Programmable DNA proteolytic target chimeras and methods of use thereof

Abstract

Described herein are programmable DNA proteolytic target chimera complexes that can be used both for the direct treatment of cancer by inhibiting biochemical pathways that are overexpressed in cancer cells, and for the indirect treatment of cancer by recruiting the E3 ligase complex to engage with a protein of interest or a mutant thereof and initiating proteolysis. Also described herein are methods of using the complexes in the treatment of cancer, as well as compositions comprising the complexes.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to compositions comprising programmable DNA proteolysis targeting chimeras (DNA-PROTACs) and methods of use thereof. Also described herein are methods of killing hyperproliferative cells using the DNA-PROTACs and DNA-PROTAC compositions.

[0002] RELATED APPLICATIONS

[0003] This disclosure claims priority to U.S. Provisional Application No. 63 / 441,956, filed January 30, 2023, the contents of which are incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing submitted electronically via EFS-Web and is hereby incorporated by reference in its entirety. The file was created on January 22, 2024, is named G8118-03701_SL.xml, and is 112,824 bytes in size. BACKGROUND

[0006] The human proteome comprises approximately 20,000 proteins, of which more than 600 proteins are estimated to be functionally important for various types of cancer, including nearly 400 non-enzymatic proteins that are challenging to target through traditional occupancy-driven pharmacology. Occupancy-driven pharmacology using small molecule inhibitors relies on the prerequisite that the target protein has a compatible binding pocket, which renders approximately 85% of the proteome “undruggable” and amplifies the risk of cumulative toxicity and drug resistance due to the need for high working concentrations.

[0007] Proteolytic processes are essential for maintaining cellular homeostasis and are tightly regulated. Through the ubiquitin-proteasome pathway (UPP), damaged, misfolded, or excess proteins can be recognized and selectively removed. Virtually every cellular process is regulated by the UPP. As the ubiquitin system controls the basic elements of cellular function, mutations in this process are the cause of a broad range of human diseases, including cancer. The three stages of ubiquitination are as follows: (i) addition of a ubiquitin polypeptide moiety to activate an enzyme known as ubiquitin-activating enzyme (El); (ii) transfer of the ubiquitin moiety to a cysteine residue on an enzyme known as ubiquitin-conjugating enzyme (E2); and (iii) catalysis by a ubiquitin ligase (E3) of the formation of an isopeptide bond between the ubiquitin moiety and a lysine in the target protein. The specificity of the process is controlled by the E3 enzyme, which recognizes and interacts with the target protein to be degraded.

[0008] Small molecule PROTACs (proteolysis targeting chimeras) represent a new paradigm in pharmacology that has the potential to be as transformative for cancer treatment as targeted kinase inhibitors, therapeutic antibodies, or immunotherapies (Bekes, M.; Langley, D. R.; Crews, C. M., PROTAC Targeted Protein Degraders: The Past Is Prologue, Nature Reviews Drug Discovery. Nature Research March 1, 2022, pp 181-200). A typical small molecule PROTAC is composed of a target protein binding ligand, an E3 ligase recruiting ligand, and a chemical linker connecting the two ligands, such as a polyethylene glycol (PEG) or an alkyl chain. The small molecule PROTAC-mediated recruitment of E3 ligases to the target protein induces ubiquitination and subsequent protein degradation by the proteasome. Unlike traditional small molecule-based drugs used in the clinic that directly inhibit the enzymatic function of the target protein, PROTACs block both the enzymatic and non-enzymatic functions of the target protein and induce degradation of the entire protein. Following promising results from the first two PROTAC anti-cancer clinical trials, several PROTAC degraders have been developed and entered into clinical trials.

[0009] PROTACs can revolutionize the field of drug discovery and lead to a specific “event-driven” pharmacology. However, considerable challenges associated with traditional small molecule PROTACs have hindered their use in clinical translation. Low solubility, low permeability, and off-target toxicity limit the applicability of current chemical linker-based PROTACs.

[0010] Recently, nucleic acid nanotechnology has emerged as a promising approach for cancer targeting and therapy. SUMMARY

[0011] As described in the present disclosure, the present invention provides a programmable DNA proteolysis targeting chimera complex comprising: (a) a first DNA strand containing one or more independent E3 ligase ligands; and (b) a second DNA strand comprising one or more independent protein of interest (POI) targeting ligands. At least a portion of the first DNA strand is complementary to a portion of the second DNA strand, and the first and second DNA strands form a DNA duplex. The one or more independent E3 ligase ligands are connected to the first DNA strand at selected positions on the first DNA strand. The one or more independent POI targeting ligands are connected to the second DNA strand at selected positions on the second DNA strand.

[0012] In some aspects, the programmable DNA proteolysis target chimera complex further comprises a targeting moiety selected from a cell penetrating peptide or a blood brain barrier traversing agent. In some aspects, the blood brain barrier traversing agent is a lipid or a cholesterol derivative.

[0013] In some aspects, the plurality of independent POI targeting ligands target different proteins. In some aspects, the plurality of independent POI targeting ligands target different portions of the same protein. In some aspects, there are at least two independent POI targeting ligands. In some aspects, there are at least two independent E3 ligase ligands.

[0014] In some aspects, the E3 ligase ligand is covalently linked to the first DNA strand. In some aspects, the E3 ligase protein is complexed with the one or more E3 ligase ligands.

[0015] In some aspects, the POI targeting ligand is covalently linked to the second DNA strand. In some aspects, the protein of interest is complexed with the one or more POI targeting ligands.

[0016] In some aspects, the selected location on the first DNA strand and the selected location on the second DNA strand are separated by a distance of about 0.99 nm to about 7 nm. In some aspects, the selected location on the first DNA strand and the selected location on the second DNA strand are separated by a rotational angle of about 36 degrees to about 180 degrees around the double stranded DNA complex. In some aspects, the selected location on the first DNA strand and the selected location on the second DNA strand are separated by a distance of about one minor groove to about one major groove.

[0017] In some aspects, the first DNA strand and second DNA strand independently comprise a nuclease resistance feature. In some aspects, the nuclease resistance feature is selected from a sugar modification or an internucleoside linkage modification. In some aspects, the sugar modification is selected from a locked nucleic acid, a threose nucleic acid, or a 2’-alkoxy modification. In some aspects, the internucleoside linkage modification is a phosphorothioate, a phosphoroselenoate, or a phosphoramidate.

[0018] In some aspects, the protein of interest is selected from CDK6, CDK4, BCR-Abl, EGFR, BTK, BRD4, HDAC6, STAT3, BCL-Xl, FAK, P38-alpha, myc, Arora, Ras, and Jak.

[0019] In some aspects, the present disclosure provides a method of killing a cancer cell, the method comprising contacting a programmable DNA proteolysis target chimera complex described herein with the cancer cell. In some aspects, the cancer cell is a glioblastoma cancer cell.

[0020] In some aspects, the present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of a programmable DNA proteolysis target chimeric complex described herein.

[0021] In some aspects, the present disclosure provides a method of treating a proliferative disease or disorder in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a complex described herein. In some aspects, the proliferative disease or disorder is a cancer.

[0022] In some aspects, the present disclosure provides a method of reducing proliferation of a cancer tumor cell, the method comprising contacting the cancer tumor cell with a complex described herein.

[0023] In some aspects, the present disclosure provides use of a complex described herein in the manufacture of a medicament for treating a cancer in a subject.

[0024] In some aspects, the present disclosure provides a composition comprising a complex described herein and a pharmaceutically acceptable carrier.

[0025] In some aspects, the present disclosure provides use of a composition comprising a complex described herein in the manufacture of a medicament for treating a proliferative disease or disorder in a subject. In some aspects, the disease or disorder is a cancer.

[0026] In some aspects, the present disclosure provides a composition comprising a complex described herein for use in the prophylactic or therapeutic treatment of a disease or disorder in a subject. In some aspects, the disease or disorder is a cancer.

[0027] In some aspects, the DNA-PROTAC comprises at least two DNA strands each independently about 20 nucleotides in length. In some aspects, the DNA-PROTAC comprises a DNA strand having at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99% or 100% sequence identity to SEQ ID NOs: 1-26. Sequences are described in the sequence ID list provided in the present disclosure. In some aspects, the DNA strand is further modified with a functional linker group. In some aspects, the functional linker group comprises an alkyne moiety, an azide moiety, or a dibenzocyclooctyne (DBCO) moiety. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1ADesign of DNA-PROTAC conjugate is shown. The ssDNA strand is functionalized with an E3 ligase binding ligand and another complementary ssDNA is functionalized with a POI (protein of interest) target binding ligand at its specific site (different positions are shown with blue balls). After successful validation of DNA-PROTAC in in vitro system, it can be functionalized with small molecules that target cells / organs (e.g. lipid molecules that cross the blood brain barrier) to avoid off-target toxicity. In some embodiments, the DNA duplex is chemically modified at the end positions with phosphorothioate nucleic acids to resist in vivo degradation.

[0029] Figure 1B Design of DNA-PROTAC for simultaneous multi-targeted protein degradation is shown.

[0030] Figure 1C Design of spatially programmable DNA-PROTAC is shown, demonstrating the spatial programmability of the complex. The distance "x" and angle of two ligands - E3 inhibitor (E3-i) and protein of interest (POI-i) along the DNA duplex can be adjusted.

[0031] Figure 1D Table showing the distance and angle obtained from a representative embodiment of spatially programmable DNA-PROTAC of the present disclosure is shown.

[0032] Figure 1E One embodiment of multi-E3 recruiting DNA-PROTAC conjugate (multi-EDPC) utilizing DNA Holliday junction binding is depicted. The DNA nanostructure can be modified with two E3 ligase binding ligands and one target ligand.

[0033] Figure 1F One embodiment of multi-target protein recruiting DNA-PROTAC conjugate (multi-TPDPC) utilizing DNA Holliday junction binding is shown. The DNA nanostructure can be modified with two different target ligands and one E3 ligase binding ligand.

[0034] Figure 2A Screened spatially programmable DTAC library is shown. From version 01 to 05, DNA-PROTAC designs with increasing distance between E3 ligase and protein of interest were prepared.

[0035] Figure 2B 8% non-denaturing PAGE gel electrophoresis of versions 01 to 05 is shown, confirming the formation of well-structured DNA-PROTAC library.

[0036] Figure 3Western blot analysis showing CDK6 degradation in U251 cells treated with distance-based DNA-PROTAC. The upper panel shows the levels of CDK6 protein after treatment with a series of DNA-PROTAC at different concentrations, and the lower panel shows β-tubulin as a loading control.

[0037] Figure 4A Dose-dependent and time-dependent degradation of CDK6 and CDK4 in U251 cells. Levels of CDK6 and CDK4 protein under different doses of DNA-PROTAC-V02 treatment.

[0038] Figure 4B Levels of CDK6 and CDK4 protein in cells after treatment with 50 nM DNA-PROTAC-V02 for the indicated time periods.

[0039] Figure 5 Quantitative confocal analysis showing CDK6 protein degradation in U251 cells, with CDK6 degradation most significant in the DNA-PROTAC group compared to control sequences of DNA-E3i and DNA-CDK6i and scrambled dsDNA, clearly demonstrating the synergistic effect of the DNA-PROTAC of the present disclosure on CDK6 degradation.

[0040] Figure 6 Proteasome-dependent CDK6 degradation in U251 cells. Proteasome inhibitor MG-132 was pre-incubated with cells before DNA-PROTAC-V02 transfection, and CDK6 levels were evaluated after 16 h.

[0041] Figure 7A DNA-PROTAC-V02-mediated CDK6 degradation at the protein level. Western blot analysis illustrates the effect of DNA-PROTAC-V02 and control treatment on CDK6 expression.

[0042] Figure 7B Levels of CDK6 and CDK4 mRNA under DNA-PROTAC-V02 treatment.

[0043] Figure 7C Western blot analysis confirming the effect of BSJ-03-123 treatment on CDK6 expression.

[0044] Figure 7D Levels of CDK6 and CDK4 mRNA under BSJ-03-123 treatment.

[0045] Figure 8Western blot analysis showing different angle-based DNA-PROTACs. Western blot analysis illustrating the effect of different angle-based DNA-PROTACs and control treatments on CDK6 expression.

[0046] Figure 9A and 9B H1 and C13 NMR spectra of selected E3 ligand pomalidomide of the present disclosure are shown.

[0047] Figure 10 Mass spectra of selected E3 ligand pomalidomide of the present disclosure are shown.

[0048] Figure 11A and 11B H1 and C13 NMR spectra of selected POI palbociclib of the present disclosure are shown.

[0049] Figure 12 Mass spectra of selected POI palbociclib of the present disclosure are shown.

[0050] Figure 13 Mass spectra of selected DNA strands of the present disclosure are shown. The figures disclose SEQ ID NOs 1-4, 3, and 5, respectively, in order of appearance.

[0051] Figure 14 Mass spectra of selected DNA strands of the present disclosure are shown. The figures disclose SEQ ID NOs 3, 6, 1, and 7, respectively, in order of appearance.

[0052] Figure 15 Chemical structure of iNH2 modifier used in the present disclosure is shown. DETAILED DESCRIPTION

[0053] The entire document is intended to be one unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if such combinations are not found together in the same sentence, paragraph or section of this document. The present disclosure illustratively described herein can suitably be practiced in the absence of any element or elements described in this document that are not specifically disclosed herein.

[0054] The present disclosure provides a chemical strategy to facilitate ligand-dependent degradation of target proteins through chemical coupling with the function of the Cereblon E3 ubiquitin ligase complex. When an E3 ubiquitin ligase covalently attaches many ubiquitin molecules to terminal lysine residues, the protein is tagged for proteasomal degradation. This process breaks the protein down into smaller peptides and ultimately into its constituent amino acids for the manufacture of new proteins.

[0055] DNA-PROTACs of the present disclosure include an E3 ligase ligand that binds to an E3 ubiquitin ligase (typically through cereblon), and a ligand for a protein of interest (POI) (also referred to as a "targeting ligand"). DNA-PROTACs can be used for therapeutic purposes by the methods described herein. Compositions thereof are also provided herein, as well as methods of making and manufacturing.

