A platform for targeted nuclear delivery of therapeutic proteins

Abstract

This application describes a novel approach to precise transcriptional control of disease-associated genes using chimeric proteins. Specifically, this disclosure provides chimeric proteins comprising a nuclear-trafficking carrier and a transcription-controlling effector, pharmaceutical composition comprising the chimeric proteins, use of the chimeric proteins and pharmaceutical compositions thereof for treatment of diseases. The novel chimeric proteins offer therapeutic potential across a broad spectrum of diseases characterized by irregularities in gene expression, especially various cancers, achieved through precise transcriptional control.

Description

[0001]Docket No.: 374819.00003 A PLATFORM FOR TARGETED NUCLEAR DELIVERY OF THERAPEUTIC PROTEINS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 554,315, filed on February 16, 2024, which is incorporated herein by reference in its entirety. FIELD OF THE DISCLOSURE The present disclosure relates to a novel platform for targeted nuclear delivery of therapeutic proteins, and methods thereof. BACKGROUND OF THE DISCLOSURE Transcription factors are a highly diverse group of proteins characterized by their DNA binding domains, which attach to specific gene promoter or enhancer regions. They play vital roles in regulating cellular gene expression, thereby governing various cellular processes and phenotypes. Additionally, given their substantial influence in driving cellular transformation, mutations and deregulation of transcription factors contribute to numerous pathological conditions, including cancer (Hanahan, D., & Weinberg, R. A. Cell, 2011.153(6), 1111-1123; Vaquerizas, J. M. et al, Cell, 2009. 139(1), 4-6. Weinberg, R. A. Cell, 2013. 153(6), 1111- 1123). Consequently, targeting transcription factors has been a longstanding pursuit in drug discovery. Despite their attractiveness as therapeutic targets in diseases like cancer, metabolic disorders, and autoimmune conditions, the endeavor to develop drugs targeting transcription factors has proven exceptionally challenging (Lambert M. et al., Molecules, 2018.23(6):1479). There are several reasons for this. Unlike enzymes, which have well-defined active sites, transcription factors often lack easily targetable pockets or binding sites. Their interaction with DNA is typically extensive and relatively flat, making the identification of suitable binding sites for small molecule drugs difficult (Citron, M., & Tjian, R. (2022). Nature Reviews Drug Discovery, 21(12), 1062-1081). Furthermore, many transcription factors exert their function through interactions with other proteins rather than direct DNA binding. Targeting these protein-protein interactions (PPIs) with small molecules is complicated because the interface between two interacting Docket No.: 374819.00003 proteins can be extensive, involving numerous amino acids. This complexity makes designing a small molecule that binds to the interface with both high affinity and specificity a formidable challenge. Moreover, PPIs are often dynamic and transient, meaning that the interaction between two proteins can be weak and fleeting, making it difficult to find a small molecule capable of disrupting the interaction (Zhang S. et al., Biophys Rev., 2018.11(4): 559–581). Additionally, transcription factors exert their regulatory function by binding to specific DNA sequences of genes, which are localized in the nuclear compartment of cells. The lack of effective and selective delivery systems for large biological molecules to traverse cell membranes and reach the nucleus has hindered the development of targeted therapies for precise transcriptional control. Despite current methods such as cell-penetrating peptides (CPPs), viruses, nanoparticles, and liposomes showing promise in facilitating nuclear sub-cellular delivery of polypeptides into cells, they have their limitations (Meunier A, et al., Mol Ther., 2007. 15(4):687-697; Wilson AA. et al., Mol. Ther., 2013. (4):825-33; Farkhani S. et al., Peptides, 2014.57:78-94). One notable weakness is their lack of specificity in targeting particular cells and tissues, resulting in indiscriminate effects on both target and non-target cells. This not only leads to side effects and potential toxicity but also diminishes therapeutic efficacy. With the rise of precision and personalized medicine, there is promise for enhanced treatment outcomes in cancer therapy. Recent strides in targeted therapies have led to the approval of numerous drugs tailored to address specific molecular irregularities fueling cancer proliferation. Despite these achievements, there remain substantial hurdles in effectively targeting the diverse array of transcription factors pivotal in the development and progression of cancer. A significant challenge in targeting these genes lies in the constraints of drug delivery technology. Traditional systems frequently fall short in efficiently delivering therapeutic agents to the nuclear compartment, where many vital genes are under the regulation of transcription factors. SUMMARY OF THE DISCLOSURE The present disclosure provides an effective solution to surmount the abovementioned obstacle. It introduces a novel construct that integrates a carrier domain for internalization and nuclear transport, along with an effector domain that demonstrates a strong binding affinity to specific DNA sequences, including those encoding transcription factors, allowing for their transcriptional control. Docket No.: 374819.00003 The present disclosure pertains to an innovative design of chimeric proteins tailored for precise transcription modulation of specific genes. These novel chimeras encompass the following distinctive attributes: Carrier Domain: The chimeric proteins are endowed with a specialized carrier domain, acting as a molecular vehicle for ferrying the attached effector domain to the target cells. This carrier domain, meticulously chosen from secreted factors outlined in the provided List, facilitates uptake and intracellular trafficking to the cell nuclei. This ensures the effective delivery of the effector domain, precisely targeting the gene of interest. Effector Domain: The effector domains consist of polypeptides proficient in binding to specific DNA sequences, thereby exerting transcriptional control over particular genes. These effectors can be further equipped with short peptides, transcription activators, repressors, or custom-designed zinc finger transcription factor motifs, ensuring a high degree of specificity in gene targeting. Functionally, the absorption of chimeric proteins is orchestrated through specialized membrane receptors, enabling the discerning delivery of the proteins exclusively to receptor- positive cells. This targeted approach results in diminished side effects and heightened therapeutic efficacy. Specifically, innovative chimeric proteins designed selectively targets tumors while sparing healthy immune cells. These therapeutics paired with immune checkpoint inhibitors offer potential for improved outcomes in treatment-resistant patients. Therapeutic Applications: This particular embodiment holds immense promise for treating a wide array of genetic disorders and diseases arising from irregular gene expression. Design of multiple novel constructions of chimeric therapeutic were described for targeting critical genes for cancer cell proliferation and progression. Moreover, innovative chimeric proteins were also presented herein as transcriptional activators for induction of Induced pluripotent stem (iPS) cells. This disclosure describes a novel approach to precise transcriptional control of disease- associated genes using chimeric proteins. These chimeric proteins are composed of two main components: a nuclear-trafficking carrier and a transcription-controlling effector. The nuclear- trafficking carrier ensures delivery of the transcription-controlling effector to the nucleus, where it can exert its desired effect. The transcription-controlling effector can be a variety of molecules, including active or dominant-negative transcription factors, polypeptides and peptides, or custom designed zinc- finger domains. This allows for a wide range of potential applications, such as gain-of-function Docket No.: 374819.00003 control of cell phenotypes, blocking of transcription factor functions, and precise transcriptional control of specific genes or regulatory elements. The novel chimeric proteins offer therapeutic potential across a broad spectrum of diseases characterized by irregularities in gene expression, achieved through precise transcriptional control. In one aspect, the present disclosure provides a chimeric protein for targeted transcriptional control of a disease-causing gene, comprising: a. a carrier domain that is specifically recognized by a receptor expressed on a population of cells, thereby facilitating sub-cellular delivery; b. an effector domain comprising a polypeptide (artificially designed zinc-finger) motif capable of specifically binding to a nucleotide sequence of the disease- causing gene or a promoter / enhance site of the gene, thereby modulating transcription of the disease-causing gene. c. a linker that is a flexible peptide connecting the carrier domain