WO2025199263A1 - Polyphosphodiester based polymer library and combinatorial approach to identify bioactive sequences - Google Patents

Polyphosphodiester based polymer library and combinatorial approach to identify bioactive sequences

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Publication number
WO2025199263A1
WO2025199263A1 PCT/US2025/020602 US2025020602W WO2025199263A1 WO 2025199263 A1 WO2025199263 A1 WO 2025199263A1 US 2025020602 W US2025020602 W US 2025020602W WO 2025199263 A1 WO2025199263 A1 WO 2025199263A1
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polymer
backbone
conjugate
seq
cancer
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French (fr)
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Jiachen LIN
Ke Zhang
Yun WEI
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Northeastern University Boston
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Northeastern University Boston
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/605—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the macromolecule containing phosphorus in the main chain, e.g. poly-phosphazene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the ASO and polymer backbone can both be assembled on the solid support.
  • the synthesis allows the incorporation of different units such as carbon lipids, spermine, or cholesterol into the backbone in specific patterns to modulate the cell-materials interactions.
  • PEG side chains were grafted onto the polymer segment of the resulting polymer-oligonucleotide - 1 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 conjugate, yielding bottlebrush polymers.
  • pacDNA oligonucleotide therapeutic delivery platform
  • pacDNA overcomes many challenges associated with oligonucleotide delivery, including carrier-mediated toxicity or immunogenicity, poor delivery to non-liver sites, and non-hybridization side effects. It is discovered that the interactions between pacDNA and cells are strongly dictated by the polymer backbone, which opens a door to explore a range of biological properties such as pharmacokinetics and biodistribution by carefully designing the backbone.
  • a library of “digital” bottlebrush polymers those with precise backbone arrangement of various monomers within the backbone, was designed and synthesized. The biodistribution, plasma pharmacokinetics, and cellular uptake of these digitized bottlebrush polymers were explored, and interesting structure-property relationships were seen. ln some embodiments, a novel drug delivery system can be tailored to each specific disease or disease type based upon digital polymers.
  • One embodiment disclosed herein is a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.
  • Another embodiment disclosed herein is a conjugate, comprising a polymer of the present disclosure and an agent conjugated to the polymer.
  • Another embodiment disclosed herein is a composition comprising a conjugate of the present disclosure and a pharmaceutically acceptable carrier.
  • Another embodiment disclosed herein is a method of modulating or altering the expression of a gene product encoded by a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate of the present disclosure. - 2 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0010]
  • Another embodiment disclosed herein is a method of delivering an agent to a subject in need thereof, the method comprising: administering a conjugate or a composition of the present disclosure to the subject.
  • Another embodiment disclosed herein is a method of delivering an agent to a cell, the method comprising: contacting the cell with a conjugate of the present disclosure.
  • Another embodiment disclosed herein is a method of detecting a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate of the present disclosure, thereby forming a polynucleotide-conjugate hybrid; and detecting a signal from the polynucleotide-conjugate hybrid.
  • a method of treating a disease the method comprising administering a conjugate of the present disclosure or a composition of the present disclosure to a subject in need thereof.
  • a method of making a bottlebrush polymer comprising: combining one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.
  • FIGs.1A-D Design of ribose-based pacDNAs.
  • FIG.1A Synthetic scheme of pacDNAs.
  • FIG.1B Backbone designs of pacDNAs. The gray and red circles represent the R- NH2 unit and R-C18 units, respectively. ASO: antisense oligonucleotide.
  • FIG.1C Schematics of the PEGylation process of ribose-based pacDNA.
  • FIG.1D Molecular dynamics (MD) simulation of ribose-based pacDNA with four C 18 modifiers (cyan: PEG; gray: backbone; red: C18; yellow: ASO). [0018]
  • FIGs.2A-D Characterization of ribose-based pacDNAs.
  • FIG.2A Aqueous GPC chromatograms of pacDNAs after two-stage PEGylation.
  • FIG.2B pacDNA Z-average (the intensity-weighted mean) molecular size in Nanopure water as determined by DLS.
  • FIG.2C Representative TEM image of pacDNA (pac-4 ⁇ 1C18-ASO1) with negative staining using 2% uranyl acetate.
  • FIG.2D pacDNA ⁇ potential in Nanopure water.
  • FIGs.3A-G In vitro properties of ribose-based pacDNAs.