[0056] By recruiting to an E3 ubiquitin ligase and subsequent ubiquitination, DNA-PROTACs facilitate proteasome-mediated degradation of specific proteins. These compounds resemble drugs, exhibiting the potential to control the temporal levels of selected proteins of interest. By eliminating pathogenic or oncogenic proteins, DNA-PROTACs can render proteins of interest inactive when added to cells or administered to animals or humans, providing a new paradigm for disease treatment.

[0057] The human proteome comprises approximately 20,000 proteins, of which more than 600 proteins are estimated to be functionally important for various types of cancer, including nearly 400 non-enzymatic proteins that are challenging to target through traditional occupancy-driven pharmacology. Occupancy-driven pharmacology using small molecule inhibitors relies on the prerequisite that the target protein has a compatible binding pocket, which renders approximately 85% of the proteome “undruggable” and exacerbates the risks of cumulative toxicity and drug resistance due to the need for high working concentrations. To address these challenges, a new drug discovery strategy called targeted protein degradation (TPD) has been developed that requires the degradation rather than just the inhibition of proteins (Pettersson, M.; Crews, C. M., PROteolysis TArgeting Chimeras (PROTACs) — Past, Present and Future, Drug Discovery Today: Technologies. 2019. doi.org / 10.1016 / j.ddtec.2019.01.002; Li, X.; Pu, W.; Zheng, Q.; Ai, M.; Chen, S.; Peng, Y., Proteolysis-Targeting Chimeras (PROTACs) in Cancer Therapy, Molecular Cancer. 2022. doi.org / 10.1186 / s12943-021-01434-3). By enlisting protein degradation pathways, TPD facilitates the complete removal of protein molecules from within or outside of cells. While pioneering proteolysis-targeting chimeras (PROTAC) technology and molecular glia hijack the ubiquitin-proteasome system (UPS), newer modalities synergistically exploit the autophagy or endo-lysosomal pathways. With this mechanism, TPD holds the promise of expanding the druggable space far beyond the reach of small molecule inhibitors.The event-driven mechanism of action (MOA) of TPDs offers several advantages over traditional occupancy-driven small molecule inhibitors, such as catalytic properties, dose reduction, and efficacy against resistance mechanisms (Martin-Acosta, P.; Xiao, X., PROTACs to Address the Challenges Facing Small Molecule Inhibitors, European Journal of Medicinal Chemistry. 2021. doi.org / 10.1016 / j.ejmech.2020.112993; Winter, G. E. et al., Phthalimide Conjugation as a Strategy for in Vivo Target Protein Degradation, Science 2015, 348 (6241). doi.org / 10.1126 / science.aab1433).

[0058] A key focus of TPDs is the development of heterobifunctional small molecule degraders, including PROTACs, which contain two linked moieties, one that binds a protein of interest (POI) and the other that binds an E3 ligase. When the PROTAC is active, the target protein ligand binds to the POI and the E3 ligase ligand binds to the E3, facilitating the formation of a ternary complex of the POI and E3 through a flexible linker. At the same time, the POI is tagged with ubiquitin, leading to the degradation of the POI by the proteasome (Burslem, G. M.; Crews, C. M., Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell. 2020. doi.org / 10.1016 / j.cell.2019.11.031; Schapira, M.; Calabrese, M. F.; Bullock, A. N.; Crews, C. M., Targeted Protein Degradation: Expanding the Toolbox, Nat Rev Drug Discov 2019, 18 (12). doi.org / 10.1038 / s41573-019-0047-y).

[0059] Since the first development of PROTAC, several key discoveries have been made. So far, a variety of PROTAC-like molecules have entered clinical trials. However, there are still considerable challenges and some limitations that hinder the clinical application of PROTACs, for example: 1) Bioavailability: PROTACs with large size (>800 Da) and high polarity exhibit limited water solubility, hindering their passage through physiological barriers and cell membranes. 2) Side effect risk: Non-selective expression of E3 ligases in both disease and normal tissues can cause serious side effects when PROTACs are widely distributed. 3) E3 ligand limitation: Most PROTACs rely on CRBN or VHL ligands, which limit their therapeutic efficacy due to cell-specific E3 ligase expression and drug resistance issues. 4) Conditional activation: Due to the limited selectivity of existing PROTRACs, the requirement for selective degradation of POIs in diseased cells while avoiding affecting normal cells poses a major challenge to existing PROTRACs.

[0060] Nucleic acid-based drugs have become an exciting new frontier in therapeutics. This emerging class of therapeutic agents encompasses clinically available nucleic acid drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), aptamers, and mRNA vaccines that are currently undergoing clinical trials. These nucleic acid drugs offer compelling advantages, including low toxicity and exceptional specificity, which highlight their prospects for precision medicine. In recent years, there has been a rapidly growing interest in nucleic acid-based TPD strategies.

[0061] While existing nucleic acid-based TPD technologies focus on using aptamers or oligonucleotides as binding motifs, they often rely on liposome transfection and typically require high concentrations to achieve efficient protein degradation. However, the properties of DNA materials, i.e., the high precision of Watson-Crick base pairs, have not been fully utilized for TPD. With the rapid development of nucleic acid nanotechnology and the ability to design DNA nanostructures, various design principles have been used to achieve structural construction in various dimensions by rationally self-assembling nanoparticles or biomolecular scaffolds, such as planar tiles, origami structures, and dynamic nanomechanical systems.

[0062] Described herein is a DNA-based programmable proteolysis targeting chimera (DNA-PROTAC) that can be conditionally activated for efficient delivery and highly specific protein degradation. This DNA nanostructure-based innovation offers key advantages over existing PROTACs, including efficient intracellular delivery, multi-targeting, conditional activation, and enhanced protein degradation. Described herein is the development of a DNA-based protein degradation system that, by hitchhiking E3 and ligands for CDK6, leads to efficient degradation of CDK4 / 6 proteins. The present disclosure provides a programmable DNA platform based on allosteric effects that can be modulated to develop conditional DNA-PROTACs. The present disclosure includes: elucidation of the programmability of DNA duplex-based protein degradation systems by transitioning from DNA duplexes to branched DNA nanostructures; demonstration of protein degradation differences caused by spatial distance and evaluation of the efficiency of multi-target degradation; integration of selected DNA nanostructures for direct cytoplasmic delivery systems with the DNA-PROTACs described herein; and implementation of conditional activation of protein degradation using two different design approaches: i) toe-end mediated conditional activation and ii) allosteric effect-mediated conditional activation of DNA-PROTACs.

[0063] DNA-PROTACs can be used alone or in combination with therapeutic agents for specific target proteins for therapeutic applications. Compositions, modes of use, and manufacturing processes for them are also provided herein.

[0064] In one embodiment, the protein of interest is a protein that is not druggable in the classical sense because it has no binding pocket or active site that can be inhibited or otherwise bound, and cannot be easily allosterically regulated. In another embodiment, the protein of interest is a protein that is druggable in the classical sense. Examples of proteins of interest are provided herein.

[0065] The present application relates to DNA-PROTACs that are covalently linked to target protein ligands through chemical coupling (e.g., click chemistry), and can optionally further comprise linkers of different lengths and functionalities. The present application also relates to a technology platform that uses the DNA-PROTACs of the present disclosure to bring target proteins of interest to E3 ligases, such as CRBN, for ubiquitination and subsequent proteasomal degradation.

[0066] This technology platform provides therapies based on reducing the levels of selected proteins of interest through degradation. This new technology allows for a wider applicability of targeted degradation than existing methods, in terms of possible targets and different cell lines or different in vivo systems.

[0067] The DNA-PROTACs of the present application can provide important clinical benefits to patients, particularly for the treatment of disease states and conditions modulated by the protein of interest.

[0068] Without wishing to be bound by any theory, it is believed that the present disclosure is based at least in part on the discovery that novel programmable DNA-PROTACs that degrade selected target proteins and / or mutants thereof are useful for treating diseases mediated by the protein or mutant thereof, particularly non-small cell lung cancer, colorectal cancer, gastric cancer, liver cancer, infiltrating breast cancer, lung adenocarcinoma, uterine cancer, adrenal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, bladder cancer, endometrial cancer, prostate cancer, low-grade glioma, glioblastoma, spindle cell carcinoma, soft tissue sarcoma, thyroid papillary carcinoma, head and neck squamous cell carcinoma, congenital fibrosarcoma, congenital mesoblastic nephroma, secretory breast cancer, breast analog secretory carcinoma, acute myeloid leukemia, ductal carcinoma, lung neuroendocrine tumor, pheochromocytoma, and Wilms tumor. In some embodiments, the cancer is glioblastoma.

[0069] Certain Definitions

[0070] Unless otherwise indicated, technical terms have their usual meanings. If certain terms are given specific definitions, the definitions will be given below in the context in which the term is used.

[0071] Unless otherwise expressly specified, a reference to a number that is not a specific number can be a reference to a plural number.

[0072] The term "about" as used herein means ± 10%.

[0073] The chemical structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a 13 C or 14 C by 12 C are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0074] When a range of values is listed, it is intended to encompass each value and sub-range within the range.

[0075] The term "operably linked" as used herein refers to the association of two chemical moieties such that the function of one is affected by the other, e.g., the manner in which the components described are arranged so as to perform their usual function.

[0076] The term "nucleic acid" as used herein refers to a polymer of deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, comprising monomers (nucleotides) containing a sugar, a phosphate, and a base which is either a purine or a pyrimidine. 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

[0077] The terms "nucleotide sequence" and "nucleic acid sequence" as used herein refer to the sequence of bases (purines and / or pyrimidines) in a DNA or RNA polymer, which can be single- or double-stranded. In some embodiments, the nucleotide sequence comprises synthetic, non-natural, or modified nucleotide bases and / or backbone modifications (e.g., modified oligomers, which can include or exclude morpholino oligomers, phosphorodiamidate morpholino oligomers, or in vivo morpholino oligomers). The terms "oligomer," "oligonucleotide," and "oligomer" can be used interchangeably and refer to such sequences of purines and / or pyrimidines. The terms "modified oligomer," "modified oligonucleotide," or "modified oligomer" can similarly be used interchangeably and refer to such sequences containing synthetic, non-natural, or modified bases and / or backbone modifications (e.g., chemical modifications to the internucleotide phosphate linkages and / or backbone sugars).

[0078] Modified nucleotides can include or exclude: alkylated purines, alkylated pyrimidines, acylated purines, and acylated pyrimidines. These classes of pyrimidines and purines can include or exclude: pseudoisocytosine, N4,N4-ethanol cytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxy carbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid methylester, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2,6-diaminopurine, methylpseudouracil, 1-methylguanine, 1-methylcytosine. Backbone modifications can include or exclude chemical modifications to the phosphodiester linkage. Chemical modifications to the phosphodiester linkage can include or exclude, for example, phosphorodiamidate, phosphorothioate (PS), N3' phosphoramidate (NP), boranophosphonate, 2',5'-phosphodiester, amide linkage, phosphonoacetate (PACE), morpholino, peptide nucleic acid (PNA), inverted linkages (5'-5' and 3'-3' linkages), and sugar modifications (e.g., 2'-O-Me, UNA, LNA).

[0079] The oligonucleotides described herein can be synthesized using solid phase or solution phase synthesis methods. In some embodiments, the oligonucleotides are synthesized using solid phase phosphoramidite chemistry (U.S. Patent No. 6773885, which is incorporated herein by reference) and an automated synthesizer. Chemical synthesis of nucleic acids allows production of a variety of forms of nucleic acids that have modified linkages, chimeric compositions, and non-standard bases or modifying groups attached at selected positions throughout the length of the nucleic acid. In some embodiments, the oligonucleotides described herein can be synthesized using enzymatic methods, which can include the addition of individual bases by enzymes.

[0080] Some embodiments of the application include isolated or substantially purified nucleic acid compositions. As used herein, an "isolated" or "purified" DNA molecule or RNA molecule means a DNA molecule or RNA molecule that exists apart from its natural environment, and thus is not a product of nature. An isolated DNA molecule or RNA molecule can exist in a purified form, or in a non-natural environment. In some embodiments, the non-natural environment can include or exclude a transgenic host cell. In some embodiments, the term "isolated" or "purified" includes a nucleic acid molecule that is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid was derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0081] A "portion" in relation to a nucleic acid molecule, sequence or segment of the application means a sequence of at least 3 nucleotides to at most 20 nucleotides (and any number of nucleotides therebetween).

[0082] "Homology" refers to the percentage of identity between two polynucleotide or two polypeptide sequences. Two DNA or polypeptide sequences are "homologous" to one another when they exhibit at least about 75% to 85% (including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%), at least about 90%, or at least about 95% to 99% (including 95%, 96%, 97%, 98%, 99%) of continuous sequence identity over a specified length of the sequences.

[0083] The terms "sequence identity" or "identity" or "homology" as used herein in the context of two nucleic acid or polypeptide sequences refers to a specified percentage of residues that are the same between the two sequences when aligned for maximum correspondence by a sequence alignment algorithm or visual inspection of an alignment window. In some embodiments, the identity between any two nucleic acid sequences is 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%, or 99%.

[0084] As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percent sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity.

[0085] For sequence comparison, typically one sequence acts as the reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the program parameters.

[0086] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Typically, stringent conditions will be those in which the particular nucleic acid sequence in question hybridizes to itself, but not to other sequences. Stringent conditions will be those in which the particular nucleic acid sequence in question hybridizes to itself but not to, for example, a different nucleic acid sequence, and will typically be those in which the particular nucleic acid sequence hybridizes to a perfectly matched sequence, but not to an altered sequence. Stringent conditions can be defined in terms of melting temperatures (Tm) under various conditions. Tm is the temperature (under a defined ionic, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence specifically. Stringent conditions can be those in which the salt and temperature are selected to permit binding of nucleic acid sequences of relatively low sequence similarity. Stringent conditions can include salt concentrations of less than about 1.0 M Na ion concentration (e.g., about 0.01 to 1.0 M Na ion concentration), and / or temperature conditions of less than about 30°C (e.g., about 30°C to about 68°C). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Moderate stringency conditions can be identified as including a salt concentration of about 0.1 M to 0.3 M Na ion; a temperature of about 30°C to about 37°C; and / or an equivalent moderate stringency condition. High stringency conditions can be identified as including a salt concentration of about 0.01 M to 0.2 M Na ion; a temperature of about 37°C to about 68°C; and / or an equivalent high stringency condition. Very high stringency conditions can be identified as including a salt concentration of about 0.0 M to 0.1 M Na ion; a temperature of about 42°C to about 68°C; and / or an equivalent very high stringency condition. Extremely high stringency conditions can be identified as including a salt concentration of about 0.0 M Na ion; a temperature of about 68°C; and / or an equivalent extremely high stringency condition.

