanning

[0319] In-vivo and in-vitro slides were scanned using Axioscan7. The entire tissue section or well was captured according to a standardized protocol for either in-vitro culture or tissue section in order to make image quantification comparable between slides. Image captures included DAPI, mCherry, EGFP, and Alexa647 channels.QuPath quantification

[0320] Quantification was performed blinded to image and sample identities. Images of in vitro slides were loaded onto a project file in QuPath 0.4.0. Appropriate ROIs were selected such that as many cells as possible were included per well, excluding the well border. All image manipulation was carried out blinded to sample identities. The cell-detection protocol was run through QuPath by detecting cells via DAPI fluorescence with a detectable threshold of 50% intensity and applied to all images. All scans were visually verified for appropriate and reasonable detection of cell boundaries. Next, object classifier scripts were run with mCherry or GFP fluorescence such that each cell was marked with their appropriate fluorescent identity using fluorescence intensity as a threshold marker. The settings for this depended on the specific slide and sample identity. Delauney clustering was used to determine distance to nearest neighbors. Annotation measurements were then recorded for cells positive for Alexa 647 signal by adjusting detection threshold based on mean intensity for each slide. Only neighboring cancer cells and hepatocytes within a 30um distance were included in the final analysis.Cell adhesion assay

[0321] 96 well plate was coated with Fibronectin (Millipore Sigma #F4759) at 37°C for 1 hr. Plates were washed with washing buffer (0.1% BSA in RPMI media). The plate was then blocked in blocking buffer (0.5% BSA in RPMI media) at 37°C for 45 mins. The plate was washed with washing buffer and then left on ice. Cells were then counted to 4x105 / mL and 50uL of cells were added in each well. Cells were incubated at 37°C for 30 mins. The plate was then shaken vigorously at 2000 RPM for 15 secs to induce mechanical stress and then washed with washing buffer 3x times. Cells were fixed with 4% PFA for 15 mins at room temperature. Cells were then stained with 0.1% crystal violet. The plate was washed with water 2x times to get rid of extra crystal violet residue. Once the plate was dry, 1% SDS in water was added in each well and incubated for 30 mins. The plate was read at 550pm.A A V generation

[0322] PAAV.TBG.PI.eGFP.WPRE.bGH (AAV8-TBG-GFP), pAAV2 / 8, and pAdDeltaF6 were obtained (Addgene™ plasmid #105535; #112864; #112867). AAV8-TBG-GFP virus was produced using AAV-Pro 293T cells (Takara™ #632273). 5x106cells were plated in a 15 cm plate one day before transfection. For transfection of one 15cm dish, 10pg AAV8-TBG-GFP, 10pg pAAV2 / 8, and 20pg pAdDeltaF6 plasmids were mixed with 1 ml Opti-MEM™ medium in one eppendorf tube. The inventors then added 75 pL of TransIT-VirusGEN® Transfection Reagent (Mirus #6700) and DNA complex was left to incubate at room temperature for 30 minutes. Immediately prior to transfection, media was changed and 25mL of DMEM with 1 % FBS and 1 % PenStrep was added to cells. DNA complex solution was then added to cell culture plates. 48 hours after transfection, the cells were scraped off the dish and collected by centrifugation at 500g for 10 mins. The supernatant was disinfected and discarded in bleach. The collected cell pellets were lysed in 1.5ml lysis buffer (PBS supplemented with NaCI powder to final concentration of 200mM, and with CHAPS powder to final concentration of 0.5% (w / v)). The cell lysis suspension was then incubated in ice for 10 mins with intermittent vortexing every 2min, and then centrifuged at 20,000g for 10 mins at 4°C. The supernatant was then collected. The inventors then set up the gravity column for AAV purification with 0.5ml of AAV8-binding slurry beads (ThermoFisher #A30789), which was enough to purify AAV from up to eight 15cm dishes, loaded into an empty column (Bio-Rad #731-1550). Beads were tightly packed at the bottom and then washed with 5ml of wash buffer (PBS supplemented with NaCI powder to a final concentration of 500mM). Supernatant was then loaded onto the column. After all of the supernatant flowed through, the beads were washed 2x times with 10ml wash buffer. AAV was then eluted with 3ml of elution buffer (100mM glycine, 500mM NaCI in water, pH 2.5) and the eluate was neutralized with 0.12ml 1M Tris- HCI (pH 7.5-8.0). AAV was concentrated by centrifugation at 2000g for 5 mins at 4°C using an 100k Amicon Ultra Centrifugal Filter Unit (Millipore #UFC810024). The concentrated AAV was diluted with 4ml AAV dialysis buffer (PBS supplemented with powders to final concentrations of 212mM NaCI and 5% sorbitol (w / v)) and centrifuged at 2000g for 3 mins at 4°C. The dilution and centrifugation processes were repeated 3 times. The final concentrated AAV was transferred into a 1 ,5ml tube and stored at -80°C.