and the effector domain. In another aspect, the present disclosure provides a chimeric protein according to any embodiments disclosed herein for use in the treatment of a cancer or an immune-related disease. In another aspect, the present disclosure provides a therapeutic composition comprising a carrier domain and an effector domain, wherein: (a) the carrier domain is selected from a group of secreted factors with binding receptors that are present on cancer cell membranes but undetectable or very low on immune cells, such as T cells; and (b) the effector domain is functionally active to modulate transcription of a specific gene after being transported to the nucleus of cancer cells. In another aspect, the present disclosure provides a pharmaceutical composition comprising a chimeric therapeutic protein according to any embodiment disclosed and a pharmaceutically acceptable carrier. In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a chimeric therapeutic protein, a therapeutic composition, or a pharmaceutical composition according to any one of the embodiments disclosed. In another aspect, the present disclosure provides a method for modulating transcription of a disease-associated gene, comprising the steps of: (a) providing a population of target cells; Docket No.: 374819.00003 (b) introducing the chimeric protein according to any embodiment disclosed into the target cells, allowing the chimeric protein to deliver the effector domain to the nuclei of the target cells; and (c) observing the modulation of transcription of the disease-associated gene. In another aspect, the present disclosure provides a method for inducing mammalian cells into induced pluripotent stem cells (iPSCs), comprising introducing a chimeric protein according to any embodiment disclosed into said mammalian cells. In another aspect, the present disclosure provides a kit incorporating a chimeric protein according to any embodiment disclosed. In another aspect, the present disclosure provides a method for producing a chimeric protein according to any embodiment disclosed, comprising expression of a recombinant DNA construct encoding the chimeric protein in host cells, which can be bacterial or mammalian in nature. In another aspect, the present disclosure provides a process for producing a chimeric protein according to any embodiment disclosed, comprising: (a) synthesizing polypeptide fragments that make up the carrier domain and effector domain protein motifs; and (b) covalently conjugating the polypeptide fragments through a chemical reaction or enzymatic ligation. In another aspect, the present disclosure provides a method for enhancing pharmacokinetic and / or pharmacodynamic properties of a chimeric therapeutic according to any one embodiment disclosed, comprising a step of modifying the protein by covalently attaching it to a polymer to improve its half-life in bloodstream. Other aspects or advantages of the disclosure can be better understood in view of the following drawings, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 illustrates the Disclosure Related to Construction of Chimeric Proteins. A. The chimeric protein comprises a carrier domain, an effector domain, and a linker connecting the two domains; B. The construction of effector domain can be a DNA binding motif alone for inhibiting gene transcription, or fused to an activator or repressor motif via a linker. FIG.2 illustrates the in vivo therapeutic efficacy of H-E2F against non-small cell lung cancer (NSCLC) xenografts in mice. H-E2F is a peptide that competes the bindings of E2F transcription factors to the specific DNA binding sites, thereby blocking the E2F activity and leading to stop the cell cycle. FIG.2A depicts the change of tumor size of the mice treated with Docket No.: 374819.00003 H-E2F versus control. The nude mice (n=7) bearing H1299 xenografts were treated with H- E2F at 5mg / kg via i.p. injection. Arrows bar indicates the injections. FIG.2B indicates that H- E2F showed no toxicity, as there was no significant weight loss in the H-E2F treated mice as compared with the control. DETAILED DESCRIPTION OF THE DISCLOSURE In light of the current shortcomings in delivery methods, which frequently lack the necessary precision and efficiency for nuclear localization, the present disclosure provides a novel delivery technology, which enables internalization facilitated by membrane-anchored receptors, followed by subsequent transport to the nuclear compartment within cells. This breakthrough has the potential to surmount these limitations and offers a versatile solution for delivering a diverse array of therapeutic proteins to specific cells, ensuring their effective transportation to the nuclear compartment. In one aspect, the present disclosure provides a chimeric protein for targeted transcriptional control of a disease-causing gene, comprising: d. a carrier domain that is specifically recognized by a receptor expressed on a population of cells, thereby facilitating sub-cellular delivery; e. an effector domain comprising a polypeptide (artificially designed zinc-finger) motif capable of specifically binding to a nucleotide sequence of the disease- causing gene or a promoter / enhance site of the gene, thereby modulating transcription of the disease-causing gene. f. a linker that is a flexible peptide connecting the carrier domain and the effector domain. In some embodiments of this aspect, the carrier domain, effector domain, and linker of the chimeric protein are arranged in one of the following orders (see FIG.1(A)): a. carrier domain - linker - effector domain; or b. effector domain - linker - carrier domain. In some embodiments of this aspect, the carrier domain is derived from a secreted factor capable of efficient intracellular trafficking and nuclear localization. In some embodiments of this aspect, the carrier domain is selected from BDNF, Cyclophilin B, EGF, CCN1 (CYR61, GIG1, IGFBP10), CCN2, CTGF, CCN3, CCN4, CCN5, CCN6, FGF-1, FGF2, FGF-3, FGF13, FGF21, FGF7, FGF19, HDGF, HDGFL1, HDGFL2, HDGFL3, HMGB1, HMGB2, IBFBP, IGFBP1, IGFBP2 (IBP2), IGFBP3, IGFBP4, IGFBP5, Docket No.: 374819.00003 IL1RN, IL15, GDF, GDNF, LTBP2, NRG2, Netrin-1, NRP2, PTHLH, PSIP1, SEMA3E, VASN, and VEGFA. In some embodiments of this aspect, the effector domain comprises a polypeptide with amino acid residues arranged to recognize and bind to the nucleotide sequence of the target gene or the promoter / enhancer site of the target gene for transcriptional modulation. In some embodiments of this aspect, the effector domain comprises an activator or suppressor motif connected to the DNA binding motif via a linker for transcriptional modulation of a specific gene (see FIG.1(B)). In some embodiments of this aspect, the chimeric protein comprises: (a) a hepatoma derived growth factor (HDGF) as the carrier domain; (b) a peptide, BPE2F, which exhibits a specific binding affinity to a promoter site recognized by E2F for inhibiting transcription of E2Fs responsive genes; and (c) an amino acid sequence of HHHRSLH, or a derivative thereof. In some embodiments of this aspect, the effector domain comprises a sequence of designed ZFTF motif with specific amino acid residues arranged to recognize and bind to the DNA sequence of the target gene or the promoter / enhancer site of the target gene. In some embodiments of this aspect, the transcription activator in the effector domain is VP60 or p65AD. In some embodiments of this aspect, the effector domain comprises a native transcription factor, variant of a native transcription factor, and / or a designed Zinc Finger transcription factor (ZFTF). In some embodiments of this aspect, the DNA binding motif included in the effector domain specifically recognizes the promoter / enhancer sequence of a gene of interest for transcriptional modulation. In some embodiments of this aspect, in the chimeric protein: (a) the carrier domain is CypB, a binding ligand of CD147, of which the over-expression is induced by lactate overproduction in cancer cells; (b) the effector domain is capable of blocking peptide of E2F (BP-E2F) or c-Myc promoter-binding protein-1 (MBP-1) in cancer cells; and (c) the linker connects the carrier domain and the effector domain. In some embodiments of this aspect, in the chimeric protein: (a) the carrier domain is selected from NRG1 and FGF2 for their nuclear trafficking activity within tumor cells; and Docket No.: 374819.00003 (b) the effector domain comprises specially designed zinc finger nuclear transport factors (ZNTFs) fused with a repressor motif (KRAB) for specific binding to genes encoding enzymes crucial for the glycolytic pathways of glucose and glutamine. In some embodiments of this aspect, in the chimeric protein: (a) NRG1 binds to its receptor including EGFR, HER2 and HER3 that are overexpressed in various tumors, including leukemia, lymphoma, glioblastoma, neuroblastoma, sarcomas, renal, esophageal, head and neck, melanoma, breast, colorectal, bladder, gastric, lung, liver, ovarian, endometrial and pancreatic cancers, facilitating internalization and nuclear transport within target tumor cells; (b) the repressor motif Krüppel-associated box repressor (KRAB) in the effector domain allows for specific binding to and transcription inhibition of the target gene(s) involved