[0087] The phrase "specifically hybridizes" refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent conditions, when that sequence is present in a complex background of other sequences. The term "substantially binds" as used herein refers to the complementary hybridization between a probe nucleic acid and a target nucleic acid and includes a small number of mismatches in certain embodiments, which are encompassed by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0088] The term "complementary" as used herein refers to the general concept of complementary base pairing between two nucleic acids aligned with each other in an anti-parallel orientation. Two nucleic acids are considered to be complementary to each other at a position when the nucleotide position in each molecule is occupied by a nucleotide that is generally capable of base pairing with the other. Two nucleic acids are substantially complementary when at least about 50%, at least about 60%, or at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that are generally capable of base pairing with each other (e.g., A:T (A:U for RNA) and G:C nucleotide pairs).

[0089] The term "derived" or "directed" as used herein with respect to a nucleotide molecule refers to the sequence identity of the molecule with a particular molecule of interest.

[0090] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and quaternary alkylammonium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate. In some embodiments, the DNA-PROTAC of the present disclosure can be in the form of a sodium salt, potassium salt, ammonium salt, or quaternary ammonium salt.

[0091] The term “administering” means implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein or a composition thereof into or onto a subject.

[0092] The term “subject” as used herein refers to a human, a higher non-human primate, a rodent, a livestock, a dairy cow, a horse, a pig, a sheep, a dog, and a cat. In one embodiment, the subject is a human.

[0093] The term "therapeutically effective amount" in the context of treating a disease state / condition means an amount of a therapeutic agent that, when administered in a single dose or multiple doses, is capable of producing any detectable, positive effect on any symptom, aspect, or characteristic of the disease state / condition. Such effect need not be absolute to be beneficial.

[0094] In certain embodiments, the therapeutically effective amount is an amount effective to promote degradation of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of a target protein of interest in a selected tissue. In certain embodiments, the effective amount is an amount effective to promote degradation of 10% to 99% (inclusive) of a target protein of interest in a selected tissue.

[0095] The term "treatment" refers both to therapeutic treatment, as well as prophylactic or preventative measures, wherein the object is to prevent or lessen an undesired physiological change or disorder. For the purposes of this disclosure, beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented or pre-empted.

[0096] The term "inhibit" or "reduce" or any variation of these terms includes any measurable decrease or complete inhibition in order to achieve a desired result. The term "promote" or "increase" or any variation of these terms includes any measurable increase or production of a protein or molecule in order to achieve a desired result.

[0097] The term "prevent" or any variation of this term means to slow down, stop, or reverse the progression toward a result. Prevention can be any slowing down of the progression toward a result.

[0098] As used herein, the term "proliferative disease" refers to a disease that occurs as a result of abnormal growth or expansion due to cell proliferation (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases can be associated with 1) pathological angiogenesis, such as proliferative retinopathies and tumor metastasis; 2) pathological migration of cells from their normal location (e.g., tumor cell metastasis); 3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) pathological proliferation of normally quiescent cells. In some embodiments, the proliferative disease includes cancer (i.e., "malignancy"), benign tumors, angiogenesis, inflammatory diseases, and autoimmune diseases.

[0099] As used herein, the term "cancer" refers to a large family of various diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and growth lead to malignant tumors, which invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancer, metastatic cancer, and recurrent cancer.

[0100] The tumor microenvironment is an important aspect of cancer biology that is involved in tumorigenesis, tumor progression, and therapeutic response. The tumor microenvironment is composed of a heterogeneous cell population, including malignant cells and cells that support the tumor proliferative, invasive, and metastatic potential through extensive interactions.

[0101] In some embodiments, the cancers treatable by the DNA-PROTAC of the present disclosure can include or exclude: lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); kidney cancer (e.g., nephroblastoma (also known as Wilms' tumor), renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast carcinoma, breast medullary carcinoma); brain cancer (e.g., meningioma, glioblastoma, neuroglioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain disease (e.g., alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumor; immunocytic amyloidosis; liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), agnogenic myeloid metaplasia (AMM) (also known as myelofibrosis (MF)), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., ovarian cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasmacytoma; paraneoplastic syndrome; intraepithelial neoplasm; prostate cancer (e.g., prostate adenocarcinoma);rectal cancer; rhabdomyosarcoma; salivary gland carcinoma; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small bowel cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., thyroid papillary carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). In some embodiments, the DNA-PROTAC of the present disclosure can treat a glioblastoma. In some embodiments, the DNA-PROTAC of the present disclosure can kill a cancer cell, wherein the cancer is any of the above cancers. In some embodiments, the DNA-PROTAC of the present disclosure can kill a glioblastoma cancer cell.

[0102] Programmable DNA-PROTACs

[0103] For the purposes of the present disclosure, the terms “DNA-PROTAC” and “programmable DNA-PROTEC” and “DNA proteolysis targeting chimera” can be used interchangeably.

[0104] In some embodiments, the present disclosure provides a DNA-PROTAC comprising a ligand targeting a protein of interest (POI) and a ligand targeting an E3 ligase and a double-stranded DNA segment. Each of the ligands can be attached to the DNA segment by conjugation to opposite DNA strands (the “first DNA strand” and the “second DNA strand,” respectively). The attachment can occur through various bioconjugation chemistries, including click chemistry, strained polycyclic click chemistry, and the like. In some embodiments, the attachment can occur through a spacer selected from an alkyl chain or a PEG (polyethylene glycol) chain.

[0105] In some embodiments, the present disclosure provides a programmable DNA proteolysis targeting chimera complex (DNA-PROTAC) comprising: a first DNA strand comprising one or more independent E3 ligase ligands; and a second DNA strand comprising one or more independent protein of interest (POI) targeting ligands. At least a portion of the first DNA strand is complementary to a portion of the second DNA strand, and the first and second DNA strands form a DNA duplex. The one or more independent E3 ligase ligands are attached to the first DNA strand at one or more independently selected positions on the first DNA strand. The one or more independent POI targeting ligands are attached to the second DNA strand at one or more independently selected positions on the second DNA strand.

[0106] In some embodiments, the DNA-PROTAC can further comprise a targeting moiety. The targeting moiety can be selected from a cell penetrating peptide or a blood brain barrier traversing agent. The blood brain barrier traversing agent can be a lipid, a neutral amino acid, a hormone, a vitamin, a cholesterol derivative (a hydroxyl or a transacetyl ester at each hydroxyl moiety on the cholesterols). The vitamin can be a vitamin B, a vitamin D, or a vitamin E. The hormone can be a cortisol, an aldosterone, a DHEA, an androgen, an epinephrine, a norepinephrine, an estrogen, a progesterone, or a testosterone. The lipid can be a C6-C18 fatty acid or a glycerol conjugate thereof. The cell penetrating peptide can be any cell penetrating peptide listed in US Patent Nos. US10288601, US10967000, US10626147, US10300118, US10253099, US10421784, and US9303076, each of which is incorporated herein by reference.

[0107] To improve degradation efficiency in the presence of E3 ligase, in some embodiments, the DNA-PROTAC can comprise two or more POI targeting ligands targeting different proteins. The two or more POI targeting ligands can be the same or different. The DNA-PROTAC can recruit two different proteins for degradation by E3 ligase tagging, thereby disrupting or reducing the levels of two or more different proteins. When two different proteins are degraded by the same DNA-PROTAC, multiple pathways can be reduced or inhibited, thereby producing a synergistic effect in disrupting cellular functions of target cells (e.g., proliferative cells (especially cancer cells)).

[0108] In some embodiments, to improve avidity for a target protein of interest, the DNA-PROTAC can comprise two or more targeting ligands for the same protein of interest. The targeting ligands can be the same or different. When the targeting ligands are the same, avidity is enhanced. When the targeting ligands are different, avidity is enhanced. The different targeting ligands can be configured to bind different portions of the same protein.

[0109] In some embodiments, to improve affinity for recruiting E3 ligase, the DNA-PROTAC can comprise two or more E3 ligase ligands. When a protein of interest binds to a DNA-PROTAC comprising at least two bound E3 ligases, the statistical degradation rate of the protein can increase, resulting in a faster overall protein degradation rate.

[0110] The one or more independently selected positions on the first or second DNA strand where the E3 ligase or POI targeting ligand is located can be configured to vary in space and angle. Using a double-stranded DNA helix, the distance and orientation between the E3 ligase ligand and POI targeting ligand can be controlled. Furthermore, using a Holliday junction can enhance structural rigidity to ensure the positions between the E3 ligase ligand site and POI targeting ligand site are structurally rigidly separated (Chem. Soc. Rev., 2021, 50, 11966-11978, doi.org / 10.1039 / D1CS00250C). Additionally, the design of the DNA double-stranded sequence can be selected to modulate the persistent length of the double-strand, which further maintains the helix rigidity to keep the positions of the POI targeting ligand and E3 ligase ligand.

[0111] In some embodiments, the selected position on the first DNA strand and the selected position on the second DNA strand can be separated by a distance of about 0.99 nm (99 Angstroms) to about 7 nm (inclusive of the end values). In some embodiments, the selected position on the first DNA strand and the selected position on the second DNA strand can be separated by a rotational angle of about 36 degrees to about 180 degrees (inclusive of the end values) around the double-stranded DNA complex. In some embodiments, the selected position on the first DNA strand and the selected position on the second DNA strand can be separated by a distance of about one minor groove to about one major groove.

[0112] In some embodiments, the sequence of the DNA strand can be modified to include a nuclease resistance feature. Avoiding premature degradation by nucleases increases the circulating half-life of the DNA-PROTAC after administration to a subject. The nuclease resistance feature can be a sugar modification or an internucleoside linkage modification. The sugar modification can be a locked nucleic acid, a threose nucleic acid, or a 2’-alkoxy modification. The internucleoside linkage modification can be a phosphorothioate, a phosphoroselenoate, or a phosphoramidate.

[0113] As used herein, the terms "targeting ligand" and "protein targeting ligand" and "protein of interest (POI) targeting ligand" are used interchangeably and shall be construed to encompass any molecule, from small molecules to large proteins, that associates or binds with a protein of interest. In certain embodiments, the targeting ligand and corresponding target protein of interest are listed in Table 1 and include CDK6, CDK4, BCR-Abl, EGFR, BTK, BRD4, HDAC6, STAT3, BCL-Xl, FAK, P38-alpha, myc, Arora, Ras, and Jak as the target protein of interest. In certain embodiments, the target protein of interest is a mutant of CDK6, CDK4, BCR-Abl, EGFR, BTK, BRD4, HDAC6, STAT3, BCL-Xl, FAK, P38-alpha, myc, Arora, Ras, and Jak.

[0114] In one embodiment, the mutant of the selected target protein of interest can include or exclude translocation, deletion, or inversion events that cause or are caused by a medical disorder. In some embodiments, the mutation of the selected target protein of interest can include or exclude post-translational modifications selected from the group consisting of phosphorylation, acetylation, acylation (including propionylation and crotonylation), N-linked glycosylation, O-linked glycosylation, amidation, hydroxylation, methylation and polymethylation, pyroglutamylation, myristoylation, farnesylation, geranylgeranylation, ubiquitination, sumoylation, sulfation, and combinations thereof. In some embodiments, the post-translational modification is caused by a medical disorder.

[0115] In some embodiments, the target protein of interest is a mutant protein found in a cancer cell, or a protein that is partially or fully gain-of-function or loss-of-function encoded, for example, by a nucleotide polymorphism. In some embodiments, the protein targeting ligand targets an abnormal form of the protein rather than a normal form of the protein.