[0323] AAV8-SerpinE2 plasmid was generated by cloning full length SerpinE2 gene into AAV- TBG-GFP plasmid instead of GFP gene. pAAV.TBG.Pl.Null.bGH (AAV8-ctrl) was obtained (Addgene plasmid # 105536). AAV8-SerpinE2 and AAV8-ctrl virus were generated by the UTSW Translational Gene Therapy Core (TGTC).A A V mouse experiments

[0324] Mice were kept on supplemental heat during the procedure. Tail vein injections were performed by injecting ~ 5 .0 x 1010vector copies of AAV particles in 200 pL of Sterile Ringers solution. Mice were monitored daily to assess health status and body weights were recorded daily. 1 week after AAV administration mice were injected with 2.5x1054T-1 -luciferase tagged cells through HD-T.V.I. Tumor progression was tracked daily by bioluminescent imaging (BLI) using AMI-HTX. Mice were injected subcutaneously with luciferin-D (15mg / kg) and then imaged 2-5 after luciferin-D administration. Ex-vivo liver BLI imaging was performed as endpoint analysis by harvesting mice liver. BLI signal intensity was measured using Aura Spectral Viewer.RNA-seq experiment and analysisRNA sequencing

[0325] RNA was isolated as described above from 4T-1wrand 4T-1KOcells in replicates. Samples were run on the Agilent Tapestation 4200 to determine levels of degradation, thus ensuring only high-quality RNA was used (RIN Score 8 or higher). Qubit® 4.0 Fluorimeter from ThermoFisher™ was used to determine the concentration prior to starting library preparation. One microgram of total DNAse treated RNA was then prepared with the TruSeq Stranded mRNA Library Prep Kit from Illumina. Poly-A RNA was purified and fragmented before strandspecific cDNA synthesis. cDNA was then a-tailed, and indexed adapters were ligated. After adapter ligation, samples were PCR amplified and purified with AmpureXP beads, then validated again on the Agilent Tapestation 4200. Before being normalized and pooled, samples were quantified by Qubit then run on the Illumina NextSeq 2000 on a P2-100 flowcell. 150 bp paired-end reads were generated, yielding on average 35 M reads per sample. All library preparations and sequencing were performed by the McDermott Center NGS Core.Preprocessing, QC, and principal component analysis (PC A)

[0326] Fastq files were quality checked using fastqc (v0.11.2). Reads from each sample were aligned to the mouse reference genome mm10 (version GRCm38.p3, retrieved from GENCODE release M4 at gencodegenes.org / mouse / release_M4.html with annotation file gencode. vM4. annotation. gtf.gz) using STAR (v2.5.3a). Read counts were generated using featureCounts from Subread (v1.4.6). Initial processing, QC, and alignment were performed by the McDermott Center Bioinformatics Lab Core. Raw counts were normalized using variance stabilizing transformation (vst) in DESeq2 (v1.34.0). PCA was performed using the plotPCA function on vst normalized counts and plotted using ggplot2 (v3.4.1) and ggrepel (vO.9.1).Differential gene expression (DEG) analysis

[0327] Using DESeq2 (v1.34.0) and the Ifcshrink ageglm method, DEG analysis was performed on raw counts between 4T-1wrand 4T-1KOsamples. Prior to DESeq2 analysis, genes with low counts (defined as genes with less than 3 samples with counts greater than or equal to 10) were filtered out. This resulted in a lower number of genes for analysis by DESeq2, which reduced multiple testing correction burden and analysis time. Benjamini- Hochberg adjusted p-value less than 0.01 and log2FC greater than 1 or less than -1 were used as the thresholds for determining significant DEGs. Volcano plots were generated using ggplot2 (v3.4.1) and ggrepel (vO.9.1). This resulted in 1 ,730 significant DEGs. Significant DEG expression heatmaps were generated using DESeq2 normalized counts and pheatmap (vl .0.12).Gene set enrichment analysis (GSEA)

[0328] Gene set enrichment analysis (GSEA) of the significant DEGs was conducted using clusterProfiler (v4.2.2) separately for human and mouse gene expression datasets. The msigdbr (v7.5.1) package based on the MSigDB v7.5.1 release (>16,000 mouse gene signatures) was used. Canonical pathway gene sets from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were analyzed. The KEGG database is found in Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28, 27-30. 10.1093 / nar / 28.1.27, which is incorporated herein by reference in its entirety. The compareCluster function was applied to determine the top significant enrichment results, and results were visualized using a waterfall plot.Quantification of extrusion

[0329] Time lapse images of xy videos were resliced into xz orientation with outspacing microns ranging from 1.5-3 pm. Once xz videos were extracted, 3 locations per image were used to quantify (top, middle, and bottom) to achieve standard representation of events occurring in the entire image. Individual events were visually monitored, with cells going out of focus in the z direction counting as a positive extrusion event.Statistical Analysis

[0330] Statistical and graphical analysis was performed using GraphPad Prism software. Shapiro-Wilk test was used to determine if data was parametric or non-parametric. All data were presented as mean ± SD unless otherwise stated. For parametric data, differences between groups were evaluated by Welch’s t-tests. For non-parametric data sets, Mann- Whitney U test was used to determine differences between groups. Results were considered significant if p<0.05 (*=p<0.05, **=p<0.01 , ***=p<0.001).Example 16: Bioinformatics screen identifies SerpinE2 as a top gene candidate involved in metastatic competition during BCLM

[0331] Among hematoxylin and eosin-stained sections of patient BCLM samples, the inventors observed a significant increase in acidophil bodies at the tumor-hepatocyte interface compared to non-tumor adjacent hepatocytes (FIGS. 33A-33B). Acidophil bodies represent cells undergoing apoptosis. This observation suggested metastatic competition between hepatocytes and breast cancer cells is a potential mechanism governing the formation of BCLM.