in the glycolytic pathways, thereby disrupting the metabolic supply of glucose and glutamine, crucial for cancer cell growth; and / or (c) FGF2 binds to its receptor including FGFR1 and FGFR2 that are overexpressed in various tumors, including breast, colorectal, bladder, gastric, lung, and pancreatic cancers, facilitating internalization and nuclear transport within target tumor cells. In some embodiments of this aspect, the chimeric protein is designed to selectively target tumor cells while sparing immune cells due to very low levels of EGFR and HER2 and the absence of the HER3 on the membranes of the latter. In another aspect, the present disclosure provides a chimeric protein according to any embodiments disclosed herein for use in the treatment of a cancer or an immune-related disease. In some embodiments of this aspect, the cancer is selected from hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), endometrial carcinoma (EC), ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, and gastric cancer; and the chimeric protein is administered either alone or in combination with an additional cancer therapy to enhance treatment outcomes. In another aspect, the present disclosure provides a therapeutic composition comprising a carrier domain and an effector domain, wherein: (a) the carrier domain is selected from a group of secreted factors with binding receptors that are present on cancer cell membranes but undetectable or very low on immune cells, such as T cells; and (b) the effector domain is functionally active to modulate transcription of a specific gene after being transported to the nucleus of cancer cells. In some embodiments of this aspect, the carrier domain is capable of ensuring nuclear delivery and localization of the functionally active effector domain to cancer cells, while Docket No.: 374819.00003 immune cells remain underexposed or unexposed to the therapeutic due to a low level or absence of the binding receptor for the carrier domain. In some embodiments of this aspect, the polypeptide or custom designed ZFTFs are engineered to repress the transcription of genes encoding critical enzymes mediating the energy supplies from glycolytic pathways sourced from preferred glucose and glutamine within cancer cells. In another aspect, the present disclosure provides a pharmaceutical composition comprising a chimeric therapeutic protein according to any embodiment disclosed and a pharmaceutically acceptable carrier. In some embodiments of this aspect, the pharmaceutical composition is formulated for administration via routes selected from intravenous, intramuscular, subcutaneous, intra-dermal, and / or other suitable routes of administration. In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a chimeric therapeutic protein, a therapeutic composition, or a pharmaceutical composition according to any one of the embodiments disclosed. In some embodiments of this aspect, the cancer is selected from leukemia, lymphoma, breast cancer, lung cancer, colon cancer, gastric cancer, hepatoma, glioblastoma, esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, colorectal cancer, renal cancer, bladder cancer, head and neck cancer, and melanoma. In some embodiments of this aspect, the chimeric therapeutic protein is administered systemically or locally to the cancer site. In some embodiments of this aspect, the chimeric protein is administered in combination with an immunotherapy agent (e.g., an immune checkpoint inhibitor), a monoclonal antibody, an adoptive cell therapy, a chemotherapy, and / or another targeted therapy. In another aspect, the present disclosure provides a method for modulating transcription of a disease-associated gene, comprising the steps of: (a) providing a population of target cells; (b) introducing the chimeric protein according to any embodiment disclosed into the target cells, allowing the chimeric protein to deliver the effector domain to the nuclei of the target cells; and (c) observing the modulation of transcription of the disease-associated gene. Docket No.: 374819.00003 In some embodiments of this aspect, the gene is associated with specific pathologies or conditions; and wherein the modulation of gene expression leads to the amelioration of said pathologies or conditions, immune-related diseases, fibrotic diseases, diabetes, obese, sepsis, and / or aging. In another aspect, the present disclosure provides a method for inducing mammalian cells into induced pluripotent stem cells (iPSCs), comprising introducing a chimeric protein according to any embodiment disclosed into said mammalian cells, wherein the chimeric protein is capable of activating genes encoding essential transcription factors including c-Myc, klf4, Oct4, and Sox2, as well as other factors promoting cellular stemness such as Nano, Lin28, Call4, Glis1, Essrb, Elf5, UTF1, and Pou2f2. In some embodiments of this aspect, the induction of iPSCs serves as a therapeutic intervention for the treatment of diseases or disorders characterized by cellular dysfunction or depletion. In another aspect, the present disclosure provides a kit incorporating a chimeric protein according to any embodiment disclosed. In another aspect, the present disclosure provides a method for producing a chimeric protein according to any embodiment disclosed, comprising expression of a recombinant DNA construct encoding the chimeric protein in host cells, which can be bacterial or mammalian in nature. In another aspect, the present disclosure provides a process for producing a chimeric protein according to any embodiment disclosed, comprising: (a) synthesizing polypeptide fragments that make up the carrier domain and effector domain protein motifs; and (b) covalently conjugating the polypeptide fragments through a chemical reaction or enzymatic ligation. In another aspect, the present disclosure provides a method for enhancing pharmacokinetic and / or pharmacodynamic properties of a chimeric therapeutic according to any one embodiment disclosed, comprising a step of modifying the protein by covalently attaching it to a polymer to improve its half-life in bloodstream, wherein said polymer is selected from polyethylene glycol (PEGylation), poly(N-vinyl pyrrolidone) (PVP), poly(lactic acid) (PLA), poly(ethylene glycol) methyl ether acrylate (PEGMA), Poly(lactic-co-glycolic acid) (PLGA), poly(hydroxypropyl)methacrylate (HPMA), polyoxylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and water-soluble polyoxyethylated polyps. Docket No.: 374819.00003 The present disclosure aims to surmount these challenges through the creation of a recombinant chimeric protein, serving as a prototype for proof-of-concept. It is well- documented that various secreted factors and cytokines have the capacity to undergo sub- cellular trafficking to the cell nuclei after binding to membrane receptors (See List). Notable examples of these nuclear-present factors include Hepatoma derived growth factor (HDGF) (Everett A.D. et al., J Biol Chem., 2001.276(40):37564-8.). Nuclear import typically involves a nuclear localization signal present either in the secreted factor or its transmembrane receptor recognized by the importins machinery. (Johnson HM. et al., Bioessays.2004. (9):993-1004; Bryant DM. et al., Traffic., 2005.6(10):947-54.; Planque N., 2006. Cell Commun Signal., 2006. 4:7). By leveraging the nuclear transport capability displayed by these factors, they can serve as a carrier domain to facilitate internalization and subsequent nuclear transport within the protein construct. Additionally, an inhibitory peptide known as Blocking Peptide of E2F (BPE2F) has been developed. It specifically targets the E2F transcription factors, which are often over-expressed in various types of cancer, leading to heightened cell proliferation and contributing to tumor growth. This inhibition is achieved through its strong binding affinity to the DNA-binding sequence of E2F, preventing it from binding to DNA and activating the transcription of genes involved in cell growth and proliferation. Studies have demonstrated the peptide's effectiveness in restraining the growth of prostate cancer cells both in culture and in vivo in a mouse model of prostate cancer. Moreover, the peptide has shown no toxicity to normal cells, indicating its potential as a safe and efficacious new cancer treatment (Xie, Q., et al., PNAS, 2014, 111(10), 3637-3642). Given E2F's pivotal role in cell cycle regulation and its significant impact on tumor growth, targeting this transcription factor holds great potential for therapeutic intervention. A prototype of a chimeric protein was invented to demonstrate the potential of this new approach to revolutionize the development of targeted therapy. The recombinant chimeric protein structure comprises HDGF serving as the carrier domain, facilitating internalization and nuclear transport. It also integrates the BPE2F peptide for its effective inhibition of E2F transcriptional activity, with a linker connecting both functional motifs. The genetic engineering of a carrier domain to link with various polypeptides for nuclear delivery in cells for therapeutic purposes is of significant interest. Among these, the zinc finger family of transcription factors stands out due to their crucial roles in gene regulation