  • FIG.3A Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs-ASO1 containing varying numbers and arrangement of C 18 modifiers (0C 18 , 2C 18 , 4C 18 , 6C 18 , 8C 18 , 10C 18 ) after 4 h incubation, as determined by flow cytometry.
  • FIG.3B Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs-ASO1 containing varying numbers and arrangement of C18 modifiers (1 ⁇ 6C18, 2 ⁇ 3C 18 , 3 ⁇ 2C 18 , 6 ⁇ 1C 18 ) after 4 h incubation, as determined by flow cytometry.
  • FIG.3C Confocal microscopy of NCI-H358 cells treated with Cy5-labeled free ASO1 or pacDNAs- ASO1 for 8 h (additional samples: FIG.25). Cy5-labeled ASO (red), DAPI-stained nuclei (blue), and phalloidin-stained F actin (green) are presented separately.
  • FIG.3D Representative molecular snapshots from the simulation trajectory (top row is the structures of entire molecules, bottom row is the corresponding structures without PEG chains to show backbones more clearly; cyan: PEG; gray: backbone; red: C18; yellow: ASO).
  • FIG.3E Interaction energy of pacDNAs after interaction with water as determined by MD simulation.
  • FIG.3F KRAS depletion efficiency of 10 ⁇ M pacDNAs-ASO1 in NCI-H358 cells. The number shows the relative KRAS protein level analyzed by ImageJ software.
  • FIG.3G Inhibitive effect of 10 ⁇ M pacDNAs-ASO1 - 4 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 on the proliferation of NCI-H358 cells after 48 h incubation. Statistical analysis was performed using one-way ANOVA. ****P ⁇ 0.0001.
  • FIGs.4A-E Plasma PK and biodistribution of ribose-based pacDNAs.
  • FIG.4A Plasma PK of Cy5-labeled pacDNA and free DNA in C57BL/6 mice following i.v. injection. Statistical analysis was performed using two-way ANOVA. ****P ⁇ 0.0001.
  • FIG.4B Plasma PK parameters of ribose-based pacDNAs with variant numbers of the C 18 modifier.
  • FIG.4C Live animal fluorescence imaging of C57BL/6 mice bearing the K273 allograft following i.v. injection of Cy5-labeled pacDNA. Areas surrounding the allograft have been shaved to facilitate imaging.
  • FIG.4D Quantitative biodistribution of pacDNAs in major organs/tissues of tumor- bearing C57BL/6 mice 72 h post i.v.
  • FIG.4E Ex vivo imaging of pacDNAs in major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection.
  • FIGs.5A-F In vivo antitumor efficacy of ribose-based pacDNAs.
  • FIG.5A K273 allograft volume changes in 24 days with i.v. injection of PBS, ASO2, and pac-4 ⁇ 1C 18 -ASO2 at equivalent ASO doses (0.5 ⁇ mol/kg) every 4 days since day 10. Injections are indicated by black arrows.
  • FIG.5B Mean tumor growth curve.
  • FIG.5C Kaplan–Meier end point animal survival analysis.
  • FIG.5D Immunohistostaining and H & E staining of K273 tumor after treatment.
  • FIG.5E Anti-PEG IgM and IgG levels in C57BL/6 mice plasma after i.v. injection of PBS, free ASO2, pac-4 ⁇ 1C18- ASO2, and PEG-KLH (positive control) at 0.5 ⁇ mol/kg every 4 days for four doses. Plasma was collected 1 week after the last dose. Statistical analysis was performed using one-way ANOVA.
  • FIG.5F Selected cytokine and chemokine levels in C57BL/6 mice plasma 5 h after i.v.
  • FIG.6 1 H NMR spectrum of compound 1 (Scheme 1).
  • FIG.7 13 C NMR spectrum of compound 1.
  • FIG.8 1 H NMR spectrum of compound 2 (Scheme 1).
  • FIG.9 13 C NMR spectrum of compound 2.
  • FIG.10 1 H NMR spectrum of compound 3 (Scheme 1).
  • FIG.11 13 C NMR spectrum of compound 3.
  • FIG.12 1 H NMR spectrum of R-NH2 (Scheme 1).
  • FIG.13 13 C NMR spectrum of R-NH 2 .
  • FIG.14 1 H NMR spectrum of compound 4 (Scheme 2).
  • FIG.15 13 C NMR spectrum of compound 4.
  • FIG.16 1 H NMR spectrum of compound 5 (Scheme 2).