[0332] To determine potential genes involved in metastatic competition between early colonizing human breast cancer cells and hepatocytes during BCLM, the inventors developed a computational strategy to perform a three-part comparative transcriptomics screen (FIG. 29A).

[0333] First, the inventors hypothesized there are shared mechanisms between mammary gland development and early breast cancer metastasis. Prior studies demonstrate that metastatic cancer cells share features of normal organ development. In the patient samples, the inventors observed that some BCLM retain cell-of-origin tubular features (FIG. 33C). Both embryonic mammary development and development of metastasis occur in well-characterized steps that mirror one another. Mammary gland development continues when a mammary bud seeds a fat pad on embryonic day 16 (E16) and then branches and grows on embryonic day 18 (E18). In early metastasis formation, cancer cells similarly seed a distant organ and then grow in that organ. Furthermore, cell competition plays a major role in branching morphogenesis, the fundamental process underlying mammary gland development. Thus, the inventors hypothesized that shared genes between breast cancer cells and developing mammary epithelial cells at E16 and E18 may lead to early metastasis formation by primary breast cancer cells. To identify these shared genes, the inventors integrated published singlecell RNA-seq (scRNA-seq) datasets of embryonic mouse mammary epithelial cells at E16 and E18 and mouse breast cancer epithelial cells from MMTV-Neu, MMTV-PyMT, and 4T-1 transplant tumor models (FIGS. 33D-33F). The scRNA-seq datasets are found in Giraddi, R.R., et al., (2018), Single-Cell Transcriptomes Distinguish Stem Cell State Changes and Lineage Specification Programs in Early Mammary Gland Development, Cell Rep 24, 1653- 1666, and Yeo, S.K., et al., (2020), Single-cell RNA-sequencing reveals distinct patterns of cell state heterogeneity in mouse models of breast cancer, Elife 9. 10.7554 / eLife.58810, which are incorporated herein by reference in their entireties. Within this integrated dataset, unsupervised clustering revealed one mixed cluster consisting of both developing mammary epithelia and breast cancer cells (FIG. 33G). The inventors identified differentially expressed genes (DEGs) enriched in this mixed cluster (FIG. 33H), which include genes previouslyassociated with metastasis such as Mmp9, Ibsp, Sic, Fscnl and SerpinE2. This analysis identified genes associated with early metastasis formation.

[0334] Second, the inventors sought to identify genes associated with liver metastatic colonization by human breast cancer cells. To do so, the inventors used the published MetMap dataset, which provides transcriptomic and experimental characterization of human pancancer cell lines in mouse xenograft models to reveal organ-specific patterns of metastasis (FIGS. 33I-33J). The MetMap dataset is found in Jin, X., et al. (2020), A metastasis map of human cancer cell lines, Nature 588, 331-336, which is incorporated herein by reference in its entirety. The inventors re-analyzed this dataset to include only human breast cancer cell lines and identified DEGs enriched in those with high versus low liver colonization, as defined by liver metastatic potential (FIG. 33K).

[0335] Third, the inventors wanted to identify genes associated with metastatic competition between hepatocytes and breast cancer cells in human BCLM. The inventors obtained a scRNA-seq dataset published of patient liver metastases following surgical resection. The inventors re-analyzed this dataset to include only BCLM and adjacent normal liver tissue samples from 6 female breast cancer patients (FIGS. 33L-33N). The inventors then identified differentially expressed genes (DEGs) enriched in breast cancer cells in BCLM compared to hepatocytes in adjacent normal liver (FIG. 330).

[0336] Finally, the inventors assessed overlapping genes of these three orthogonal comparative transcriptomics analyses. This three-part screen identified 8 genes present across all three DEG lists: SERPINE2, CXCL2, FSCN1, MAP3K20, SLC4A7, PSAT1, SOAT1, and PPP1R18 (FIG. 29B). Among these, SERPINE2 was most consistently upregulated by mean Iog2-fold change (log2FC) of 2.65 (FIG. 29C), making it the highest ranked gene candidate involved in metastatic competition during BCLM. Out of all 8 gene candidates, only elevated SERPINE2 expression is associated with worse 10-year overall survival (OS) and relapse-free survival (RFS) in the analysis of the METABRIC breast cancer cohort (FIGS. 34A- 34H). Interestingly, differences in SERPINE2 are not significantly associated with differences in tumor stage (FIG. 34I)Example 17: Loss of SerpinE2 in breast cancer cells reduces liver metastases but does not alter cancer cell intrinsic features