and their ability to bind to DNA sequences with remarkable precision. They are named after Docket No.: 374819.00003 the zinc atoms that are present in their structure, which help to stabilize the protein and give it its characteristic shape. The DNA-binding domain of a zinc finger transcription factor is composed of one or more zinc fingers, each consisting of approximately 30 amino acids. These amino acids form a loop that binds to a specific sequence of three nucleotides on DNA. The number of zinc fingers can vary, influencing the protein's DNA-binding specificity. Those with more zinc fingers have a broader range of DNA sequences they can bind to. Zinc finger transcription factors can either activate or repress gene expression. Activating factors bind to DNA sequences near the promoter region of a gene, recruiting other proteins to initiate transcription. Repressing factors bind to sequences within the gene, preventing transcription. This innovative construct design for recombinant therapeutics holds the potential to develop a wide range of precisely targeted therapies with exceptional specificity, achieved through gene transcription modulation. Notably, recent advances in artificial intelligence (AI) technology have made it practically feasible to design zinc finger transcription factors (ZNTFs) capable of binding to universal nucleotide sequences of genes (D. Ichikawa et al., Nature Biotechnology, 2023.41: 1117–1129). This inventive approach to developing novel targeted therapies is significant, as intervening at the transcriptional level offers several advantages over targeting translational and post-translational processes, particularly in terms of therapeutic effectiveness and minimizing off-target side effects: 1). Amplification Effect at Transcription: Despite typically having only two gene copies (one from each parent), the transcriptional process can generate a substantial number of mRNA molecules. Each of these can then give rise to multiple protein molecules through translation. This means that a single intervention at the transcriptional level can significantly impact the overall production of proteins related to a specific gene. 2). Precise Targeting: Transcriptional intervention allows for the specific targeting of the gene of interest. By modulating the expression of a particular gene, it's possible to directly influence the production of the corresponding protein. This precision reduces the likelihood of affecting unintended targets. 3). Broad Downstream Effects: Altering transcription can have far-reaching effects on various cellular processes. This is particularly advantageous when a single gene plays a central role in a disease or pathway. Targeting transcription can have a cascading effect on related genes and pathways, amplifying the therapeutic impact. Docket No.: 374819.00003 4). Overcome drug resistance: Kinase inhibitors are drugs that block the activity of kinases, enzymes that play a role in cell growth, differentiation, and survival. Kinase inhibitors are used to treat many cancers, but they can lose efficacy over time due to resistance, one mechanism of which is nuclear import of the receptor. Receptor kinases are proteins on the cell surface that bind to growth factors and activate downstream signaling pathways that lead to cell growth and survival. In some cases, receptor kinases can be transported into the nucleus, where they can regulate the expression of genes involved in cell growth, survival, and migration. This can lead to resistance to kinase inhibitors, even in the presence of the drug. For instance, EGFR and HER2 can be transported into the nucleus, maintaining cell growth and survival despite inhibitors (Li C. et al., Oncogene. 2009 Oct 29;28(43):3801-13; Hwang S. et al., Cancers, 2020. 12(6):1540). Targeting receptor kinase transcription could effectively curb resistance by preventing their production, stopping nuclear transport, and downstream activation of growth and survival pathways. This is crucial, as aberrant gene expression is often a fundamental driver of disease. By addressing the root cause, transcriptional interventions can potentially provide more profound and sustained therapeutic effects. 5). Longer-Lasting Effects: Changes at the transcriptional level tend to have longer- lasting effects compared to interventions at the translational or post-translational levels. This is because alterations in gene expression can persist through multiple rounds of cell division and protein turnover. Combination with Existing Therapeutics: Transcriptional interventions can be used in combination with other existing therapeutics, offering a synergistic approach that can enhance treatment efficacy and potentially overcome drug resistance. The present disclosure introduces a method for constructing a chimeric protein designed to facilitate nuclear delivery of a peptide that intercepts E2F binding to promoter sites. This is achieved by fusing the peptide with a nuclear-trafficking-capable HDGF. The resulting chimeric protein demonstrates potent antitumor activity against non-small cell lung cancer (NSCLC) in the animal model. This prototype, serving as a proof of concept, validates a novel approach to significantly broaden the scope of targeted therapy development. It achieves this by employing specific transcription control of genes with high precision, offering a promising avenue for a wide range of diseases. Docket No.: 374819.00003 One embodiment of the present disclosure relates to a chimeric therapeutic protein for cancer therapy. The chimeric protein comprises a carrier domain, an effector domain, and a linker connecting the two domains. The first embodiment of the present disclosure related to the carrier domain is HDGF that enables the chimeric protein to internalize and be transported to the nucleus of cancer cells. Alternatively, other carrier domain can be selected from the group of secreted factor capable of nuclear trafficking (see TABLE bellow). The effector domain is a blocking peptide of E2F (BPE2F) that competes with E2F for binding to DNA. The linker is a flexible peptide that connects the carrier domain and the effector domain. The linker allows the two domains to fold independently and function properly. t. ol ., . . Docket No.: 374819.00003 FGF-3 Kiefer P.. Embo J., 1994.13(17):4126-4136; Planque N. Cell et r . ., ., . . Docket No.: 374819.00003 IGFBP5 Planque N. Cell Commun Signal., 2006.4:7. r ABBREVIATION DEFINITION, (AKA) BDNF | Brain derived neurotrophic factor CCN1 | Cellular communication network factor, (CYR61, GIG1, IGFBP10), CCN2 | Cellular communication network factor 2, (CTGF, IGFBP8), CCN3 | Cellular communication network factor 3, (IGFBP9, NOV) CCN4 | Cellular communication network factor 4, (WISP1, WISP1-OT1, WISP1-UT1) CCN5 | Cellular communication network factor 5, (WISP1) Docket No.: 374819.00003 CCN6 | Cellular communication network factor 6, (WISP3) CYPB | Cyclophilin B DNA | Deoxyribonucleic acid E2F | E2 promoter-binding factor E2FIP | E2F inhibiting peptide EGF | Epidermal growth factor FGF1 | Fibroblast growth factor 1 (AFGF, ECGF, ECGF-beta, ECGFA, ECGFB, FGF-alpha, FGFA, GLIO703, HBGF1) FGF-2 | Fibroblast growth factor 2 (FGFB) FGF-3 | Fibroblast growth factor 3 (HBGF-3, INT2) FGF7 | Fibroblast growth factor 7 (KGF) FGF21 | Fibroblast growth factor 21 GDNF | Glial cell derived neurotrophic factor (ATF1, ATF2, HFB1-GDNF) GDF15 | Growth differentiation factor 15 GDNF | Glial cell derived neurotrophic factor, (ATF1, ATF2, HFB1-GDNF) GFP | Green fluorescent protein HDGF | Hepatoma derive growth factor HDGFL1 | HDGF like 1 (dJ309H15.1, Hdgfrp1, HRP-1, PWWP1) HDGFL2 | HDGF like 2 (Hdgfrp2, HRP2) HDGFL3 | HDGF like 3 (Hdgfrp3, HRP-3) HMGB1 | High mobility group box 1 (DKFZp686A04236, HMG1, HMG3, SBP-1) HMGB2 | High mobility group box 2 HSPG2 | Heparan sulfate proteoglycan 2, (Perlecan, PRCAN, SJS1) IGF1 | Insulin growth factor 1 IGFBP1 | Insulin like growth factor binding protein 1 IGFBP2 | Insulin like growth factor binding protein 2 (IBP2) IGFBP3 | Insulin like growth factor binding protein 3 (BP-53, IBP3) IGFBP4 | Insulin like growth factor binding protein 4 (BP-4, HT29, IGFBP, IBP4) IGFBP5 | Insulin like growth factor binding protein 5 IL1RN | Interleukin 1 receptor antagonist (ICIL-1RA, IL-1RN, IL1F3, IL1RA, IRAP, MGC10430) Docket No.: 374819.00003 IL15 | Interleukin 15, (IL-15, MGC9721) KRAB | Krüppel-associated box repressor LTBP2 | Latent transforming growth factor beta binding protein 2, (C14orf141, LTBP3) NRG2 | Neuregulin 2, ((Don-1, HRG2, NTAK) NRP2 | Neuropilin-2, (VEGF165R2) PSIP1 | PC4 and SFRS1 interacting protein 1, (DFS70, LEDGF, p52, p75, PSIP2) PTHLH | Parathyroid hormone like hormone, (HHM, PLP, PTHR, PTHRP) NTN1 | Netrin-1, (NET1, NTN1L) RNA | Ribonucleic acid SEMA3E | Semaphorin 3E, (coll-5, KIAA0331, M-SemaK, SEMAH) TF | (transcription factor) ZFTF | Zinc Finger transcription factor VEGFA | Vascular endothelial growth factor A, (VEGF, VEGF-A, VPF) VASN | Vasorin, (SLITL2) The chimeric protein of the present disclosure is designed to specifically target and block E2F activity in cancer cells. E2F is a transcription factor that plays a critical role in the regulation of cell cycle progression and DNA replication. By blocking E2F activity, the chimeric protein can