[0116] In some embodiments, the target-binding ligand is a ligand, drug, antibody, aptamer, scFv, or nanobody that preferentially binds to a target protein of interest. In some embodiments, the target-binding ligand is a target-binding ligand listed in Table 1. In some embodiments, the target-binding ligand is selected from paldocicib, GNF-5, gefitinib, inotinib, OTX-015, SD-36, ABT-263, and fostamatinib, and variants thereof. In some embodiments, the target-binding ligand is an HDAC6 inhibitor. In some embodiments, the HDAC6 inhibitor is selected from vorinostat, romidepsin, panobinostat, and belinostat. In some embodiments, the HDAC6 inhibitor is selected from CAY10603, WT161, ACY-738, KA2507, Citarinostat (ACY-241), Tubacin, Ricolinostat (ACY-1215), Nexturastat A, ACY-775, Tubastatin A HCl, Tubasatin A TFA, Tubasttin A, HPOB, and SKLB-23bb (all available from Selleck Chem, USA). In some embodiments, the target-binding ligand is a BRD4 inhibitor. The BRD4 inhibitor can include a BRD4 inhibitor provided in U.S. Patent No. 10646575, which is incorporated by reference herein. In some embodiments, the BRD4 inhibitor is selected from BRD4770, BRD4 inhibitor-10, FL-411, BI 2536, I-BET151 (GSK1210151A), PFI-1 (PF-6405761), (+)-JQ1, Bromosporine, SGC-CBP30, CPI-203, MS436, Birabresib (OTX015), XMD8-92, GSK1324726A (I-BET726), I-BRD9, Pelebresib (CPI-0610), Mivebresib (ABBV-075), AZD5153 6-hydroxy-2-naphthoic acid, F2523, ABBV-744, ZL0420, INCB054329, dBET6, dBET1, PLX51107, ARV-825, A1874, SRX3207, dBET57, Y06036, ARV-771, MZ-1, GSK778, GSK046, (R)-(-)-JQ1 enantiomer, GNE-781, thalidomide-NH-C4-NH-Boc, and NHWD-870 (all available from Selleck Chem, USA).In some embodiments, the target-binding ligand is a CDK6 inhibitor. The CDK6 inhibitor can include or exclude: abemaciclib, palbociclib, and ribociclib. In some embodiments, the target-binding ligand is an EGFR inhibitor. The EGFR inhibitor can include or exclude: erlotinib, osimertinib, neratinib, gefitinib, dacomitinib, lapatinib, mobociclib, and vandetanib. In some embodiments, the target-binding ligand is a BCR-Abl inhibitor. The BCR-Abl inhibitor can include or exclude: imatinib, nilotinib, dasatinib, bosutinib, ponatinib, acsiminib, and dasatinib. In some embodiments, the target-binding ligand is a BTK inhibitor. The BTK inhibitor can include or exclude: ibrutinib, acalabrutinib, and zanubrutinib. In some embodiments, the target-binding ligand is a STAT3 inhibitor. The STAT3 inhibitor can include or exclude: HJC0152, Cucurbitacin I, Cucurbitacin IIb, APTSTAT3-9R, SC-1, SC99, Shortleaf Geranilin A, Wogonoside, GYY4137, C188-9, niclosamide, STAT3-IN-1, WP1066, Cryptotanshinone (Tanshinone C), Stattic, inS3-54-A18, Resveratrol, Moracin, NSC 74859 (S3I-201), Kaempferol-3-O-rutinoside, Ochromycinone (STA-21), Colifoskinic acid C17:1, HO-3867, Napabucasin (BBI608), Artesunate (WR-256283), Bosutinib, and TPCA-1, SC-43 (all available from Selleck Chem, USA). In some embodiments, the STAT3 inhibitor is selected from IMX-110, AZD9150, Napabucasin, bazedoxifene, siltuximab, CNTO 328, ruxolitinib, itacitinib, ponatinib, and sunitinib. In some embodiments, the target-binding ligand is a BCl-XL inhibitor. The BCl-Xl inhibitor can include or exclude: ABT-263 / "Navitoclax" and A-1331852. In some embodiments, the target-binding ligand is a FAK inhibitor.The FAK inhibitors can include or exclude: TAE226 (NVP-226), VS-6062 (PF00562271), PF-573228 (PF-228), VS-6063 (defactinib), GSK2256098, VS-4718 (PND-1186), Y15, C4, R2, BI853520 (IN10018, ifenbrotinib), CT-707 (conatumumab), AMP-945 (namofumab), and APG-2449. In some embodiments, the target binding ligand is a P38-alpha inhibitor. The P38-alpha inhibitors can include or exclude: PH797804, DBM 1285 dihydrochloride, SB 706504, AL 8697, TAK 715, AMG 548, VX 745, SB 202190, SB203580, BIRB 796, SB 203580 hydrochloride, SB 239063, EO 1428, RWJ 67657, and SCIO 469 hydrochloride (all available from R&D Systems, USA). In some embodiments, the P38-alpha inhibitors can include or exclude: ralimetinib and foretinib. In some embodiments, the P38-alpha inhibitors can be those identified in Smith et al. (Nature Communications, 10, 131 (2019), doi.org / 10.1038 / s41467-018-08027-7), incorporated herein by reference. In some embodiments, the variant is a conjugate of the above-mentioned compounds by reaction of a phenol, alcohol, amide, alkynyl, amino, carboxyl, or acrylamide functional group on the compound.

[0117] In some embodiments, the targeting ligand also includes pharmaceutically acceptable salts, prodrugs, and isotopic derivatives thereof.

[0118] In some embodiments, the target protein of interest mediates chromatin structure and function. The target protein of interest can mediate epigenetic effects, such as DNA methylation or covalent modification of histones. One example is histone deacetylase 6 (HDAC6).

[0119] In some embodiments, the target protein of interest mediates mitotic cell cycle including cell division, the protein includes cyclin-dependent kinase 6 (CDK6). Cyclin-CDK complexes are the main driving mechanism for cell cycle progression. Among the CDKs, cyclin-dependent kinases 4 and 6 (CDK4 / 6) are key orchestrators of cell cycle regulation because they control the progression of the cell cycle from the G1 to the S phase. Cell cycle dysregulation driven by upregulated and hyperactivated CDKs significantly promotes uncontrolled cell proliferation, a fundamental hallmark of cancer. CDK4 / 6 inhibitors have been approved by the US FDA for the treatment of patients with advanced or metastatic breast cancer.

[0120] In some embodiments, the target protein of interest mediates cell adhesion, mitogenic activation, or apoptosis inhibition (BCR-Abl).

[0121] In some embodiments, the target protein of interest mediates cell proliferation, invasion, metastasis, apoptosis, and angiogenesis (EGFR).

[0122] In some embodiments, the target protein of interest mediates B-cell development (BTK). In some embodiments, the target protein of interest mediates tumor proliferation, metastasis, and invasion.

[0123] In some embodiments, the target protein of interest is a lysine acetylation reader (BRD4).

[0124] In some embodiments, the target protein of interest mediates cell growth and apoptosis (STAT3).

[0125] In some embodiments, the target protein of interest mediates cell apoptosis (BCl-Xl).

[0126] In some embodiments, the target protein of interest mediates cell growth (FAK).

[0127] In some embodiments, the target protein of interest mediates proliferation, differentiation, and transcriptional regulation (P38-alpha).

[0128] In some embodiments, the target protein of interest is a modulator of a signaling cascade associated with a known disease state. In another embodiment, the target protein of interest mediates a disorder through a mechanism other than modulating a signaling cascade. Using the present application, any protein in a eukaryotic system or a microbial system is a target for proteasomal degradation. In some embodiments, the target protein of interest can be a eukaryotic protein (e.g., a human protein).

[0129] The term "E3 ligase ligand" or "ligand of an E3 ligase" or "E3 ligase binding ligand" refers to a compound that targets an E3 ligase. In certain embodiments, the E3 ligase ligand includes one or more of a cereblon E3 ligase, a VHL E3 ligase, a MDM2 ligase, a TRIM24 ligase, a TRIM21 ligase, a KEAP1 ligase, and an IAP ligase. In certain embodiments, the E3 ligase ligand is selected from pomalidomide, thalidomide, lenalidomide, VH032, adamantane, 1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112, RG7338, AMG 232, AA-115, betulinic acid, MV1, LCL161, and / or an analog thereof. In some embodiments, the E3 ligase binding ligand includes those listed in Table 1.

[0130] The term "DNA strand" as used in the present disclosure refers to a hybridizable nucleic acid strand comprising at least three nucleic acids. In some embodiments, a DNA strand of the present disclosure can have the sequence of any one of SEQ ID NOs: 1-16. In some embodiments, a DNA strand of the present disclosure can have a sequence having at least about 60% sequence identity to any one of SEQ ID NOs: 1-16. In some embodiments, a DNA strand of the present disclosure comprises a nucleic acid sequence having at least about 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% sequence identity to any one of SEQ ID NOs: 1-16. In some embodiments, a DNA strand of the present disclosure consists of a nucleic acid sequence having at least about 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% sequence identity to any one of SEQ ID NOs: 1-16. In certain embodiments, a DNA strand of the present disclosure comprises any sequence containing any one of SEQ ID NOs: 1-16.

[0131] In some embodiments, the DNA strand of the present disclosure comprises one or more modified nucleic acids. In some embodiments, the one or more modified nucleic acids is an alkyne-modified nucleotide.

[0132] In some embodiments, the alkyne-modified nucleotide is synthesized from a phosphoramidite selected from 5'-dimethoxytrityl-5-[(6-oxo-6-(dibenzo[b,f]azocin-4- yl)-decenoylamido-N-hex-6-yl)-3-propenoylimino]-2'-deoxyuridine, 3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-5-ethynyl-2'- deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-hexynyl- phosphoramidite, 5'-dimethoxytrityl-5-(oct-1,7-diynyl)-2'-deoxyuridine, 3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 10-(6-oxo-6-(dibenzo[b,f]azocin- 4-yl)-decenoylamido-N-ethyl)-O-triethyleneglycol-1 -[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 6-bromo-hex-1 -yl-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramidite, 3-dimethoxytrityloxy-2-(3-(5-hexenoylamido)propoyl)- propyl-1 -O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-3'-propargyl-5-methyl-2'-deoxycytidine-N-succinyl-long chain alkylamino- CPG.

[0133] The term "therapeutic agent" as used herein refers to an agent that provides a desired therapeutic effect when administered to an animal. The animal is a mammal, which can include or exclude humans. The therapeutic agent can be of natural or synthetic origin. In some embodiments, the therapeutic agent can include or exclude a nucleic acid, a polypeptide, a protein, a peptide, a radioisotope, a sugar or polysaccharide, or an organic compound, which can include or exclude a small molecule. The term "small molecule" includes an organic molecule having a molecular weight of less than, for example, about 1000 daltons. In one embodiment, a small molecule can have a molecular weight of less than about 800 daltons. In another embodiment, a small molecule can have a molecular weight of less than about 500 daltons.

[0134] Table 1. Target proteins and corresponding representative target ligands that can be coupled at an amino, phenolic, alkyne, acrylamido, carboxyl, or alcohol site for linkage to a DNA strand to form a DNA-PROTAC of the present disclosure.

[0135]

[0136]

[0137] Certain Methods

[0138] In some embodiments, the present disclosure provides a method of identifying a DNA-PROTAC that mediates degradation or reduction of a protein of interest, the method comprising: providing a test DNA-PROTAC comprising a ligand for a selected POI coupled to a first DNA strand, wherein the first DNA strand is hybridized to a second DNA strand having an E3 ligase ligand attached thereto; contacting the test DNA-PROTAC with a cell comprising an E3 ligase and the protein of interest; determining whether the level of the protein of interest in the cell is reduced; and identifying the test DNA-PROTAC as a DNA-PROTAC that mediates degradation or reduction of the selected protein of interest. In some embodiments, the method for identifying a DNA-PROTAC that mediates degradation or reduction of a protein of interest is performed in the presence of a proteasome inhibitor to confirm the mechanism of action is through proteolysis of the target protein of interest (when the protein of interest is not reduced in the presence of the proteasome inhibitor). In certain embodiments, the cell is a cancer cell. In certain embodiments, the cancer cell is a glioblastoma cancer cell.

[0139] In some embodiments, the DNA-PROTAC of the present disclosure is more effective at treating a disease or disorder (e.g., cancer) than the use of the targeting ligand alone or the use of the E3 ligase ligand not linked. In some embodiments, the DNA-PROTAC of the present disclosure is more effective at treating a disease or disorder than the use of the targeting ligand alone or is able to treat a disease or disorder that is resistant to the targeting ligand and is more potent at inhibiting cell (e.g., cancer cell) growth or reducing cell (e.g., cancer cell) viability than the use of the targeting ligand alone. In certain embodiments, the DNA-PROTAC has an IC50 for inhibiting cell (e.g., cancer cell) growth or reducing cell (e.g., cancer cell) viability that is lower than the IC50 for the targeting ligand for inhibiting the growth of the cell or reducing cell viability. In certain embodiments, the IC50 of the DNA-PROTAC is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone. In certain embodiments, the IC50 of the DNA-PROTAC is at most 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the use of the targeting ligand alone.In certain embodiments, the DNA-PROTAC inhibits cell (e.g., cancer cell) growth or reduces cell (e.g., cancer cell) viability with an Emax that is less than the Emax of inhibiting cell growth or reducing cell viability using the targeting ligand alone. In certain embodiments, the Emax of the DNA-PROTAC is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the Emax of using the targeting ligand alone. In certain embodiments, the Emax of the DNA-PROTAC is at most 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the Emax of using the targeting ligand alone. In certain embodiments, the Emax of the DNA-PROTAC is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the Emax of using the targeting ligand alone. In certain embodiments, the Emax of the DNA-PROTAC is at most 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the Emax of using the targeting ligand alone.

[0140] In some embodiments, the DNA-PROTAC of the present application is more effective than using the targeting ligand alone in treating a disease or disorder, or is capable of treating a disease or disorder that is resistant to the targeting ligand, wherein the disease or disorder is a cancer (e.g., a cancer described herein). In other embodiments, the cancer is a glioblastoma.

[0141] In some embodiments, the DNA-PROTAC of the present disclosure promotes degradation of a target protein of interest by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 99%, or up to 100% at a concentration of 100,000 nM or less, 50,000 nM or less, 20,000 nM or less, 10,000 nM or less, 5,000 nM or less, 3,500 nM or less, 2,500 nM or less, 1,000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

[0142] In certain embodiments, the DNA-PROTAC of the present disclosure increases the rate of degradation of a target protein of interest by up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 99%, or up to 100% at a concentration of 100,000 nM or less, 50,000 nM or less, 20,000 nM or less, 10,000 nM or less, 5,000 nM or less, 3,500 nM or less, 2,500 nM or less, 1,000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

[0143] Certain embodiments of the present application also provide a method of treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0144] In some embodiments, the method of the present application further comprises administering to the subject at least one therapeutic agent. In some embodiments, the at least one therapeutic agent is administered in combination with the DNA-PROTAC. The phrase “in combination” as used herein refers to the simultaneous or sequential administration of the DNA-PROTAC and the at least one therapeutic agent. For simultaneous administration, the DNA-PROTAC and at least one therapeutic agent are present in a single composition or are separate. In some embodiments, when the DNA-PROTAC and at least one therapeutic agent are administered simultaneously, they are administered by the same or different routes.

[0145] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a DNA-PROTAC or a composition comprising a DNA-PROTAC described herein.

[0146] In some embodiments, the disease or disorder is cancer.

[0147] In some embodiments, the cancer is glioblastoma.

[0148] In some embodiments, the method further comprises administering to the subject at least one therapeutic agent.