[0337] SerpinE2 is a secreted protein that belongs to a large family of serine protease inhibitors and is involved in lung metastasis across different cancers, including breast cancer. To study whether SerpinE2 expression in breast cancer cells correlates with their ability to form liver metastases, the inventors used a CRISPR-Cas9 system to knock-out (KO) SerpinE2 in 4T-1 cells. Complete KO of SerpinE2 in 4T-1KOcells was validated by quantifying mRNAexpression (FIG. 34J) and secreted SerpinE2 levels relative to parental 4T-1WT(FIG. 34K). The inventors first directly engrafted livers of NOD-SCID-gamma (NSG) mice with 4T-1WTor 4T-1KOcells using hydrodynamic tail vein injection (HD-TVI) and assessed for the number of liver metastases. Mice injected with 4T-1KOcells showed a 4.98-fold decrease in liver metastases burden (FIG. 29D). Representative H&E-stained images of mouse liver show that 4T-1KOinjected mice had fewer and smaller liver metastases relative to 4T-1wrinjected mice (FIG. 29E). The inventors then assessed whether primary tumor growth is affected by SerpinE2 loss in cancer cells. The inventors implanted mammary fat pad of NOD-scid-gamma (NSG) mice with 4T-1wror 4T-1KOcells and monitored tumor formation. Over 26 days, no significant difference in tumor size and growth rate was observed between 4T-1WTand 4T-1KOinjected mice (FIG. 29F). These observations to the conclusion that SerpinE2 expression does not affect primary tumor growth, but it is crucial for the formation of liver metastases.

[0338] Next, the inventors tested whether cancer cell intrinsic properties that govern metastasis were altered in 4T-1KOcells, to explain in vivo differences in liver metastases. In a Transwell migration assay which models invasion, intravasation, and extravasation, the inventors observe no significant difference between 4T-1KOand 4T-1WTcells (FIGS. 29G-29H). Since metastatic colonization is an essential step to form metastases in distant organs, the inventors tested whether loss of SerpinE2 decreases colony formation ability. The inventors observe no significant difference in colony formation ability between 4T-1KOand 4T-1wrcells (FIGS. 29I-29J). The inventors further assessed changes in proliferation rate defined by counting cell nuclei over time. Over 72 hours, the inventors observed no significant difference in proliferation between 4T-1KOand 4T-1wrcells (FIG 29K). In summary, loss of SerpinE2 did not result in cancer cell intrinsic differences across the metastatic cascade that could impact liver metastasis formation. This led us to hypothesize SerpinE2 mediates other cancer cell extrinsic interactions between breast cancer cells and hepatocytes during BCLM formation.Example 18: Metastatic competition occurs between breast cancer cells and hepatocytes

[0339] Given these clinical observations, the inventors tested whether these interactions between breast cancer cells and hepatocytes result in metastatic competition. The inventors first developed a metastatic competition assay using lineage traced mCherry+ breast cancer cells and GFP+ hepatocytes co-cultured at equal ratios (FIG. 30A). The inventors first used 4T-1WTcells and H2.35wrcells, a well-characterized mouse hepatocyte cell line model, to test for metastatic competition. After 72 hours of co-culture, a higher population of 4T-1WTcells were found in co-cultures than H2.35WTcells (FIG. 30B). No differences in proliferation in monoculture were observed between 4T-1wrand H2.35WTcells (FIG. 35A), nor were any differences observed in total surface area occupied by 4T-1WTand H2.35wrcells grown inmonoculture over 7 days (FIG. 35B). These findings excluded differences in proliferation and cell size as an explanation for the observed co-culture results.

[0340] Through time-lapse imaging of co-cultures, it was observed that hepatocytes neighboring cancer cells underwent blebbing and cell fragmentation, suggesting that cells were undergoing apoptosis. Therefore, it was hypothesized that breast cancer cells compete for space by eliminating neighboring hepatocytes through apoptosis, a common mode of cell competition. To test this hypothesis, the co-cultures of 4T-1wrand H2.35WTwere stained for cleaved caspase-3 (CC3). A computational analysis pipeline identifying neighboring cancer cells and hepatocytes was used to detect CC3 expression within these cells. A higher percentage of CC3+ H2.35WTcells compared to 4T-1wrcells were detected in co-cultures (FIGS. 30C-30D). In contrast, no significant difference was detected in percentage of CC3+ cells between 4T-1WTand H2.35wrcells in monocultures (FIG. 35C). Because there is a higher density of hepatocytes to cancer cells during BCLM, it was tested whether hepatocyte density affects competition. The inventors plated co-cultures with higher ratios of hepatocytes to breast cancer cells. In co-culture with 1 :2 and 1 :5 ratios of 4T-1wrto H2.35wrcells, a higher percentage of CC3+ H2.35wrcells compared to 4T-1wrcells was observed (FIG. 35D). These observations parallel the trend seen in 1 : 1 co-culture and suggest breast cancer cells compete with hepatocytes independent of cancer cell or hepatocyte density. Next, the inventors treated monocultures of 4T-1WTcells and H2.35WTcells with conditioned media (CM) derived from the other cell type and observe no difference between percentage of CC3+ 4T-1WTand H2.35wrcells (FIG. 35E). Collectively, these results indicate that metastatic competition leading to increased apoptosis in neighboring hepatocytes requires direct interactions with breast cancer cells.Example 19: SerpinE2 mediates metastatic competition between breast cancer cells and hepatocytes