inhibit the growth and proliferation of cancer cells. One embodiment of the present disclosure relates to a chimeric therapeutic protein for cancer therapy, wherein the carrier domain is HDGF or its variant that enables the chimeric protein to internalize and be transported to the nucleus of cancer cells. Alternatively, the carrier domain can also be selected from the group of secreted factor capable of nuclear trafficking (see TABLE). The effector domain can be c-Myc binding protein-1(MBP-1), a repressor of c- Myc, that suppresses the transcription of the c-Myc gene. The chimeric protein of the present disclosure is designed to specifically target and suppress the c-Myc gene in cancer cells. The c-Myc gene is an oncogene that plays a critical role in the development and progression of many types of cancer. By suppressing the c-Myc gene, the chimeric protein can inhibit the growth and proliferation of cancer cells. Another embodiment of the present disclosure relates to a chimeric therapeutic protein for cancer therapy, wherein the carrier domain is cyclophilin B (CypB) that interacts with a CD147 that is overexpressed in cancer cells due to lactate overproduction as a result of Docket No.: 374819.00003 anaerobic glycolysis metabolism (Warburg effect) of the cells. As such, CypB chimeric proteins have a propensity to target those cancer cells. The effector domain is a blocking peptide of E2F (BP-E2F) that competes with E2F for binding to DNA. The linker is a flexible peptide that connects the carrier domain and the effector domain. The linker allows the two domains to fold independently and function properly. Another embodiment of the disclosure of the chimeric therapeutic of which the effector domain can be a repressor of c-Myc (MBP- 1). This chimeric protein is designed to specifically target and block E2F or c-Myc activity in cancer cells. E2F and c-Myc are transcription factors that play critical roles in the regulation of cell cycle progression, DNA replication, and metabolism (Wise DR. et al., Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18782-7). By blocking E2F or c-Myc activity, the chimeric protein can inhibit the growth and proliferation of cancer cells. In a preferred embodiment of the present disclosure, the chimeric protein incorporates a carrier domain, specifically identified as FGF2 or its variant form, known for its demonstrated nuclear trafficking capabilities within tumor cells. FGF2 effectively binds to its cognate receptor, FGFR3, which is notably overexpressed in a range of tumors including breast, colorectal, ovarian, gastric, lung, cholangiocarcinoma and endometrial cancers. This ligand thus serves as an efficient carrier domain for internalization and subsequent nuclear transport within target tumor cells. The effector domain of this chimeric protein features specially designed Zinc finger transcription factors (ZNTFs) fused with a repressor motif, Krüppel-associated box repressor (KRAB), allowing for specific binding to genes responsible for key enzymatic processes in the glycolytic pathways of glucose and glutamine metabolism. This targeted action disrupts the metabolic pathways, thereby depriving cancer cells of essential fuels for growth. A unique advantage of this chimeric protein construct is its selectivity, as it avoids targeting immune cells due to the absence of FGFR2 on their membranes. FGF2 exhibits selective binding to various FGFR dimers, displaying distinct binding affinities. Specifically, its binding affinity (Kd) for FGFR1-FGFR2 is higher than that for FGF2-FGF2 (Ornitz DM. et al., J Biol Chem. 1996. 271(25):15292-7). Leveraging this difference in binding affinity, therapeutic targeting of FGFR2 is expected to exert a considerably greater impact on tumor cells compared to immune cells lacking FGFR2. This indicates that employing this targeted therapy approach could significantly broaden the therapeutic window, enhancing its effectiveness. Docket No.: 374819.00003 Results of previous studies have shown that combining the glycolysis inhibitor 2- deoxyglucose with the immunotherapy drug pembrolizumab improved the survival of mice with melanoma that was resistant to pembrolizumab alone (Zhang, T. et al., (2022). Cancer Letters, 550, 39-52.), suggesting that combining glycolysis pathway inhibition with immunotherapy is a promising approach for treating cancer that is resistant to these treatments. Consequently, this chimeric construct holds immense clinical potential, particularly when combined with immunotherapy, thereby enhancing treatment outcomes for patients. In another preferred embodiment, wherein this chimeric protein incorporates a carrier domain, designated as NRG1 or its variant form, known for its nuclear trafficking abilities within tumor cells. NRG1 binds to its receptors, including EGFR, HER2, and HER3, all of which are notably overexpressed in various types of tumors. The following types of cancer commonly exhibit elevated levels of EGFR, HER2, and HER3, respectively: lung cancer, colorectal cancer, head and neck cancer, bladder cancer, pancreatic cancer, glioblastoma for EGFR; breast cancer, ovarian cancer, gastric cancer, endometrial cancer, esophageal cancer for HER2; and breast cancer, lung cancer, colorectal cancer, ovarian cancer, gastric cancer, esophageal cancer for HER3. As a result, this ligand effectively functions as a carrier domain, enabling internalization and subsequent nuclear transport within target tumor cells. The effector domain of this chimeric protein incorporates specially designed Zinc Finger Nuclear Transport Factors (ZNTFs) coupled with a repressor motif (KRAB). These ZNTFs are tailored for specific binding to genes responsible for key enzymatic processes within the glycolytic pathways of glucose and glutamine metabolism. Cancer cells undergo a metabolic shift, adopting a "glycolysis-dominant" metabolic profile to enhance their survival and fulfill their energy and macromolecule demands. This phenomenon, also known as the "Warburg effect," confers a survival advantage upon cancer cells (Wise DR. & Thompson CB. Trends Biochem Sci., 2010.35(8):427-33; DeBerardinis RJ. et al., Proc Natl Acad Sci U S A, 2007.104(49):19345-50). Therefore, disrupting the transcription of critical enzymes involved in the glycolytic pathways for glucose and glutamine can cripple the cancer cell's essential energy supply (Chelakkot C. et al., Int J Mol Sci., 2023.24(3):2606). One of the key benefits of this unique chimeric protein construct is its selectivity. It is designed to avoid targeting immune cells because very low levels of both EGFR and HER2 expression, and absence of HER3 on the surface of these cells. As a result, this chimeric protein has significant clinical promise, especially when used in combination with immunotherapy. It has the potential to improve treatment outcomes for patients. Docket No.: 374819.00003 One of the embodiments of the current disclosure pertains to the development of chimeric proteins with nuclear targeting capabilities. These proteins consist of a carrier domain linked to an effector domain through a connector. The carrier domain is chosen from a selection of secreted protein factors or their variants (please refer to the provided TABLE) that is capable of internalization through interaction with their receptor, and subsequent nuclear transport. As used herein, the term “variants” used as carrier domains refers to a variety of the versions encompasses iso-forms or truncated versions resulting from alternative splicing, post- transcriptional and post-translational modifications. Additionally, the term of “variants” refer to the analogs of the selected secreted factors with one or more changes in the amino acid sequence of polypeptides. These protein variants may be generated by site-directed mutagenesis having additions, deletions, or substitutions of amino acid residues that remain intact activity of nuclear transport. A preferred embodiment of this disclosure involves chimeric fusion proteins that incorporate a carrier domain and an effector domain. These two polypeptide chains are connected together by a linker. Additionally, the effector domain may incorporate a DNA binding motif (see FIG.1(B)), with or without a transcriptional activator or repressor. The two polypeptide motifs within the effector domain are joined by a linker. This linker comprises a sequence of amino acids selected from a range including (GGGGS)n, (GGSGGSGGS)n, (EAAAK)n, and (AAEEAAK)n, where n can be 1, 2, 3, 4, 5, or a combination of these distinct sets of linkers. The chimeric therapeutics or compositions may also be administered to a mammal subcutaneously or even by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. In general, the dosage of an administered chimeric therapeutic will vary depending upon such factors as the patients age, weight, height, sex, general medical condition and previous medical history. Additional pharmaceutical methods may be employed to control the duration of action of the therapeutic. Control release preparations can be prepared through the use of polymers to complex or adsorb the chimeric therapeutic. The chimeric therapeutics portions thereof of this disclosure are administered at a concentration that is therapeutically effective to prevent or treat any