[0149] In some embodiments, the therapeutic agent is a chemotherapeutic drug. In some embodiments, the chemotherapeutic drug is selected from Abraxane (chemical name: albumin-bound or nanoparticle albumin-bound paclitaxel), Adriamycin (chemical name: doxorubicin), Paraplatin (trade name: carboplatin), Cytoxan (chemical name: cyclophosphamide), Cerubidine, DaunoXome (chemical name: daunorubicin), Doxil (chemical name: doxorubicin), Ellence (chemical name: epirubicin), fluorouracil (also known as 5-fluorouracil or 5-FU; trade name: Adrucil), Gemzar (chemical name: gemcitabine), Halaven (chemical name: eribulin), Ixempra (chemical name: ixabepilone), methotrexate (trade names: Amethopterin, Mexate, Folex), mitomycin (chemical name: mitomycin), mitoxantrone (trade name: Novantrone), Navelbine (chemical name: vinorelbine), Taxol (chemical name: paclitaxel), Taxotere (chemical name: docetaxel), Thioplex (trade name: thiotepa), vincristine (trade names: Oncovin, Vincasar PES, Vincrex), and Xeloda (chemical name: capecitabine). In some embodiments, the chemotherapeutic agent is selected from Abraxane (albumin-bound paclitaxel injection), Adriamycin (doxorubicin), Afinitor (everolimus), Alecensa (alectinib), Alimta (pemetrexed), Aliqopa (cypatinib), Alkeran injection (melphalan), Alunbrig (brigatinib), Aredia (pamidronate), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arzerra (ofatumumab), Avastin (bevacizumab), Bavencio (avelumab), Beleodaq (belinostat), Besponsa (inotuzumab ozogamicin), Bexxar (tositumomab), BiCNU (carmustine), Blenoxane (bleomycin), Blincyto (blinatumomab), Bosulif (bosutinib), Braftovi (encorafenib), Busulfex (busulfan), Cabometyx (cabozantinib), Calquence (acalabrutinib), Campath (alemtuzumab), Camptosar (irinotecan), Caprelsa (vandetanib), Casodex (bicalutamide), CeeNU (lomustine), CeeNU DosePack, Cerubidine, Cinqair, Clolar, Cometriq, Copiktra, Cosmegen, Cotellic, Cyramza, Cytosar U, Cytoxan, Cyclophosphamide, Dacogen, Darzalex, DaunoXome, Daurismo, Decadron, DepoCyt, Dexamethasone Intensol, Dexpak Taperpak, Docefrez, Doxil, DTIC, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Emend, Empliciti, Erbitux, Erivedge, Erleada, Erwinaze, Ethyol, Etopophos, Eulexin, Fareston, Farydak, Faslodex, Femara, Firmagon, FloPred, Fludara, Folex, Folotyn, FUDR, Gazyva, Gemzar, Gilotrif, Gleevec, Halaven, Herceptin, Hexalen, Hycamtin, Hydrea, Ibrance, Iclusig, Idamycin, Ifex, Intron A, Invanz, Iressa, Jakafi, Kadcyla, Kepivance, Keytruda, Kyprolis, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, Kyowa Hakko Kirin, Kyowa, KyowaPFS (Idarubicin), Idhifa (Enasidenib), Ifex (Ifosfamide), Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin), Ixempra (Ixabepilone), Jakafi (Ruxolitinib), Jevtana (Carfilzomib), Kadcyla (Ado-trastuzumab emtansine), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kyprolis (Carfilzomib), Lanvima (Lenvatinib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lorbrena (Lorlatinib), Lupron (Leuprolide), Lynparza (Olaparib), Lysodren (Mitotane), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mektovi (Binimetinib), Mesnex (Mesna), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab ozogamicin), Navelbine (Vinorelbine), Nerlynx (Neratinib), Neulasta (Filgrastim), Neulasta (Filgrastim pegol), Neupogen (Filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (Nilutamide)), Ninlaro (Istirratuzumab), Nipent (Pentostatin), Nolvadex (Tamoxifen), Odomzo (Sonidegib), Oncaspar (Pegaspargase), Oncovin (Vincristine), Opdivo (Nivolumab), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab), Platinol (Cisplatin), Platinol AQ (Cisplatin), Pomalyst (Pomalidomide), Portrazza (Necitumumab), Proleukin (Interleukin-2), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rituxan (Rituximab), Roferon A (Interferon alfa-2a), Rubraca (Rucaparib), Rubidiun (Rubidomide), Rubitecan (Ranimustim), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil), Ruboxidil (Ruboxidil),alfaa (interferon alfa-2a), Rubex (doxorubicin), Rubraca (rucaparib), Rydapt (midostaurin), Sandostatin (octreotide), Soltamox (tamoxifen), Sprycel (dasatinib), Stivarga (regorafenib), Sutent (sunitinib), Sylvant (siltuximab), Synribo (omacetaxin), Tabloid (tioguanine), Taflinar (dabrafenib), Tagrisso (osimertinib), Talzenna (talazoparib), Tarceva (erlotinib), Targretin capsules (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), Tepadina (thiotepa), Thioplex (thiotepa), Tibsovo (ivosidenib), Toposar (etoposide), Torisel (temsirolimus), Treanda (bendamustine hydrochloride), Trelstar (triptorelin), Tykerb (lapatinib), Unituxin (dinutuximab), Valstar (valrubicin), Varubi (rolapitant), Vectibix (panitumumab), Velban (vinblastine), Velcade (bortezomib), Venclexta (venetoclax), Vepesid (etoposide), Vepesid (etoposide injection), Verzenio (abemaciclib), Vesanoid (tretinoin), Vidaza (azacitidine), Vincasar PFS (vincristine), Vincrex (vincristine), Vistogard (uridine triacetate), VitrakviI (larotrectinib), Vizimpro (dacomitinib), Votrient (pazopanib), Vumon (teniposide), Wellcovorin (leucovorin), Xalkori (crizotinib), Xgeva (denosumab), Xofigo (radium-223 dichloride), Xeloda (capecitabine), Xineom (peginterferon alfa-2b), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), Xofigo (radium-223 dichloride), XofigIV (leucovorin), Xalkori (crizotinib), Xeloda (capecitabine), Xospata (gilteritinib), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagene ciloleucel), Yondelis (trabectedin), Zaltrap (afilbercept), Zanosar (streptozocin), Zejula (niraparib), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zoladex (goserelin), Zolinza (vorinostat), Zometa (zoledronic acid), Zortress (everolimus), Zydelig (idelalisib), Zykadia (ceritinib), Zytiga (abiraterone), or combinations thereof. In some embodiments, the chemotherapeutic drug is selected from Abraxane (albumin-stabilized paclitaxel nanoparticle formulation), Afinitor (everolimus), erlotinib hydrochloride, everolimus, gemcitabine hydrochloride, irinotecan hydrochloride, Lynparza (olaparib), mitomycin, olaparib, cyclophosphamide, doxorubicin, oxaliplatin, mitoxantrone, sunitinib malate, or combinations thereof. In some embodiments, the chemotherapeutic drug is a combination of any of the above chemotherapeutic drugs.

[0150] In some embodiments, a pharmaceutical composition comprising a DNA-PROTAC of the present disclosure is administered (depending on the mode of administration) at a dosage level sufficient to deliver about 0.001 mg / kg to about 200 mg / kg in one or more doses over the course of a day or days, e.g., parenterally. In certain embodiments, the effective amount per dose is about 0.001 mg / kg to about 200 mg / kg, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 40 mg / kg, preferably about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, more preferably about 1 mg / kg to about 25 mg / kg, per day in one or more doses to achieve the desired therapeutic and / or prophylactic effect, per body weight of the subject. In certain embodiments, the dosage level of the compounds described herein can be sufficient to deliver about 0.001 mg / kg to about 200 mg / kg, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 40 mg / kg, preferably about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, more preferably about 1 mg / kg to about 25 mg / kg, per day in one or more doses to achieve the desired therapeutic and / or prophylactic effect, per body weight of the subject. The desired dose can be delivered three times a day, twice a day, once a day, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In certain embodiments, the compositions described herein are administered at a dose below that at which the agent causes nonspecific effects.

[0151] In some embodiments, the pharmaceutical composition comprising the DNA-PROTAC of the present disclosure is administered at a dose of about 0.001 mg to about 1000 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 200 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 100 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 50 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 10 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dose of about 0.1 mg to about 10 mg per unit dose.

[0152] The pharmaceutical compositions described herein can be prepared by any of the methods known in the art of pharmacology. In general, such preparative methods include the step of bringing into association the composition comprising the DNA-PROTAC of the present disclosure with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit. The carrier serves to carry the DNA-PROTAC of the present disclosure, which is in the form of a hybridization complex.

[0153] Certain Uses

[0154] In certain embodiments, the present application provides the use of a DNA-PROTAC described herein, or a composition comprising the DNA-PROTAC, in the manufacture of a medicament for inducing a tumor necrosis response in a subject.

[0155] In certain embodiments, the present application provides the use of a composition described herein for inducing a tumor necrosis response.

[0156] In certain embodiments, the present application provides the use of a composition described herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0157] Compositions

[0158] Certain embodiments of the present application provide a composition described herein for use in medical therapy.

[0159] Certain embodiments of the present application provide the use of a composition described herein in combination with at least one therapeutic agent in the manufacture of a medicament for inducing an immune response in a subject. In some embodiments, the subject is a mammal, which can include or exclude humans.

[0160] Certain embodiments of the application provide a composition described herein for use in inducing an immune response in combination with at least one therapeutic agent.

[0161] Certain embodiments of the application provide use of a composition described herein in the manufacture of a medicament for treating a disease or disorder in a subject.

[0162] Certain embodiments of the application provide use of a composition described herein in the manufacture of a medicament for treating a disease or disorder in a subject in combination with at least one therapeutic agent.

[0163] Certain embodiments of the application provide a composition described herein for use in the prophylactic or therapeutic treatment of a disease or disorder.

[0164] Certain embodiments of the application provide a composition described herein for use in the prophylactic or therapeutic treatment of a disease or disorder in combination with at least one therapeutic agent.

[0165] Formulations

[0166] The pharmaceutical combinations of the application can be formulated with a "carrier." As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The pharmaceutical combinations can be specially formulated for administration in solid or liquid form, including those adapted for the following routes of administration: (1) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or intradermal injection, for example, as a sterile solution or suspension or sustained release formulation; (2) topical administration, e.g., as a cream, lotion, gel, ointment for application to the skin, or a controlled release patch or spray; (3) intravaginal or intrarectal administration, e.g., as a pessary, cream, suppository or foam; (4) sublingual administration; (5) ocular administration; or (6) nasal administration. The carriers of the present disclosure do not include denaturing agents to preserve the hybrid structure of the DNA-PROTAC.

[0167] The combinations of the present disclosure can be provided in a single formulation. In other embodiments, the pharmaceutical combinations of the present disclosure can be provided in separate formulations. The pharmaceutical combinations can be formulated in a variety of and / or multiple forms suitable for one or more preferred routes of administration. Thus, the pharmaceutical combinations can be administered by one or more known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical combinations, or a portion thereof, can be administered to a mucosal surface, for example, by administration to, for example, the nasal mucosa or respiratory mucosa (e.g., by a spray or aerosol). The pharmaceutical combinations, or a portion thereof, can also be administered by sustained or delayed release.

[0168] The pharmaceutical compositions of the present disclosure can be conveniently presented in unit dosage form and can be prepared by methods well known in the art of pharmacy. Methods of preparing the combinations of the present disclosure in combination with a pharmaceutically acceptable carrier comprise the step of bringing into association the combination of the present disclosure and a carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions of the present disclosure can be prepared by uniformly and / or intimately bringing into association the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0169] In some embodiments, the methods can comprise administering a sufficient amount of the pharmaceutical compositions of the present disclosure to provide a dose of, for example, about 0.1 mg / kg to about 1000 mg / kg to the subject.

[0170] Administration

[0171] As described herein, the methods of the present application comprise administering a composition comprising the compositions described herein. In some embodiments, such compositions are formulated as pharmaceutical compositions and administered to a mammalian host (which can include or exclude human patients) in a variety of forms adapted to the chosen route of administration (i.e., oral or parenteral, intravenous, intramuscular, intraperitoneal, or topical or subcutaneous routes).

[0172] In some embodiments, the compositions are administered systemically in combination with a pharmaceutically acceptable carrier. In some embodiments, such compositions and formulations comprise at least 0.1% of the active DNA-PROTAC. Of course, the percentage of the compositions and formulations can vary and can conveniently be between about 0.1% to about 60% of the weight of a given unit dosage form. The amount of active DNA-PROTAC in such therapeutically useful compositions is such that an effective dosage level is achieved. Furthermore, the active compounds can be incorporated into sustained- release preparations and devices.

[0173] The active compounds can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compounds or salts thereof can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0174] Pharmaceutical formulations suitable for injectable or infusible use can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. In some embodiments, the liquid carrier or vehicle can be a solvent or a liquid dispersion medium comprising a liquid, which can include, but is not limited to: water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of required particle size in the case of dispersions or by the use of surfactants.

[0175] Sterile injectable solutions are prepared by incorporating the active DNA-PROTAC in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0176] Useful dosages of the compounds can be determined by comparison with in vitro activity and in vivo activity in an animal model. Methods for extrapolating effective dosages in mice and other animals to humans can include U.S. Patent No. 4938949, incorporated herein by reference.

[0177] The amount of DNA-PROTAC required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient, and will be ultimately at the discretion of the attendant physician or clinician.

[0178] The DNA-PROTAC of the present disclosure can be conveniently formulated into unit dosage forms. In one embodiment, the present application provides a composition comprising a compound formulated in such unit dosage form. The desired dose can conveniently be presented in a single dose or in divided doses administered at appropriate intervals. In some embodiments, the dose interval is selected from twice, three times, four times or more divided doses per day.

[0179] Examples

[0180] The application will now be demonstrated by the following non-limiting examples.

[0181] Materials and Methods

[0182] In this application, the techniques used are found in several well-known references, for example: Molecular Cloning: A Laboratory Manual (Sambrook et al. 1989, Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (Ausubel et al. Wiley-Interscience, 1988. New York) and PCR Protocols: A Guide to Methods and Applications (Innis et al. 1990. Academic Press, San Diego, Calif.), unless otherwise stated.

[0183] Small molecule synthesis and characterization

[0184] General methods: All non-aqueous reactions were carried out under an argon or nitrogen atmosphere in oven-dried glassware unless otherwise stated. Reagents were commercially available and used without further purification, anhydrous solvents were purchased from Sigma-Aldrich as the highest grade solvents. Thin layer chromatography was used to monitor reactions using 0.25 mm Silicycle silica gel 60 F254 plates. Flash column chromatography was used to purify small molecules using commercially available 40-60 mesh silica gel columns. Yields are isolated yields of pure compounds verified by spectroscopy (NMR and LC / MS).1H and13C NMR spectra were obtained using 400 and 500 MHz Varian spectrometers. Chemical shifts are expressed in parts per million (ppm, δ) with reference to the residual1H resonance of the solvent (CDCl3, 7.26 ppm, DMSO-d6, 2.49 ppm).13C spectra were referenced to the residual13C resonance of the solvent (CDCl3, 77.16 ppm, DMSO-d6, 39.52 ppm). Splitting patterns are indicated as follows: s, singlet; br, broad; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet.