[0341] To test the role of SerpinE2 in metastatic competition between breast cancer cells and hepatocytes, the inventors co-cultured 4T-1KOand H2.35wrcells. By 72 hours, a higher percentage of H2.35WTcells compared to 4T-1KOcells was observed (FIG. 30E). Further, 4T- 1KOneighboring H2.35WTcells underwent blebbing and cell fragmentation and there was an observed increase in the percentage of CC3+ 4T-1KOcells compared to H2.35wrcells (FIG. 30F-30G). These observations contrast co-cultures with 4T-1WTcells. The inventors also measured sensitivity of 4T-1KOand 4T-1wrcells to clinically used chemotherapies and observed no difference between the two conditions (FIG. 35F). This indicates that loss of SerpinE2 did not increase susceptibility to cell-intrinsic apoptotic deficits. Next, the inventors sought to restore SerpinE2 expression in 4T-1KOcells by overexpressing SerpinE2 in 4T-1KOcells (4T-1Res). These SerpinE2 cells were then co-cultured with H2.35WTcells resulting in ahigher percentage of CC3+ H2.35WTcells in co-cultures with 4T-1Rescells, similar to co-cultures with 4T-1vn’ cells (FIG. 30H). Restoring SerpinE2 levels in 4T-1Rescells rescued cancer cells from undergoing apoptosis during metastatic competition, while increasing hepatocyte apoptosis activity. Similar to previous monocultures, no differences were observed between percentage of CC3+ 4T-1KO, 4T-1Res, and H2.35WTcells (FIG. 30I).

[0342] To test whether competition was specific to hepatocytes or liver epithelial cells in general, the inventors co-cultured 603B cells, a cholangiocyte cell line, with 4T-1WTcells. In contrast to hepatocytes, there was a lower percentage of CC3+ cholangiocytes than 4T-1wrcells (FIG. 35G). To further understand these differences with hepatocytes, the inventors assessed SerpinE2 expression. 603B cells secrete SerpinE2 at similar concentrations as 4T- 1wrcells, whereas H2.35wrcells have no detectable expression of SerpinE2 (FIG. 35H). These observations suggest cancer cells create space by specifically inducing hepatocyte apoptosis through SerpinE2-mediated metastatic competition.

[0343] Next, whether SerpinE2 mediates cancer cell-hepatocyte competition in human models was tested by characterizing SerpinE2 expression in a panel of four human breast cancer cell lines. Among these cell lines, HCC1954 secreted the highest concentration of SerpinE2, followed by MDA-MB-231 , and MCF7, while MDA-MB-436 secreted negligible concentrations of SerpinE2 (FIG. 30J). These human breast cancer cell lines were then cocultured at equal ratios with THLE2, a human hepatocyte cell line. By 48 hours, HCC1954 had the highest population of cells in co-culture with THLE2 cells, followed by MD-MB-231. In contrast, MDA-MB-436 cells had the lowest population of cells in co-culture with THLE2, followed by MCF7 (FIG. 30K). These observations showed an association between levels of SerpinE2 secreted by cancer cells and the population of cancer cells in co-cultures with THLE2. These cross-species findings in mouse and human models support the role of SerpinE2-mediated metastatic competition between breast cancer cells and hepatocytes.Example 20: Loss of SerpinE2 increases susceptibility to extrusion by disrupting focal adhesions in breast cancer cells

[0344] To understand the mechanism by which SerpinE2 loss results in reduced cancer cell expansion during competition with hepatocytes, the inventors further probed interactions using time-lapse imaging. The inventors captured cancer cells and hepatocyte movement in the z- direction by projecting an xz plane of the co-cultures. During co-cultures of 4T-1WTand H2.35WTcells, the inventors observe that mainly hepatocytes and few cancer cells are extruded (FIG. 31 A). However, in co-cultures with 4T-1KOand H2.35wrcells the inventors observed that many more cancer cells are extruded (FIG. 31 B). The inventors assessed the frequency of cancer cell extrusion in co-cultures with hepatocytes and observed significantlyhigher extrusion events of 4T-1KOcells compared to 4T-1WTcells (FIG. 31C). The inventors then performed a cell adhesion assay which subjects cells to mechanical stress and demonstrated that 4T-1KOcells have decreased attachment compared to 4T-1WTcells (FIG. 31 D). These findings indicate that 4T-1KOcells are more susceptible to extrusion due to reduced adhesive properties.

[0345] To understand which cell intrinsic factors may increase susceptibility of 4T-1KOcells to extrusion, the inventors performed bulk RNA-sequencing (RNA-seq) of 4T-1wrand 4T-1KOcells and investigated transcriptomic differences associated with loss of SerpinE2. Principal component analysis (PCA) showed principal component 1 (PC1) stratifies the 6 samples based on loss of SerpinE2 (FIG. 31E). Next, DEGs between 4T-1WTand 4T-1KOcells were identified (FIG. 31 F), and gene set enrichment analysis (GSEA) was performed to determine KEGG canonical pathways associated with differences between the 4T-1wrand 4T-1KOphenotypes. The inventors observe that focal adhesion, actin cytoskeleton regulation, and ECM receptor interaction pathways are significantly enriched and are likely disrupted by loss of SerpinE2 (FIG. 31G).