of the aforementioned diseases states. To accomplish this goal the chimeric therapeutics may be formulated using a variety of acceptable excipients known in the art. Typically, the chimeric therapeutics are preferably administered by injection, either intravenously or intra-peritoneally. Methods to accomplish this administration are known to those of ordinary skill in the art. The compositions Docket No.: 374819.00003 may also be topically or orally administered or be capable of transmission across mucous membranes. Before administration to patients, formulants may be added to the chimeric therapeutics. A liquid formulation is preferred. For example, these formulants may include oils, polymers, Vitamins, carbohydrates, amino acids, salts, buffers, albumin, Surfactants, or bulking agents. Preferably carbohydrates include Sugar or Sugar alcohols, such as mono-, di- or polysaccharides, or water-soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha-and beta-cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose, or mixtures thereof. To improve the half-life of the chimeric therapeutics in the bloodstream and enhance their pharmacokinetic and pharmacodynamic properties, a preferred embodiment of the present disclosure involves modifying the proteins by attaching a polymer. Preferred polymers include polyethylene glycol (PEGylation), poly(N-vinyl pyrrolidone) (PVP), poly(lactic acid) (PLA), poly(ethylene glycol) methyl ether acrylate (PEGMA), Poly(lactic-co-glycolic acid) (PLGA) and poly(hydroxypropyl)methacrylate (HPMA), and the like. Water soluble polyoxyethylated polyps are also useful in the present disclosure. They include polyoxylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. As a person of ordinary skill in the art would understand, the present disclosure encompasses any reasonable combinations of the embodiments disclosed. Unless defined otherwise, all the terms and notations, and all the scientific terms or terminology, used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. As used herein, the singular forms “a,” “an,” and “the” include plural reference, and vice versa, any plural forms include singular reference, unless the context clearly dictates otherwise. The terms "comprising", "having", "including", and "containing", or the like, are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. An “effective amount” or “an amount effective” refers to an amount of the pharmaceutical formulation of the disclosure which is effective, upon single or multiple dose Docket No.: 374819.00003 administrations to a subject, in treating a cell, or curing, alleviating, relieving or improving a symptom of a disorder. An effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. As used herein, the term “prevent” or “preventing,” as used in the context of the administration of an agent to a subject, refers to subjecting the subject to a regimen, e.g., the administration of a pharmaceutical formulation of the disclosure such that the onset of at least one symptom of the disorder is delayed as compared to what would be seen in the absence of the regimen. As used herein, the term “subject,” “patient,” or the like, is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein, or a normal subject. The term “non-human animals' includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals. Such as non-human primates, domesticated and / or agriculturally useful animals, e.g., horse, sheep, dog, cat, cow, pig, etc. As used herein, the term “treatment” of, or “treat” or “treating,” a subject having a disorder refers to subjecting the subject to a regimen, e.g., the administration of a pharmaceutical formulation of the disclosure such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder. The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner. The term “non-small cell lung cancer” (NSCLC) refers to any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are several other types that occur less frequently, and all types can occur in unusual histologic variants. Docket No.: 374819.00003 The term “pharmaceutically acceptable carrier / excipient” means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe non- toxic and neither biologically nor otherwise undesirable, and includes a carrier or an excipient that is acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable carrier / excipient" as used in the specification and claims includes both one and more than one such excipient. EXAMPLE Construct of Chimeric Proteins To commence the evaluation of the therapeutic effectiveness of the innovative chimeric protein construct (designated as H-E2F), which encompasses HDGF, a linker, and BPE2F. This prototypical chimeric protein was encoded by a synthetic fusion gene, designed based on the provided amino acid sequences (Sequences List). The fusion gene was constructed and expressed in bacteria (Genscript, Piscataway, NJ) to generate the chimeric protein. The amino acid sequence of H-E2F as present in the construct vector pET-41a(+) for bacterial expression is shown below: SEQ ID NO: 1 MGSRSNRQKEYKCGDLVFAK 20 MKGYPHWPARIDEMPEAAVK 40 STANKYQVFFFGTHETAFLGP 60 KDLFPYEESKEKFGKPNKRK 80 GFSEGLWEIENNPTVKASGY 100 QSAQKKSCVEEPEPEPEAAE 120 GDGDKKGNAEGSSDEEGKLV 140 IDEPAKEKNEKGALKRRAGD 160 LLEDSPKRPKEAENPEGEEK 180 EAATLEVERPLPMEVEKNST 200 PSEPGSGRGPPQEEEEEEDE 220 EEEATKEDAEAPGIRDHESL 240 GGGSGGRQIKIFFQNRRMKF 260 KKCHHHRLSHCLE Docket No.: 374819.00003 SEQ ID NO: 2 (Amino acid sequences of BP-E2F that binds to the DNA binding site of E2F) HHHRLSH HDGF is a mitogen, and plays important roles in tumor progression. HDGF is associated tumor progression, including PI3K / AKT and ERK signaling pathway activation (Bao C. et al., Future Oncol., 2014.10: 2675-2685). Moreover, Its specific involvement lies in modulating the Zeb transcription factor within tumors. Zeb constitutes a crucial family of transcription factors with significant roles in cellular differentiation and development. In cancer, levels of Zeb proteins are often elevated, which can lead to tumor progression and metastasis. HDGF promotes tumor activity by interacting with Zeb proteins and enhancing their activity. This can lead to the suppression of genes that are important for cell differentiation and apoptosis. HDGF also promotes tumor growth and angiogenesis (Xiao YY. et al., Am J Cancer Res., 2019.9(11):2314-2330). Furthermore, studies have shown that HDGF overexpression is associated with poor prognosis in patients with a variety of cancers, including hepatocellular carcinoma (HCC) (Zhou, Y. Et al., Diagnostic Pathology, 2010.5(58), 1-8.), non-small cell lung cancer (NSCLC) (Zhang, H., et al., Tumor Biol., 2015. 36(2), 1261-1268.), endometrial carcinoma (EC) (Liu, X. Et al., Intern. J. of Clin. and Exper. Path, 2017.10(7): 9896), breast cancer, colorectal cancer, and gastric cancer Since the nuclear localization of HDGF has been demonstrated by staining its fusion protein with GFP (Kishima Y. et al., J Biol Chem., 2002, 277(12):10315-22), it is possible that HDGF could be used to deliver effector motifs that can interact with target genes by binding to specific DNA sequences. To achieve this, a new recombinant chimeric protein was constructed by fusing HDGF with BPE2F using a linker, as described. Assessment of Chimeric Protein Antitumor Activity The assessment of antitumor activity of the chimeric proteins H-E2F was conducted on non-small cell lung cancer. The antitumor activity of H-E2F was examined in H1299 xenograft models. The results are shown in FIG.2. The graph depicts the mean estimated tumor volume (n = 6) for each treatment group and its corresponding standard error. Treatment with H-E2F at a dosage of 5 mg / kg was initiated when the average tumor sizes became palpable. Subsequently, the mice were administered the agent via the tail vein, as indicated by the arrows. Docket No.: 374819.00003 The results demonstrated a remarkable reduction in tumor sizes after one week of agent administration compared to the control group. Compared to the control group, the growth of tumor volumes of treated mice was markedly repressed during the treatment course (FIG.2A). These findings provide insights into the potential therapeutic efficacy of the chimeric protein in combating non-small cell lung cancer. Furthermore, the toxicity was not shown as the treatment did not cause significant changes in weights of mice (FIG.2B). These results underscore the potent anti-tumor efficacy, indicating the successful delivery of this chimeric therapeutic to inhibit E2F function within the nuclei of tumor cells. This innovative prototype chimeric protein is a new type of targeted therapeutics, offering significant potential to enhance treatment efficacy across a diverse range of diseases due to its heightened effectiveness and remarkably broad spectrum of targeted genes. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present disclosure without departing from the spirit and scope of the disclosure. Thus, the present disclosure is intended to encompass the modifications and variations of this disclosure, provided they fall within the scope of the appended claims and their equivalents. All patent or non-patent literature cited are incorporated herein by reference in their entireties for all purposes without admission of them as prior art.