[0185] General biological methods

[0186] Cell culture

[0187] U-251 MG glioblastoma cell line was grown in Dulbecco's Modified Eagles Medium (DMEM) containing 10% heat inactivated fetal bovine serum (FBS), streptomycin (5 pg / mL) and 5 U / mL penicillin (95 U / mL). All cell lines were maintained and cell culture experiments were performed in a humidified incubator at 37 °C and supplemented with 5% CO2.

[0188] DNA-PROTAC transfection

[0189] One day prior to DNA-PROTAC transfection, cells were propagated in 6 cm cell culture dishes containing the appropriate complete growth medium. Prior to transfection, the complete medium was replaced with transfection medium (5% FBS, no penicillin-streptomycin). DNA-PROTAC transfection was performed using lipofectamine 3000 reagent according to the manufacturer's provided protocol. All transfections were performed in 6 cm dishes with 3 mL of medium and the concentration of DNA-PROTAC was calculated according to this volume (3 mL). Briefly, for a concentration of 50 nM, 5 pi of 30 pM DNA-PROTAC stock and 12 pi of p3000 reagent were added to a tube containing 125 pi of OPTIM-MEM and 8 pi of Lipofectamine 3000 reagent was added to a separate tube containing 125 pi of OPTIM-MEM. Both tubes were incubated at room temperature for 5 minutes and then the DNA-PROTAC containing OPTI-MEM was slowly added to the second tube containing the lipo3000. The solution in the tubes was mixed thoroughly by pipetting up and down a few times. After 15-20 minutes of incubation at room temperature, 250 pi of the DNA-PROTAC-lipofectamine complex was added dropwise to the cells containing the transfection medium. The transfection medium was mixed thoroughly and then the plate was transferred to the incubator. After the appropriate time, the cells were harvested or replaced with fresh medium and incubated to the desired time point before harvesting. For proteasome inhibition assays, 5 pM MG-132 was incubated with the cells for 2 h prior to DNA-PROTAC transfection. Then, the cells were incubated for an additional 12 h before lysing the cells. Cell lysates were prepared by incubating the cells in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail from Roche and 1 mM PMSF) for 30 minutes on ice and the cell lysates were clarified by high speed (15000 rpm) centrifugation for 15 minutes. The clarified supernatant was collected for further experiments.

[0190] Annealing reaction

[0191] RP-HPLC purified single stranded oligos coupled to CRBN ligand (DNA-E3i) and their reverse complementary oligos coupled to CDK6 ligand (cDNA-CDK6i) were dissolved in ultrapure water. Single stranded DNA-E3i and single stranded reverse complementary oligos were mixed in a 1 : 1 molar ratio in IX annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl and 1 mM EDTA) (final concentration of DNA-PROTAC was set to 30 mM) and incubated in a water bath at 95 °C for 5 min and then cooled to 4 °C. Double stranded DNA-PROTAC was mixed by gentle vortexing, aliquoted and stored at -20 °C.

[0192] Western blotting

[0193] Protein concentration in all cell lysates was measured by BCA protein assay kit and equal amounts of each lysate were mixed with 4X loading dye, boiled for 5 min and then centrifuged for 2 min before loading into SDS-PAGE gels. Next, proteins on SDS-PAGE gels were transferred to nitrocellulose membranes by western blotting and membranes were blocked with 5% milk in TBST (0.05% Tween 20) for 1 h. Primary antibodies were prepared in 2.5% milk in TBST (all Abeam antibodies were diluted 1 :5000) and membranes were incubated overnight at 4 °C. The next day, membranes were washed for 15 min (incubate three times for 5 min each), appropriate secondary antibodies were prepared in TBST (1 :5000) and incubated with membranes for 1 h at room temperature (RT). Membranes were washed with TBST for 15 min (incubate three times for 10 min each) before imaging.

[0194] Immunofluorescence staining

[0195] U-251 MG cells were seeded in 10 mm confocal dishes (Ibidi) and incubated overnight. Subsequently, cells were rapidly washed three times with ice-cold PBS and then fixed with 4% paraformaldehyde for 20 min. After fixation, cells were washed with PBS and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Samples were then incubated in blocking solution for 1 h at room temperature before incubation with primary antibodies overnight at 4°C. The primary antibody used was mouse CDK6 antibody (Abcam, 1 : 100). After washing three times with PBST (PBS containing 0.1% Tween 20), samples were incubated with secondary antibodies for 1.5 h at room temperature in the dark. Cells were washed 3 times in PBST for 5 minutes each before incubation in the appropriate secondary antibody supplemented with CoraLite 488 conjugated goat anti-rabbit IgG (H+L) antibody (Proteintech, 1 : 100) for 3 hrs. Nuclei were counterstained with DAPI (Hoechst 3342, lug / mL) for 20 minutes before washing 3 times with PBST. Finally, the slides were imaged by a Nikon X confocal microscope.

[0196] Methods of mouse models

[0197] In certain embodiments, the methods of the present disclosure can treat cancer in relevant models. A mouse model of pancreatic cancer that the methods of the present disclosure are contemplated to demonstrate their cancer treatment effects is described in Herreros-Villanueva et al., Mouse models of pancreatic cancer, World J Gastroenterol. (2012) Mar 28; 18(12): 1286-1294. doi: 10.3748 / wjg.v18.i12.1286, PubMed ID: 22493542, incorporated herein by reference.

[0198] Example 1. Synthesis of representative embodiments of DNA-based chimeras for targeted protein degradation of CDK6 (DNA-PROTACs)

[0199] Optimization of the spatial conformational relationship of the DNA-PROTAC library: The success of PROTACs based on small molecules depends largely on optimal linker length. (Cyrus, K.; Wehenkel, M.; Choi, E. Y.; Han, H. J.; Lee, H.; Swanson, H.; Kim, K. B., Impact of Linker Length on the Activity of PROTACs, Mol Biosyst 2011, 7(2), doi.org / 10.1039 / c0mb00074d; Chen, Y.; Tandon, I.; Heelan, W.; Wang, Y.; Tang, W.; Hu, Q., Proteolysis-Targeting Chimera (PROTAC) Delivery System: Advancing Protein Degraders towards Clinical Translation, Chemical Society Reviews. Royal Society of Chemistry June 17, 2022, pp 5330-5350. doi.org / 10.1039 / d1cs00762a; Poongavanam, V.; Atilaw, Y.; Siegel, S.; Giese, A.; Lehmann, L.; Meibom, D.; Erdelyi, M.; Kihlberg, J., Linker-Dependent Folding Rationalizes PROTAC Cell Permeability, J Med Chem 2022, 65(19). doi.org / 10.1021 / acs.jmedchem.2c00877). However, controlling the spatial orientation using flexible chemical linkers remains challenging.Orientation control of the ligands can influence the relative spatial position of the protein to the E3 ligase complex, which can contribute to ubiquitination, and in the case of protein complexes such as CDK6 / CDK4, selective ubiquitination of a single protein (Lebraud, H.; Wright, D. J.; Johnson, C. N.; Heightman, T. D. Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras. ACS Cent Sci 2016, 2 (12). doi.org / 10.1021 / acscentsci.6b00280; Li, B.; Ran, T.; Chen, H. 3D Based Generative PROTAC Linker Design with Reinforcement Learning). Therefore, in this example, it is of interest to explore a small library of DNA-PROTAC constructs that are synthesized with spatial control of the distance and orientation between the two small molecules. The key assumption of the work is to position the small molecules on a DNA scaffold platform with a predetermined spatial organization and orientation control. As a starting test module, a small DNA duplex (20 bp; ~6.8 nm) was utilized as a scaffold that was functionalized with small molecules through strain-promoted azide-alkyne click (SPAAC) chemistry, coupling small molecule ligands pre-modified with polyethylene glycol (PEG) azides to the synthesized single strands (ssDNA). The small molecules used for this work were palbociclib (a targeting ligand for E3 ligases) and pomalidomide (a targeting ligand for CDK4 / CDK6). The organization (distance control) and orientation (angle control) of the small molecule ligands in the DNA-PROTAC was achieved by manipulating the position of the internal amino-threose phosphoramidite during DNA synthesis. This position provided an attachment site for DBCO-NHS ester, which was linked to the azide handle on the targeting ligand on either ssDNA of the DNA-PROTAC duplex through SPAAC reaction. The inventors’ data showed that the yield of this coupling reaction was over 98% regardless of whether it occurred at the internal or terminal position of the DNA scaffold. Figure 2B). Thus, the inventors utilized this strategy to prepare various DNA-PROTAC constructs for investigating: 1) distance effect: investigating the increase in the average distance (nm) between the POI-i (0, 7) and E3-i (1-6) ligands, and 2) angle effect: exploring the influence of the orientation change between the two ligands from 0º (parallel) to 180º (perpendicular) in increments of 36° per base Figure 2A ). To investigate the distance effect, five different versions were synthesized using the general scheme described below Figure 2A ). The small molecule ligands pre-modified with PEG-azide were coupled to the synthesized ssDNA via SPAAC. The small molecules used for this work were palbociclib (E3 ligase-targeting ligand; E3-i) and pomalidomide (CDK4 / CDK6-targeting ligand; POI-i) which were comprehensively tested by the inventors in preliminary studies. The distance control of the small molecule ligands in the different versions of DNA-PROTAC was achieved by manipulating the position of the internal amino-thymidine phosphoramidite during the solid-phase DNA synthesis Figure 2A ). This position provided an attachment site for DBCO-NHS ester which was connected to the azide handle on the targeting ligand on either ssDNA of the DNA-PROTAC duplex via SPAAC. The obtained each ssDNA strand was purified using RP-HPLC (reverse phase high-performance liquid chromatography), lyophilized, re-dissolved in Milli-Q water and characterized by native polyacrylamide gel electrophoresis (PAGE) Figure 2B ) and quadrupole time-of-flight liquid chromatography mass spectrometry (QTOF LC / MS) (Figures 9-14). The above constructs were chosen to systematically screen the position (distance control) of the two targeting ligands along the major and minor grooves on the opposite and same ends of the DNA scaffold, covering a range from 9.9 Å to 64.2 Å distance. Moreover, in the future, comparison with the angle effect will allow the inventors to determine the most critical factor leading to protein degradation.

[0200] Example 2. Evaluation of the distance effect of DNA-PROTACs in reducing CDK6 protein levels

[0201] The degradation efficiency of the various DNA-PROTACs was evaluated by transfecting them simultaneously into U251 cells to ensure consistency of experimental conditions. The double-stranded DNA sequence without any ligand was used as a negative control to provide a baseline for comparison. After a treatment period of 14 hours (duration chosen based on preliminary time course studies), the cells were harvested using trypsin digestion. The obtained lysate was then subjected to SDS-PAGE followed by Western blot analysis to assess protein degradation. As Figure 3As shown in FIG. 4, DNA-PROTAC version 02 (V02) exhibited the most pronounced CDK6 degradation. This was evidenced by a significant reduction in the visibility of the CDK6 band on the gel at 50 nM and 100 nM concentrations, in stark contrast to the controls. This finding highlights the efficacy of DNA-PROTAC V02 in targeting and reducing CDK6 protein levels. In comparison, DNA-PROTAC versions 03 and 04 also facilitated protein degradation, albeit with varying efficiencies. The 03 version was observed to significantly reduce CDK6 protein levels at 100 nM. However, the effect was not as pronounced as V02. On the other hand, the 04 version showed more pronounced reduction in protein levels at the lower concentration of 50 nM. Interestingly, version 05, characterized by a maximum ligand distance of about 60.2 Å, did not exhibit any appreciable protein degradation at either concentration. Likewise, version 06, which introduced the E3 ligand and the POI ligand (CDK6 inhibitor) simultaneously at the ends of the DNA duplex, also did not show significant protein degradation. These observations suggest that the spatial arrangement, particularly the distance and angle, between the E3 ligand and the POI ligand can be modulated to influence the protein degradation efficiency of the DNA-PROTAC. Based on these results, the structural configuration of the DNA-PROTAC, particularly the spatial relationship between its constituent ligands, can be modulated to mediate the degradation of the CDK6 protein.

[0202] Using other cell lines to model other cancer types is expected to have similar results to demonstrate that the methods described herein can be used to treat a wide range of cancers. For example, human Ewing’s sarcoma cell line A-673 and human leukemia cell lines THP-1 and HL-60 can also be used to demonstrate that the DNA-PROTACs described herein can be used to kill cancer cells and, therefore, treat subjects with cancer, by methods similar to those described herein.

[0203] Example 3. Assessing the angle effect of DNA-PROTACs in reducing CDK6 protein levels

[0204] To investigate the effect of ligand orientation on protein degradation efficiency, a series of DNA-PROTACs named A-minor-00, A-minor-01, A-minor-02, and A-minor-03, as well as a variant DNA-PROTAC-VO2 were designed. The orientation between the E3 ligand and the protein of interest (POI) ligand in these compounds ranged from 0º (parallel) to 180º (perpendicular). As shown in FIG. 4, DNA-PROTAC version 02 (V02) exhibited the most pronounced CDK6 degradation. This was evidenced by a significant reduction in the visibility of the CDK6 band on the gel at 50 nM and 100 nM concentrations, in stark contrast to the controls. This finding highlights the efficacy of DNA-PROTAC V02 in targeting and reducing CDK6 protein levels. In comparison, DNA-PROTAC versions 03 and 04 also facilitated protein degradation, albeit with varying efficiencies. The 03 version was observed to significantly reduce CDK6 protein levels at 100 nM. However, the effect was not as pronounced as V02. On the other hand, the 04 version showed more pronounced reduction in protein levels at the lower concentration of 50 nM. Interestingly, version 05, characterized by a maximum ligand distance of about 60.2 Å, did not exhibit any appreciable protein degradation at either concentration. Likewise, version 06, which introduced the E3 ligand and the POI ligand (CDK6 inhibitor) simultaneously at the ends of the DNA duplex, also did not show significant protein degradation. These observations suggest that the spatial arrangement, particularly the distance and angle, between the E3 ligand and the POI ligand can be modulated to influence the protein degradation efficiency of the DNA-PROTAC. Based on these results, the structural configuration of the DNA-PROTAC, particularly the spatial relationship between its constituent ligands, can be modulated to mediate the degradation of the CDK6 protein. Figure 6As shown in Figure 7, their ability to form DNA duplexes was confirmed by natural gel electrophoresis. U251 cells were then treated with these angle-dependent DNA-PROTACs, using double-stranded DNA as a negative control. A treatment time of 14 hours, determined from the initial time-process experiments, was used, followed by cell harvesting via trypsin digestion. Cell lysates were analyzed by SDS-PAGE and Western blot analysis to assess protein degradation. As shown in Figure 7, A-minor-00 exhibited mild CDK6 degradation. In contrast, A-minor-01 and A-minor-02 promoted significant degradation at a concentration of 100 nM, while A-minor-03 and DNA-PROTAC-VO2 achieved significant CDK6 degradation starting from 20 nM. These findings suggest that protein degradation efficiency is modulated by the angular arrangement between E3 and POI ligands.