[0346] Given the observations, the inventors hypothesized that loss of SerpinE2 alters metastatic competition through disruption of focal adhesions. To test this, the inventors assessed differences in key proteins involved in the structure of and signaling within focal adhesions. The inventors observed that transcriptomic expression of paxillin (Pxn), vinculin (Vcl), and tensin 2 (Tns2) is decreased in 4T-1KOcells compared to 4T-1WTcells (FIG. 31 H), which supports the GSEA findings. Additionally, staining of these focal adhesion markers reveals decreased and disorganized expression in 4T-1KOcells (FIG. 311). These findings establish loss of SerpinE2 disrupts focal adhesions in cancer cells, causing them to be extruded by hepatocytes in co-cultures.Example 21 : Overexpressing SerpinE2 in hepatocytes reverses metastatic competition and reduces liver metastases

[0347] To test the sufficiency of SerpinE2 in metastatic competition between cancer cells and hepatocytes, the inventors overexpressed SerpinE2 in H2.35 cells (H2.35OE). Both mRNA and secreted SerpinE2 expression were higher in H2.35OEcompared to both parental H2.35wrand 4T-1WTcells (FIG. 36A-36B). In co-cultures of H2.35OEcells with 4T-1mcells, CC3 localization was observed in cancer cells neighboring hepatocytes (FIG. 32A). A higher percentage of CC3+ 4T-1WTcells was detected in co-culture with H2.35OEcells compared to co-cultures with H2.35WTcells (FIG. 32B). A lower percentage of CC3+ H2.35 cells was observed in co-cultures of 4T-1WTand H2.35wrcells (FIG. 32C). However, no difference in percentage of CC3+ cells was observed between monoculture of H2.35OEand H2.35WTcells (FIG. 36C).

[0348] These in vitro observations led to the hypothesis that altering SerpinE2 expression in hepatocytes in vivo can lead to a decrease in BCLM. To test this hypothesis, the inventors developed an adeno-associated virus serotype 8 (AAV8) with a TBG promoter to deliver SerpinE2 to mouse hepatocytes in NSG mice. The inventors chose NSG mice as the experimental model to eliminate confounding effects from an immune response.

[0349] To first determine whether this approach efficiently delivers SerpinE2 to hepatocytes, the inventors administered NSG mice with AAV8-TBG-GFP (FIG. 36D) and observed efficient and high GFP expression in mouse liver with no off-target specificity in the lungs or brain (FIG. 36E). The inventors then administered AAV8-Ctrl (FIG. 36F) to a control cohort and AAV8- SerpinE2 (FIG. 36G) to an experimental cohort. Mice administered with AAV8-SerpinE2 showed higher liver SerpinE2 mRNA expression (FIG. 36H) and elevated serum SerpinE2 levels relative to mice administered with AAV8-Ctrl (FIG. 361). To determine the safety of the approach, the inventors monitored whether AAV8-SerpinE2 administration could lead to liver toxicity through changes in aspartate aminotransferase (AST), and alanine transaminase (ALT) levels post AAV8-SerpinE2 administration. No significant change in AST and ALT levels was detected 7 days post AAV8 administration relative to baseline levels (FIGS. 36J-36K). No changes in body weight were observed over 7 days post AAV8 administration (FIG. 36L).

[0350] Next, the inventors administered AAV8-SerpinE2 (SEQ ID NO: 7) to an experimental cohort and AAV8-ctrl to a control cohort, where each cohort was injected with luciferase tagged 4T-1 cells either before or after AAV administration.

[0351] In a first set of experimental and control cohorts, one week post AAV8 administration, mice were injected with luciferase tagged 4T-1 cells and tumor progression was monitored through bioluminescent imaging (BLI) (FIG. 32D). The inventors observed a significant decrease in whole body BLI signal in treatment cohort compared to control cohort at Day 7 (FIG. 32E). Additionally, the inventors observe an overall decrease in tumor progression over the course of 7 days (FIG. 32F). Ex vivo imaging of livers harvested at 7 days showed an overall 7.12-fold decrease in BLI signal in treatment cohort relative to control cohort (FIGS. 32G-32H).

[0352] In a second set of experimental and control cohorts, 5 days before AAV administration, mice were injected with luciferase tagged 4T-1 cells and tumor progression was monitored through bioluminescent imaging (BLI) (FIG. 37A). The inventors observed a significant decrease in whole body BLI signal in the treatment cohort compared to control cohort at day 11 (FIG. 37B). Additionally, ex-vivo imaging of livers harvested 7 days after administering the AAV vectors showed a decrease in BLI signal in the treatment cohort relative to control cohort (FIG. 37C).

[0353] Together, these results suggest that hepatocyte specific gene delivery of SerpinE2 reduces BCLM in vivo and the AAV vectors can have a therapeutic effect whether they are delivered before or after the cancer is established.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a metastatic tumor in a subject, the method comprising administering a therapeutic agent that modulates activity of one or more cellular factors in one or more host cells and / or one or more metastasizing cancer cells, wherein the one or more host cells are located in an organ or tissue, the organ or tissue comprising a site of metastasis and / or the one or more metastasizing cancer cells, wherein the one or more cellular factors are selected from a serine protease inhibitor, C-X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20, solute carrier family 4 member 7, phosphoserine aminotransferase 1, sterol O-acyltransferase 1 , protein phosphatase 1 regulatory subunit 18, Matrix Gia protein, Parathyroid hormone-related protein, Pleiotrophin, Midkine, Secreted frizzled-related protein1. Aldehyde dehydrogenase family 1 member A3, Nucleolin, Sclerostin domain-containing protein 1, Histone-lysine N-methyltransferase EZH2, Insulin-like growth factor-binding protein 2, Transient receptor potential cation channel subfamily M member 2, Galectin-7, or any combination thereof.