Claims

Docket No.: 374819.00003 CLAIMS What is claimed is:

1. A chimeric protein for targeted transcriptional control of a disease-causing gene, comprising: g. a carrier domain that is specifically recognized by a receptor expressed on a population of cells, thereby facilitating sub-cellular delivery; h. an effector domain comprising a polypeptide (artificially designed zinc-finger) motif capable of specifically binding to a nucleotide sequence of the disease- causing gene or a promoter / enhance site of the gene, thereby modulating transcription of the disease-causing gene. i. a linker that is a flexible peptide connecting the carrier domain and the effector domain.

2. The chimeric protein of claim 1, wherein the carrier domain, effector domain, and linker are arranged in one of the following orders: c. carrier domain - linker - effector domain; or d. effector domain - linker - carrier domain.

3. The chimeric protein of claim 1, wherein the carrier domain is derived from a secreted factor capable of efficient intracellular trafficking and nuclear localization.

4. The chimeric protein of claim 1 or 2, wherein the carrier domain is selected from BDNF, Cyclophilin B, EGF, CCN1 (CYR61, GIG1, IGFBP10), CCN2, CTGF, CCN3, CCN4, CCN5, CCN6, FGF-1, FGF2, FGF-3, FGF13, FGF21, FGF7, FGF19, HDGF, HDGFL1, HDGFL2, HDGFL3, HMGB1, HMGB2, IBFBP, IGFBP1, IGFBP2 (IBP2), IGFBP3, IGFBP4, IGFBP5, IL1RN, IL15, GDF, GDNF, LTBP2, NRG2, Netrin-1, NRP2, PTHLH, PSIP1, SEMA3E, VASN, and VEGFA.

5. The chimeric protein of any one of claims 1-4, wherein the effector domain comprises a polypeptide with amino acid residues arranged to recognize and bind to the nucleotide sequence of the target gene or the promoter / enhancer site of the target gene for transcriptional modulation.

6. The chimeric protein of any one of claims 1-5, wherein the effector domain comprises an activator or suppressor motif connected to the DNA binding motif via a linker for transcriptional modulation of a specific gene.

7. The chimeric protein of any one of claims 1-6, wherein the chimeric protein comprises: (a) a hepatoma derived growth factor (HDGF) as the carrier domain;Docket No.: 374819.00003 (b) a peptide, BP-E2F, which exhibits a specific binding affinity to a promoter site recognized by E2F for inhibiting transcription of E2Fs responsive genes; and (c) an amino acid sequence of HHHRSLH, or a derivative thereof.