[0205] Example 4. DNA-PROTAC-VO2 effectively induces CDK4 and CDK6 degradation in a dose- and time-dependent manner.

[0206] Given the significant CDK6 degradation efficiency observed using DNA-PROTAC-VO2, and considering the binding affinity of CDK6 inhibitors to CDK4, representative DNA-PROTACs were prepared to measure their effects on the CDK4 protein. Figures 6-8 As shown, DNA-PROTAC-V02 induces dose-dependent degradation of CDK6, starting from a significantly low concentration of 10 nM and increasing in effect with increasing concentration. Notably, CDK4 protein levels also decrease simultaneously starting at 40 nM, highlighting the dual protein degradation capability of DNA-PROTAC-V02. In summary, DNA-PROTAC-V02 exhibits the ability to induce dual degradation of CDK4 and CDK6 proteins in a dose-dependent manner. To further elucidate the functional characterization of DNA-PROTAC-V02, immunofluorescence staining was performed under various treatment conditions, including control conditions (dsDNA only, dsDNA with a CDK6 inhibitor, and dsDNA with an E3 ligand) and concentrations of DNA-PROTAC-V02 at 20 nM and 100 nM. Figure 5As shown in FIG. 3B, immunofluorescence imaging showed that DNA-PROTAC-V02 induced a decrease in intracellular green fluorescence compared to controls (dsDNA, dsDNA-CDK6-i, dsDNA-E3-i). This observation provides additional evidence for the efficacy of DNA-PROTAC-V02 in mediating dual protein degradation, particularly in reducing CDK6 protein levels. Furthermore, the study showed that DNA-PROTAC-V02 induced dual degradation of CDK4 and CDK6 proteins in a time-dependent manner. As shown in FIG. 4, degradation of CDK4 / 6 proteins began after 48 hours of treatment and continued to degrade up to 48 hours. These findings provide valuable insights into the multi-faceted degradation capabilities of DNA-PROTAC-V02, demonstrating its utility in targeted modulation of both CDK6 and CDK4 proteins.

[0207] Example 5. Verification of the mechanism of DNA-PROTAC-V02-mediated protein degradation

[0208] To fully understand the mechanism of DNA-PROTAC-V02-induced protein degradation, a chemical linker targeting CDK6 known as BSJ-03-123 was used in U251 cells. Figure 7A Subsequent results shown in FIG. 3B demonstrated that protein levels were perceptibly reduced at 160 nM treatment. Nonetheless, the exceptional efficacy of DNA-PROTAC-V02 became apparent at the lowest concentration of 50 nM, achieving over 80% degradation ( Figure 7A ). To determine the specificity of the protein degradation mechanism of DNA-PROTAC-V02 and its potential impact on mRNA levels, U251 cells were treated with DX, DX-E3i, DX-CDK6i, and DNA-PROTAC / V02 (100 nM, 6 hours). Subsequent RNA isolation and RT-PCR detection enabled quantitative assessment of CDK6 and CDK4 mRNA expression levels. Figure 7B, D clearly indicates that DNA-PROTAC-V02 treatment does not cause a reduction in CDK6 and CDK4 mRNA levels, a finding that is consistent with observations in the chemical PROTAC treatment groups. This highlights the ability of DNA-PROTAC-V02 to selectively induce protein degradation without affecting mRNA expression. To further confirm the mechanistic complexity of DNA-PROTAC-V02, its interaction with the proteasome pathway was probed. U251 cells were pre-treated with a proteasome pathway inhibitor (MG132) for 1 hour to temporarily impede proteasome function prior to transfection with DNA-PROTAC-V02 (50 nM). Following the 8-hour treatment period, cells were harvested and subjected to Western blot analysis. Reduction of CDK6 protein was observed in the absence of MG132 treatment, while MG132 intervention rescued CDK6 protein in both experimental trials. In summary, DNA-PROTAC-V02 as a representative embodiment of the present disclosure operates by selectively utilizing the proteasome pathway, providing a nuanced understanding of its protein degradation mechanism.

[0209] Example 6: Multi-targeting DNA-PROTAC

[0210] In complex diseases such as cancer, the universal limitation of single-target drugs is their susceptibility to rapid development of drug resistance, leading to further morbidity and tumor recurrence. DNA scaffolds can be used to functionalize multi-targeting DNA-PROTAC ligands—targeting multiple proteins as well as multiple domains within a single protein. With the hierarchical self-assembly properties of DNA scaffolds from basic monomer units, the advantage of higher synthetic complexity than traditional small molecules as target ligands can be achieved. Different arms can strategically place DNA-PROTAC targeting ligands, cell-directing agents, and cell-targeting moieties. To evaluate the multi-targeting ability, DNA-PROTACs for simultaneous targeting of different CDK protein family members, specifically CDK6, CDK9 (THAL-SNS-032 or B03), and CDK2 (AZD5438 or AT7519-7) can be prepared. Small molecule inhibitors designed against CDK proteins are modified with azido handles, purified, and subsequently linked to DNA strands containing DBCO groups. DNA nanostructures can be thermally annealed using component functionalized single-stranded DNA strands, which are then purified using well-established techniques, including PAGE gel electrophoresis, centrifugal filtration, and centrifugal gradient methods. Another aspect of placing multiple single-protein targeting ligands within DNA nanostructures is to overcome the hook effect. This approach can be useful when using high concentrations of DNA-PROTAC and can counteract the formation of E3 ligase:DNA-PROTAC (1:1) and POI:DNA-PROTAC (1:1) complexes, also known as the “hook effect.”

[0211] DNA-PROTACs can be developed based on similar principles described herein to modify different ligands to degrade different target proteins. Here, two basic designs of DNA-PROTACs can be generated, one is to recruit multiple E3 ligases to degrade target proteins as shown in Figure 1E Figure 6B. Based on similar DNA designs, multi-target degradation can also be achieved by changing the DNA sequence to couple with the same or different protein ligands (Figure 6C). Figure 1F More importantly, due to the high programmability and biocompatibility of DNA nanostructures, it will be possible to design degradation machines with different modules. To verify the universal application of DPCs to different targets, some new DPCs for well-known oncogenes such as bromodomain-containing protein 9 (BRD9), epidermal growth factor receptor (EGFR), BTK, and those listed in Table 1 will be synthesized and the degradation efficacy will be evaluated by the methods described herein.

[0212] Example 7. Development of a DNA scaffold-based targeted protein degradation platform (DNA-PROTAC)

[0213] Structure-activity relationship of DNA-PROTAC library.

[0214] The success of small molecule-based PROTACs relies heavily on optimal linker length. However, controlling spatial orientation remains challenging when using flexible chemical linkers. Orientation control of ligands can influence the relative spatial position of the protein to the E3 ligase complex, which can contribute to ubiquitination, and selective ubiquitination of individual proteins in the case of protein complexes. The inventors developed a small library of DNA-PROTAC constructs that were synthesized with spatial control over the distance and orientation between two small molecules to place small molecules on a DNA scaffold platform and precisely control pre-defined spatial arrangement and orientation. The small DNA duplex (20 bp; ~6.8 nm) as a scaffold can be functionalized with small molecules through a two-step procedure: 1) insertion of amino-modified phosphoramidites into ssDNA during solid-phase oligonucleotide synthesis; and 2) coupling of small molecule ligands pre-modified with PEG-azide to the synthesized ssDNA through SPAAC. The small molecules used in this example are pomalidomide (targeting ligand for E3 ligase) and palbociclib (targeting ligand for CDK4 / CDK6). The arrangement (distance control) and orientation (angle control) of small molecule ligands in DNA-PROTACs were achieved by manipulating the position of internal amino-threitol phosphoramidites during DNA synthesis. This position provides an attachment site for DBCO-NHS ester, which connects to azido handles on the targeting ligands on either ssDNA of the DNA-PROTAC duplex through SPAAC. This coupling reaction occurs with a yield of over 98% whether at internal or terminal positions of the DNA scaffold. This strategy demonstrates the preparation of selected DNA-PROTAC constructs for the study of: 1) distance effect: investigating the increase in average distance (nm) between POI-i (0, 7) and E3-i (1-6) ligands, and 2) angle effect: exploring the influence of orientation changes from 0º (parallel) to 180º (perpendicular) between the two ligands in increments of 36° per base. After coupling, each ssDNA chain obtained was purified using RP-HPLC, lyophilized, re-dissolved in Milli-Q water, and characterized by QTOF LC / MS. DNA-PROTACs were selected for systematic screening of the positions (distance control) of two targeting ligands along the major and minor grooves on the DNA scaffold at opposite and identical ends, covering distances ranging from 9.9 Å to 64.2 Å.

[0215] Example 8. Validation of target selectivity of DNA-PROTACs

[0216] U251 cells treated with DNA-PROTACs showed a significant and dose-dependent reduction in both CDK4 and CDK6 protein levels. In contrast, treatment with DNA duplex alone, DNA-E3 ligase, or DNA-CDK6 ligand did not induce significant protein degradation. Given that CDK4 / 6 forms a protein complex with cyclin D1, cyclin D1 protein can also undergo degradation by a proximity-induced mechanism. Western blot screening assays can be used to detect the response of several other CDKs and kinases to DNA-PROTACs, including the response of cyclin D3, CDK1, CDK2, CDK4, CDK5, and CDK9 to DNA-PROTAC treatment. In addition, other proteins that can be degraded by DNA-PROTACs due to potential non-specific binding and regulatory pathways need to be identified. To further characterize the formation of the functional ternary complex of DNA-PROTACs with CDK4 / 6 proteins and E3 ligases simultaneously, surface plasmon resonance (SPR) assays can be used to assess the binding affinity of different DNA-PROTAC variants to their corresponding target proteins. Since the degradation process is dependent on the ubiquitin-proteasome pathway, proteasome inhibitors including MG132 can be used to reverse target degradation. In addition, ligand competition assays are used to compete with DNA-PROTACs for binding to the target or E3 ligase, thereby blocking degradation and confirming the formation of a functional ternary complex in cells.

[0217] Example 9. Construction of representative DNA-PROTACs against undruggable targets

[0218] Not all proteins have conventional active sites suitable for binding to small molecule-based drugs, these are often referred to as“undruggable” protein targets. The DNA PROTACs of the present disclosure comprise small molecule ligands known to interact with specific protein targets. To overcome the limitations imposed by the lack of suitable ligands for certain proteins, in some embodiments, surrogate binders are used as targeting ligands, including macrocyclic peptides, staple peptides, and monobody proteins. These novel binders can target proteins when traditional small molecules are not a viable option. In some embodiments, the surrogate binders can include or exclude: a cyclic peptide that shows preferential binding to GTP-bound KRAS (G12D) protein, or a monobody (12VC1) that targets KRAS (G12V) and KRAS (G12C) mutant active states. Coupling of these surrogate binders to DNA can be achieved by two different methods. First, cysteine-modified surrogate binders can be modified with a heterobifunctional small molecule-based linker such as 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl ester (SMCC). Second, for surrogate binders containing intrinsic cysteine functional groups, coupling by click chemistry can be used, introducing functional groups such as azides or alkynes through non-canonical amino acids. Peptide synthesis can be performed using a peptide synthesizer, while micro-binding proteins can be recombinantly expressed in E. coli. After coupling, purification can be performed by PAGE, FPLC, or ion exchange chromatography, and full characterization using mass spectrometry. As a representative embodiment of an undruggable target, cyclin D1 will be used, which is traditionally considered undruggable. However, within the nucleus, cyclin D1 forms a complex with CDK6 and CDK4. Through detailed spatial structure-activity relationship studies, ubiquitination of cyclin D1 can be achieved while using palbociclib, a traditional CDK6 inhibitor.

[0219] Example 10. Stability studies of representative DNA-PROTACs under biological conditions

[0220] Assessing the stability of the branched DNA-PROTAC can be performed using PAGE gel migration and melting curve analysis. To enhance the stability of the branched structure, flexible linkers can be incorporated into the DNA-PROTAC design. These linkers can include alkyl carbon chains or PEG spacers. The self-assembly of the DNA-PROTAC can be accomplished from its individual component strands in a one-pot reaction. The flexible linkers at the junctions are also expected to improve resistance to nuclease degradation. To apply the DNA-PROTAC in vitro and in vivo, it is necessary to stabilize the DNA scaffold against nuclease degradation and chemical degradation characteristic of physiological conditions. The DNA-PROTAC can be stabilized against nuclease degradation using chemical modifications such as sugar modifications (such as locked nucleic acids, threose nucleic acids) and backbone modifications (such as phosphorothioates). The integrity of the DNA scaffold consisting of modified DNA strands can be assessed under physiological salt concentrations (1 mM Mg2+, 100 mM Na+) and in the presence of different concentrations of DNA nucleases. Stability kinetics is assessed by time-dependent reverse phase HPLC and ESI-MS analysis.