2. The method of claim 1 , wherein the one or more cellular factors comprise the serine protease inhibitor (Serpin), Matrix Gia protein, Parathyroid hormone-related protein, C-X-C motif chemokine ligand 2, fascin actin-bundling protein 1 , mitogen-activated protein kinase kinase kinase 20, solute carrier family 4 member 7, phosphoserine aminotransferase 1, or any combination thereof.

3. The method of claim 1 or claim 2, wherein the one or more cellular factors comprise at least one serine protease inhibitor (Serpin) selected from SerpinE2, SerpinEI, SerpinE3, SerpinAI, SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAIO, SerpinA11, SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinB11, SerpinB12, SerpinB13, SerpinCI, SerpinDI, SerpinFI, SerpinF2, SerpinGI , SerpinHI, Serpinll, and Serpinl2.

4. The method of claim 3, wherein the one or more cellular factors comprise SerpinE2.

5. The method of any one of claims 1 to 4, wherein the therapeutic agent increases activity and / or expression levels of the cellular factor in the one or more host cells.

6. The method of claim 5, wherein the therapeutic agent comprises a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a gene therapy vector, a nanoparticle, a liposome, a polysaccharide or any combination thereof.

7. The method of claim 6, wherein the therapeutic agent comprises a gene therapy vector.

8. The method of claim 7, wherein the gene therapy vector comprises an expression construct encoding the one or more cellular factors.

9. The method of claim 8, wherein the gene therapy comprises an expression construct of any one of claims 21 to 31 and / or the vector of any one of claims 32 to 40.

10. The method of any one of claims 1 to 4, wherein the therapeutic agent decreases activity and / or expression levels of the cellular factor in the one or more metastatic cancer cells.

11. The method of claim 10, wherein the therapeutic agent comprises a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a gene therapy vector, a nanoparticle, a liposome, a polysaccharide or any combination thereof.

12. The method of claim 11, wherein the therapeutic agent comprises a small molecule inhibitor of the cellular factor or an interfering nucleic acid that reduces expression of the cellular factor.

13. The method of claim 12, wherein the therapeutic agent comprises an interfering nucleic acid that specifically targets a 10-50 nucleotide sequence region on a sequence as set forth in SEQ ID NO: 2, or a sequence at least about 60% identical thereto.

14. The method of any one of claims 1 to 13, wherein the metastatic tumor comprises a breast tumor, a liver tumor, a pancreatic tumor, squamous cell tumor, prostate tumor, melanoma, skin tumor, urothelial tumor, kidney tumor, colon tumor, gastric tumor, gastrointestinal tumor, a lung tumor, melanoma, a sarcoma, an adnexal tumor, or a tumor of a peritoneum.

15. The method of any one of claims 1 to 14, wherein the metastatic tumor comprises a breast cancer liver metastasis (BCLM).

16. The method of any one of claims 1 to 15, wherein the organ or tissue comprising the site of metastasis is selected from liver, bone, lungs, brain, and adrenal glands.

17. The method of any one of claims 1 to 16, wherein the one or more metastatic cancer cells comprise metastatic breast cancer cells.

18. The method of any one of claims 1 to 17, wherein the one or more host cells comprise hepatocytes.

19. The method of any one of claims 1 to 18, further comprising reducing or preventing metastasis in the subject.

20. The method of any one of claims 1 to 19, wherein the subject is human.

21. An expression construct comprising a nucleic acid, the nucleic acid comprising a nucleic acid sequence encoding for a serine protease inhibitor operably linked to a promoter sequence.

22. The expression construct of claim 21, wherein the serine protease inhibitor comprises SerpinE2, SerpinEI, SerpinE3, SerpinAI, SerpinA2, SerpinA3, SerpinA4, SerpinA5, SerpinA6, SerpinA7, SerpinA8, SerpinA9, SerpinAIO, SerpinA11, SerpinA12, SerpinBI , SerpinB2, SerpinB3, SerpinB4, SerpinB5, SerpinB6, SerpinB7, SerpinB8, SerpinB9, SerpinBIO, SerpinBH, SerpinB12, SerpinB13, SerpinCI , SerpinDI, SerpinFI , SerpinF2, SerpinGI , SerpinHI, Serpinll, or Serpinl2.

23. The expression construct of claim 22, wherein the serine protease inhibitor comprises SerpinE2.

24. The expression construct of claim 23, wherein the nucleic acid sequence encoding for the serine protease inhibitor has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

25. The expression construct of any one of claims 21 to 24, wherein the promoter is a constitutive promoter or an inducible promoter.

26. The expression construct of any one of claims 21 to 25, wherein the promoter is a tissue specific promoter.

27. The expression construct of claim 26, wherein the tissue specific promoter is a liver specific promoter.

28. The expression construct of claim 27, wherein the liver specific promoter is a TBG promoter or an hAAT promoter.

29. The expression construct of claim 28, wherein the promoter comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence to any one of SEQ ID NOs: 3 to 6.