8. The chimeric protein of any one of claims 1-6, wherein the effector domain comprises a sequence of designed ZFTF motif with specific amino acid residues arranged to recognize and bind to the DNA sequence of the target gene or the promoter / enhancer site of the target gene.

9. The chimeric protein of any one of claims 1-6, wherein the transcription activator in the effector domain is VP60 or p65AD.

10. The chimeric protein of any one of claims 1-6, wherein the effector domain comprises a native transcription factor, variant of a native transcription factor, and / or a designed Zinc Finger transcription factor (ZFTF).

11. The chimeric protein of claim 9 or 10, wherein the DNA binding motif included in the effector domain specifically recognizes the promoter / enhancer sequence of a gene of interest for transcriptional modulation.

12. The chimeric protein of any one of claims 1-6, wherein: (a) the carrier domain is CypB, a binding ligand of CD147, of which the over-expression is induced by lactate overproduction in cancer cells; (b) the effector domain is capable of blocking peptide of E2F (BP-E2F) or c-Myc promoter-binding protein-1 (MBP-1) in cancer cells; and (c) the linker connects the carrier domain and the effector domain.

13. The chimeric protein of any one of claims 1-6, wherein (a) the carrier domain is selected from NRG1 and FGF2 for their nuclear trafficking activity within tumor cells; and (b) the effector domain comprises specially designed zinc finger nuclear transport factors (ZNTFs) fused with a repressor motif (KRAB) for specific binding to genes encoding enzymes crucial for the glycolytic pathways of glucose and glutamine.

14. The chimeric protein of claim 13, wherein NRG1 binds to its receptor including EGFR, HER2 and HER3 that are overexpressed in various tumors, including leukemia, lymphoma, glioblastoma, neuroblastoma, sarcomas, renal, esophageal, head and neck, melanoma, breast, colorectal, bladder, gastric, lung, liver, ovarian, endometrial and pancreatic cancers, facilitating internalization and nuclear transport within target tumor cells; wherein the repressor motif Krüppel-associated box repressor (KRAB) in the effector domain allows for specific binding to and transcription inhibition of the targetDocket No.: 374819.00003 gene(s) involved in the glycolytic pathways, thereby disrupting the metabolic supply of glucose and glutamine, crucial for cancer cell growth; and / or wherein FGF2 binds to its receptor including FGFR1 and FGFR2 that are overexpressed in various tumors, including breast, colorectal, bladder, gastric, lung, and pancreatic cancers, facilitating internalization and nuclear transport within target tumor cells.

15. The chimeric protein of claim 13 or 14, designed to selectively target tumor cells while sparing immune cells due to very low levels of EGFR and HER2 and the absence of the HER3 on the membranes of the latter.

16. The chimeric protein of any one of claims 1-15, for use in the treatment of a cancer or an immune-related disease.

17. The chimeric protein for use of claim 16, wherein the cancer is selected from hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), endometrial carcinoma (EC), ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, and gastric cancer; and the chimeric protein is administered either alone or in combination with an additional cancer therapy to enhance treatment outcomes.

18. A therapeutic composition comprising a carrier domain and an effector domain, wherein: (a) the carrier domain is selected from a group of secreted factors with binding receptors that are present on cancer cell membranes but undetectable or very low on immune cells, such as T cells; and (b) the effector domain is functionally active to modulate transcription of a specific gene after being transported to the nucleus of cancer cells.

19. The therapeutic composition of claim 18, wherein the carrier domain is capable of ensuring nuclear delivery and localization of the functionally active effector domain to cancer cells, while immune cells remain underexposed or unexposed to the therapeutic due to a low level or absence of the binding receptor for the carrier domain.

20. The therapeutic composition of claim 18 or 19, wherein the polypeptide or custom designed ZFTFs are engineered to repress the transcription of genes encoding critical enzymes mediating the energy supplies from glycolytic pathways sourced from preferred glucose and glutamine within cancer cells.

21. A pharmaceutical composition comprising a chimeric therapeutic protein according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, formulated for administration via routes selected from intravenous, intramuscular, subcutaneous, intra-dermal, and / or other suitable routes of administration.Docket No.: 374819.00003 23. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a chimeric therapeutic protein according to any one of claims 1-15, a therapeutic composition according to any one of claims 18-20, or a pharmaceutical composition of claim 21 or 22.

24. The method of claim 23, wherein the cancer is selected from leukemia, lymphoma, breast cancer, lung cancer, colon cancer, gastric cancer, hepatoma, glioblastoma, esophageal cancer, pancreatic cancer, ovarian cancer, prostate cancer, colorectal cancer, renal cancer, bladder cancer, head and neck cancer, and melanoma.

25. The method of claim 24, wherein the chimeric therapeutic protein is administered systemically or locally to the cancer site.

26. The method of any one of claims 23-25, wherein the chimeric protein is administered in combination with an immunotherapy agent (e.g., an immune checkpoint inhibitor), a monoclonal antibody, an adoptive cell therapy, a chemotherapy, and / or another targeted therapy.

27. A method for modulating transcription of a disease-associated gene, comprising the steps of: (a) providing a population of target cells; (b) introducing the chimeric protein of any one of claims 1-15 into the target cells, allowing the chimeric protein to deliver the effector domain to the nuclei of the target cells; and (c) observing the modulation of transcription of the disease-associated gene.

28. The method of claim 27, wherein the gene is associated with specific pathologies or conditions; and wherein the modulation of gene expression leads to the amelioration of said pathologies or conditions, immune-related diseases, fibrotic diseases, diabetes, obese, sepsis, and / or aging.

29. A method for inducing mammalian cells into induced pluripotent stem cells (iPSCs), comprising introducing a chimeric protein according to any one of claims 1-15 into said mammalian cells, wherein the chimeric protein is capable of activating genes encoding essential transcription factors including c-Myc, klf4, Oct4, and Sox2, as well as other factors promoting cellular stemness such as Nano, Lin28, Call4, Glis1, Essrb, Elf5, UTF1, and Pou2f2.Docket No.: 374819.00003 30. The method of claim 29, wherein the induction of iPSCs serves as a therapeutic intervention for the treatment of diseases or disorders characterized by cellular dysfunction or depletion.

31. A kit incorporating a chimeric protein according to any one of claims 1-15.

32. A method for producing a chimeric protein according to any one of claims 1-15, comprising expression of a recombinant DNA construct encoding the chimeric protein in host cells, which can be bacterial or mammalian in nature.

33. A process for producing a chimeric protein according to any one of claims 1-15, comprising: (a) synthesizing polypeptide fragments that make up the carrier domain and effector domain protein motifs; and (b) covalently conjugating the polypeptide fragments through a chemical reaction or enzymatic ligation.

34. A method for enhancing pharmacokinetic and / or pharmacodynamic properties of a chimeric therapeutic according to any one of claims 1-15, comprising a step of modifying the protein by covalently attaching it to a polymer to improve its half-life in bloodstream, wherein said polymer is selected from polyethylene glycol (PEGylation), poly(N-vinyl pyrrolidone) (PVP), poly(lactic acid) (PLA), poly(ethylene glycol) methyl ether acrylate (PEGMA), Poly(lactic-co-glycolic acid) (PLGA), poly(hydroxypropyl)methacrylate (HPMA), polyoxylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and water-soluble polyoxyethylated polyps.