[0221] Given that many "undruggable" proteins lack ligands, targeting "linker proteins" can be a promising strategy. Potential pitfalls and alternative strategies are as follows: 1) One potential risk of using DNA-PROTAC is the binding of non-target proteins to the DNA scaffold, whether in a sequence-specific manner or through local DNA structure. Once these non-target binders are bound to the DNA scaffold and ubiquitinated by proximity to the E3 ligase, they can be degraded. To address this issue, the DNA-PROTAC sequence can be designed to ensure that only the target protein is degraded. 2) To gain a more detailed understanding of the interaction of the E3 ligase and the POI, all-atom simulations and molecular docking simulations of the DNA-PROTAC with the bound protein can be performed. 3) Coupling small molecules to DNA can potentially affect protein binding. In this case, the length of the chemical linker that links the small molecule inhibitor to the DNA scaffold can be adjusted to mitigate this pitfall. By employing surface plasmon resonance (SPR) analysis, the binding constant (kD) of the small molecule-functionalized DNA strand to the protein can be analyzed to assess the stability of the ternary complex formation. 4) Current methods for linking small molecules are primarily limited to click chemistry. Using appropriate corresponding modifiers (available from Glen Research, Trilink, etc.), small molecules can be directly attached to the DNA scaffold using phosphoramidite chemistry. This advancement will facilitate the ability to make multiple modifications of ligands on the same DNA strand, increasing resolution to less than 1 nm.

[0222] Example 11. Synthesis of 4-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-2-(2,6- dioxopiperidin-3-yl)isoindoline-1,3-dione (Azido-PEG3-Pomalidomide; E3-i)

[0223] The azido-PEG3-pomalidomide molecule was synthesized with slight modifications to the published protocol. 4-Fluorosalidomide (0.100 g, 0.362 mmol, 1 equiv), azido-PEG2-amine (0.082 g, 0.471 mmol, 1.3 equiv), and N,N-diisopropylethylamine (189 µL, 1.086 mmol, 3 equiv) were added to anhydrous dimethyl sulfoxide (1 mL) and the reaction mixture was heated at 100 °C under an argon atmosphere overnight. The solvent was then co-evaporated with ethanol several times. The crude mixture was purified by flash chromatography eluting with 1:1 hexanes: ethyl acetate (50:50) to obtain the product as a yellow-orange solid powder: yield (0.095 g, 0.220 mmol, 61.03%);1H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.10 (d, J = 7.1 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.50 (t, J = 5.6 Hz, 1H), 5.00 – 4.89 (m, 1H), 3.74 (t, J = 5.4 Hz, 2H), 3.69 (d, J = 4.1 Hz, 6H), 3.48 (q, J = 5.5 Hz, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.91 – 2.67 (m, 3H), 2.11 (ddd, J = 9.7, 7.2, 3.1 Hz, 1H) (Figure 9); QTOF-LC / MS (ESI+): m / z calculated for C19H22N6O6: 430.16. [M+Na]+; found 453.15.

[0224] Example 12. Synthesis of 6-acetyl-2-((5-(4-(2-(2-(2-azidoethoxy)ethoxy)ethyl)piperazin-1- yl)pyridin-2-yl)amino)-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (Azido- PEG3-Palbociclib; POI-i)

[0225] Palbociclib (0.100 g, 0.223 mmol, 1 equiv), azido-PEG3-iodide (0.083 g, 0.290 mmol, 1.3 equiv), potassium carbonate (0.077 g, 0.558 mmol, 2.5 equiv) and catalytic amount of tetraethylammonium bromide (0.015 g, 0.045 mmol, 0.2 equiv) were added to anhydrous dimethylformamide (2 mL) and the reaction mixture was heated at 90 °C for 6 hours under argon atmosphere (Figure 11). Then, the reaction mixture was quenched by the addition of distilled water (20 mL) and the product was extracted with ethyl acetate (20 mL). Next, the organic phase was washed with brine, water and then dried over sodium sulfate for a few minutes. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography eluting with 95:5 dichloromethane:methanol. The product was obtained as a bright yellow solid: yield (0.07 g, 0.115 mmol, 52%);1H NMR (500 MHz, CDCl3) δ 8.86 (s, 1H), 8.62 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 8.08 (d, J = 3.0 Hz, 1H), 7.33 (dd, J = 9.1, 3.0 Hz, 1H), 5.88 (q, J = 8.9 Hz, 1H), 3.68 (qd, J = 6.6, 3.6 Hz, 7H), 3.40 (t, J = 5.1 Hz, 2H), 3.22 (t, J = 5.0 Hz, 4H), 2.71 (dt, J = 15.8, 5.4 Hz, 5H), 2.55 (s, 3H), 2.38 (s, 5H), 2.17 - 2.00 (m, 2H), 1.96 - 1.83 (m, 2H), 1.75 - 1.58 (m, 2H), 1.26 (s, 1H) (Figure 11); QTOF-LC / MS (ESI+): m / z calculated for C30H40N10O4: 604.32. [M+H]+; found 605.33.

[0226] Example 13. Synthesis and characterization of amine-modified oligonucleotides

[0227] Oligonucleotides were obtained by standard solid-phase oligonucleotide synthesis on controlled pore glass (CPG, 1 μm). Standard DNA phosphoramidites, solid supports, and other reagents were purchased from Glen Research. Oligonucleotides were synthesized on an Applied Biosystems 3400 automated DNA / RNA synthesizer using standard 1.0 μmole phosphoramidite cycles that included acid-catalyzed detritylation activation and coupling, capping, and iodine oxidation. Stepwise coupling efficiency and overall yield were determined by automated trityl cation conductivity monitoring. For phosphorothioate modifications, 0.05 M sulfuration reagent II was prepared by first dissolving in 40 mL of pyridine, then adding 60 mL of acetonitrile to form a homogeneous solution. It was attached to an auxiliary port on the DNA synthesizer and can be used similar to a regular iodine oxidation. For internal amine modifications, amino-serinol phosphoramidites were dissolved in anhydrous acetonitrile to a concentration of 0.1 M just prior to use. Cleavage and deprotection of oligonucleotides from the solid support was achieved by exposure to a 30% ammonia solution at 55 °C for 120 minutes on a heating block. The cleavage solution was diluted with water and the ammonia was removed by washing with water using a 100 kDa Amicon filter. Sequences of the synthesized oligonucleotides are listed in Table 2 and Table 3 along with mass spectrometry data (where "i-NH2" indicates an internal amine (depicted in Figure 15

[0228] Table 2. Sequences of DNA strands with internal amine modifiers for subsequent coupling to small molecules

[0229]

[0230] Table 3. Sequences of DNA strands with internal amine modifiers for subsequent coupling to small molecules and protein binding sites of interest.

[0231]

[0232] Example 14. Synthesis and characterization of dibenzocyclooctyne (DBCO)-modified oligonucleotides

[0233] ​​For DBCO conjugation to oligonucleotides, amine-modified DNA strands (see DNA sequences used) were treated with a 6-fold excess of 200 mM DBCO-sulfo-NHS-ester (dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester) in 1X PBS buffer at pH ~8.5. The mixture was gently shaken at 37°C for 3 h. DNA-DBCO conjugates were purified by repeated washing (5X) with distilled water at 8000 rcf using 3 kDa amicon filters to remove excess small molecules and salts. After filtration, the mixture was purified using RP-HPLC and the conjugate peak was verified using ESI-MS.

[0234] Synthesis of E3-i and POI-i-DNA conjugates. Conjugates were synthesized using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Briefly, 2 molar equivalents of 10 mM azido-PEG3-pomalidomide (E3-i) or azido-PEG3-palbociclib (POI-i) in DMSO were added to DNA-DBCO conjugates in 1xTAE-12.5 mM MgCl2, pH 7.5. The reaction mixture was stirred and kept at 37°C overnight. DNA-drug conjugates were purified by repeated washing (5X) with distilled water at 8000 rcf using 3 kDa amicon filters to remove excess small molecules and salts. After filtration, the mixture was purified using RP-HPLC and the conjugate peak was verified using ESI-MS.

[0235] RP-HPLC purification. After reaction, small molecule-DNA conjugates were purified using a C-18 column of Agilent 1220 Infinity LC HPLC. The mixture was purified using a linear gradient method with buffer-A (50 mM triethylammonium acetate (TEAA)) and buffer-B (methanol). The gradient of buffer B was linearly increased from 10% to 100% in 60 min. The conjugates were monitored and collected by absorbance at 260 nm (for DNA) and 309 nm (for DBCO). The collected fractions were lyophilized overnight until dry.

[0236] Full mass analysis of DTAC conjugates. The collected peaks were tested for purity and identified using quadrupole time-of-flight liquid chromatography mass spectrometry (QTOF LC / MS) in negative mode. A 50 uM solution was prepared from the stock solution of small molecule-conjugated DNA strands using 0.1% ammonium hydroxide as the mobile phase for analysis. The identified peaks were deconvoluted within the expected mass range to obtain the full mass of the conjugates.

[0237] PAGE analysis of DTAC conjugates. Small molecule-DNA conjugates were analyzed using 8% non-denaturing polyacrylamide gel electrophoresis (PAGE). 15 μΐ of 2 μΜ sample was added to each lane (for single-stranded oligonucleotides, 15 μΐ of 10 μΜ sample was added), and the gel was run at 4°C, 200 V (constant voltage) for 1.5 h, then stained with ethidium bromide (EtBr) and imaged using a Bio-Rad Molecular Imager GelDOC XR+ imaging system.

[0238] The examples provided herein clearly demonstrate that the compositions of the present disclosure can be used to significantly reduce tumor burden in a subject, thereby prolonging survival time, including up to the end of the experimental study.

[0239] While the foregoing specification and examples have been sufficient to enable one of ordinary skill in the art to practice the embodiments of the present disclosure, they are not intended to limit the scope of the application as defined by the claims. It is understood that the examples and embodiments described herein are illustrative of the present disclosure and not limiting. Numerous alternative

[0240] All publications, patents, and patent applications are herein incorporated by reference. Although the application has been described in connection with certain embodiments thereof, it will be understood that it is capable of further modifications and that this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure that come within known or customary practice within the art to which the application pertains. It is intended to cover and embrace all alternatives, modifications and equivalents.

[0241] The use of the terms "including", "containing", "having", "including”, "including” and "comprising” and variations thereof herein are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Although the foregoing specification and examples have been sufficient to enable one of ordinary skill in the art to practice the embodiments of the present disclosure, they are not intended to limit the scope of the application as defined by the claims. It is understood that the examples and embodiments described herein are illustrative of the present disclosure and not limiting. Numerous alternative

[0242] Embodiments of the application are described herein, including the best mode known to the inventors of practicing the application. Variations of those embodiments can become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect professionals in the field to employ such variations as appropriate, and the inventors intend for the application to be practiced otherwise than specifically described herein. Accordingly, the application includes all modifications and equivalents of the subject matter recited in the claims as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise evident to one of ordinary skill in the art.

Claims

1. A programmable DNA proteolysis target chimera complex comprising: a. a first DNA strand comprising one or more independent E3 ligase ligands; and b. a second DNA strand comprising one or more independent protein of interest (POI) targeting ligands, wherein at least a portion of the first DNA strand is complementary to a portion of the second DNA strand, and the first and second DNA strands form a DNA duplex, wherein the one or more independent E3 ligase ligands are attached to the first DNA strand at one or more independently selected positions on the first DNA strand, and wherein the one or more independent POI targeting ligands are attached to the second DNA strand at one or more independently selected positions on the second DNA strand.

2. The complex of claim 1, further comprising a targeting moiety selected from a cell penetrating peptide or a blood brain barrier traversing agent.

3. The complex of claim 2, wherein the blood brain barrier traversing agent is a lipid or a cholesterol derivative.

4. The complex of claim 1, wherein the plurality of independent POI targeting ligands target different proteins.

5. The complex of claim 1, wherein the plurality of independent POI targeting ligands target different portions of the same protein.

6. The complex of claim 1, wherein there are at least two independent POI targeting ligands.

7. The complex of claim 1, wherein there are at least two independent E3 ligase ligands.

8. The complex of claim 1, wherein the E3 ligase ligand is covalently attached to the first DNA strand.

9. The complex of claim 1, wherein the E3 ligase ligand is complexed with one or more E3 ligase proteins.

10. The complex of claim 1, wherein the POI targeting ligand is covalently attached to the second DNA strand.

11. The complex of claim 11, further comprising a protein of interest complexed with the one or more POI targeting ligands.

12. The complex of claim 1, wherein the selected position on the first DNA strand and the selected position on the second DNA strand are separated by a distance of about 0.99 nm to about 7 nm.

13. The complex of claim 1, wherein the selected position on the first DNA strand and the selected position on the second DNA strand are separated by a rotational angle of about 36 degrees to about 180 degrees around the double stranded DNA complex.

14. The complex of claim 1, wherein the selected position on the first DNA strand and the selected position on the second DNA strand are separated by a distance of about one minor groove to about one major groove.

15. The complex of claim 1, wherein the first and second DNA strands independently comprise a nuclease resistance feature.

16. The complex of claim 16, wherein the nuclease resistance feature is selected from a sugar modification or an internucleoside linkage modification.

17. The complex of claim 17, wherein the sugar modification is selected from a locked nucleic acid, a threose nucleic acid, or a 2'-alkoxy modification.

18. The complex of claim 17, wherein the internucleoside linkage modification is a phosphorothioate, phosphoroselenoate, or phosphoramidate.

19. The complex of claim 1, wherein the protein of interest is selected from the group consisting of: CDK6, CDK4, BCR-Abl, EGFR, BTK, BRD4, HDAC6, STAT3, BCL-Xl, FAK, P38-alpha, myc, Arora, Ras, and Jak.

20. A method of killing a cancer cell, the method comprising contacting a complex according to any one of claims 1-20 with a cancer cell.

21. The method of claim 21, wherein the cancer is a glioblastoma.

22. A method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of a complex according to any one of claims 1-20.

23. A method of treating a proliferative disease or disorder in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a complex according to any one of claims 1-20.

24. The method of claim 24, wherein the proliferative disease or disorder is a cancer.

25. A method of reducing cancer tumor cell proliferation, the method comprising contacting the cancer tumor cell with a complex according to any one of claims 1-20.

26. Use of a complex according to any one of claims 1-20 in the manufacture of a medicament for treating a cancer in a subject.

27. A composition comprising a complex according to any one of claims 1-20 and a pharmaceutically acceptable carrier.

28. Use of a composition comprising a complex according to any one of claims 1-20 in the manufacture of a medicament for treating a proliferative disease or disorder in a subject.

29. A composition comprising a complex according to any one of claims 1-20 for use in the prophylactic or therapeutic treatment of a disease or disorder in a subject.

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