30. The expression construct of any one of claims 21 to 29, wherein the expression construct is optimized for expression in mammalian cells.

31. The expression construct of claim 29, wherein the expression construct is optimized for expression in human cells, mouse cells, rat cells, canine cells, non-human primate cells, or bovine cells.

32. A vector comprising the expression construct of any one of claims 21 to 31, wherein the vector is formulated for delivery to a cell, optionally wherein the promoter is heterologous to the cell.

33. The vector of claim 32, wherein the vector comprises a viral vector.

34. The vector of claim 33, wherein the viral vector comprises a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a poxviral vector, or a herpes viral vector.

35. The vector of claim 34, wherein the viral vector comprises an adeno-associated viral (AAV) vector, wherein the AAV vector is serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), or any combination thereof.

36. The vector of claim 34 or claim 35, wherein the AAV vector is an organ specific AAV vector.

37. The vector of any one of claims 34 to 36, wherein the vector is a recombinant adeno- associated viral (AAV) vector comprising: a) a first AAV ITR sequence; b) the promoter sequence; c) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises the nucleic acid sequence encoding the serine protease inhibitor; d) a polyA sequence; ande) a second AAV ITR sequence.

38. The vector of claim 37, wherein the AAV vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7 or 8.

39. The vector of claim 32, wherein the vector comprises a non-viral vector.

40. The vector of claim 39, wherein the non-viral vector comprises a lipid nanoparticle (LNP) or an antibody-oligo conjugate.

41. A cell comprising the expression construct of any one of claims 21 to 31 or the vector of any one of claims 30 to 38.

42. The cell of claim 41, wherein the cell is a mammalian cell.

43. The cell of claim 42, wherein the cell is a human cell, a mouse cell, a rat cell, a canine cell, a non-human primate cell, or a bovine cell.

44. The cell of any one of claims 41 to 43, wherein the cell is a hepatocyte or other host organ-specific non-cancerous cells.

45. The cell of any one of claims 41 to 43, wherein the cell is an induced pluripotent stem cell (iPSC).

46. A composition comprising (a) an expression construct of any one of claims 21 to 31 , a vector of any one of claims 30 to 38, or a cell of any one of claims 41 to 45 and (b) and at least one pharmaceutically appropriate carrier or excipient.

47. A kit comprising (a) an expression construct of any one of claims 21 to 31 , a vector of any one of claims 30 to 38, a cell of any one of claims 41 to 45, a composition of claim 46, or any combination thereof and (b) at least one container.

48. A method of identifying one or more cellular factors that increases competitiveness of a metastasizing tumor cell against host cells in an organ or tissue comprising a site of metastasis, the method comprising:(a) obtaining or having obtained:(i) a first gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing tumor cell and having an elevated organ-specific metastatic potential relative to other tumor cells of the same tumor type;(ii) a second gene expression dataset of one or more tumor cells of the same tumor type as the metastasizing cancer cell;(iii) a third gene expression dataset of one or more developing cells (e.g., embryonic cells) in an organ of origination of the metastasizing tumor cell;(iv) a fourth gene expression dataset of the metastasizing tumor cell or one or more metastasizing tumor cells of the same tumor type as the metastasizing cancer cell that have metastasized to the same target organ; and(v) a fifth gene expression dataset of one or more host cells in the organ or tissue comprising the site of metastasis;(b) identifying one or more gene(s) that:(i) have elevated expression in the first expression dataset; and(ii) have elevated expression in both the second gene expression dataset and third gene expression dataset; and(iii) have higher expression in the fourth gene expression dataset relative to the fifth gene expression dataset; and(c) identifying one or more protein product(s) encoded by the one or more gene(s) of (b) as the one or more cellular factors that increases competitiveness of the metastasizing tumor cells.

49. A method of treating a cancer metastasis in a subject in need thereof, the method comprising identifying at least one cellular factor that increases competitiveness of metastasizing cells in the cancer metastasis according to the method of claim 48 and administering at least one therapeutic agent that modulates activity of the cellular factor in a cell to the subject.

50. The method of claim 49, wherein the therapeutic agent decreases activity of the cellular factor in at least one metastatic cancer cell in the subject.

51. The method of claim 50, wherein the therapeutic agent comprises a gene therapy vector, a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a nanoparticle, a liposome, a polysaccharide or any combination thereof.

52. The method of claim 50, wherein the therapeutic agent comprises a small molecule inhibitor of the cellular factor or an interfering nucleic acid that reduces expression of the cellular factor.

53. The method of claim 49, wherein the therapeutic agent increases activity and / or expression levels of the cellular factor in one or more host cells in the organ or tissue comprising the site of metastasis.

54. The method of claim 53, wherein the therapeutic agent comprises a gene therapy vector, a small molecule, a protein, an antibody, a therapeutic peptide, an oligonucleotide, a nanoparticle, a liposome, a polysaccharide or any combination thereof.

55. The method of claim 54, wherein the therapeutic agent comprises a gene therapy vector encoding for the cellular factor.

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