  • FIG.17 13 C NMR spectrum of compound 5.
  • FIG.18 1 H NMR spectrum of compound 6 (Scheme 2).
  • FIG.19 13 C NMR spectrum of compound 6.
  • FIG.20 1 H NMR spectrum of R-C 18 (Scheme 2).
  • FIG.21 13 C NMR spectrum of R-C18.
  • FIG.22 DLS measurements of ribose-based pacDNAs in Nanopure water.
  • FIGs.23A-F Comparison of polynorbornene bottlebrush (pac-PN) and ribose-based pac 4 ⁇ 1C18.
  • FIG.23A The molecular weight of five batches of pac-PN and pac 4 ⁇ 1C18 brush polymers.
  • FIG.23B Descriptive statistics of the molecular weight of FIG.23A.
  • FIG.23C Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs and free oligo.
  • FIG.23D Plasma pharmacokinetics of Cy5-labeled pacDNA and free DNA in C57BL/6 mice following i.v. injection. Statistical analysis was performed using two-way ANOVA. **** P ⁇ 0.0001.
  • FIG. 23E Live animal fluorescence imaging of C57BL/6 mice bearing K273 allograft following i.v. injection of Cy5-labeled pacDNA. Areas surrounding the allograft has been shaved to facilitate imaging.
  • FIG.23F The ex vivo imaging of pacDNAs in major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection.
  • FIGs.24A-B In vitro properties of ribose-based pacDNAs without ASO.
  • FIG.24A Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs containing varying numbers and arrangement of C18 modifiers after 4 h incubation, as determined by flow cytometry.
  • MFI mean fluorescence intensity.
  • FIG.24B Cytotoxicity of 10 ⁇ M ribose-based pacDNAs (without ASO) on the proliferation of NCI-H358 cells after 48 h incubation.
  • FIG.25 Confocal microscopy of NCI-H358 cells treated with Cy5-labeled free DNA or ribose-based pacDNAs for 8 h.
  • FIG.26 Inhibitive effect of ribose-based pacDNAs-ASO1 on the proliferation of NCI-H358 cells at different concentration (from 100 nM to 5 ⁇ M) after 48 h incubation.
  • FIG.27 Fluorescence imaging of major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection of Cy5-label ribose-based pacDNAs.
  • FIGs.28A-B In vitro efficacy test of ribose-based pacDNAs on K273 cell line.
  • FIG. 28A KRAS depletion efficacy of pac-4 ⁇ 1C18-ASO2 dose-dependently.
  • FIG.28B Inhibition of K273 cell proliferation 48 h after treatment of pac-4 ⁇ 1C 18 -ASO2, pac-4 ⁇ 1C 18 -scramble and free-ASO2 at different levels of dose.
  • FIG.29 Body weight change of C57BL/6 mice bearing K273 allograft model. See FIG.5A for the administration time frame.
  • FIG.30 H&E staining of major organs and tissues of C57BL/6 mice after i.v. injection of PBS, free ASO2 and pac-4 ⁇ 1C18-ASO2 at doses 0.5 ⁇ mol/kg every four days, four doses at total.
  • FIG.31A Four different example building blocks for digital brush polymer. Example structures of cholesterol, spermine, serinol (void), and C18 alkyl phosphoramadites.
  • FIG.32A Transmission electron microscopy (TEM) for synthesized bottle brush polymers.
  • FIG.32B Gel permeation chromatography (GPC) results of brushes with different backbones.
  • FIG.32C Dynamic light scattering (DLS) reveals size similarity of polymers with different backbones.
  • FIG.32D Zeta potential reveals differences in charge on different backbones.
  • FIG.33A Cellular uptake on NCI-H358 cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells.
  • - 7 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1
  • FIG.33B Cellular uptake on DM1 fibroblast cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells.
  • FIG.33C Cellular uptake on HepG2 cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells.
  • FIG.34A Biodistribution analysis of bottlebrush polymers in various organs. IVIS fluorescence imaging of excised organs, showing distinct accumulation patterns among different polymer formulations. The epi-fluorescence intensities are shown in the heatmap legend.
  • FIG.34B Heatmap representation of quantified fluorescence intensities, with warmer colors indicating higher accumulation levels in specific organs.
  • FIG.34C Quantification bar graph illustrating the comparative distribution of polymers across different organs.
  • Oligonucleotide-based therapeutics hold immense promise for treating diseases through diverse mechanisms, such as gene regulation, receptor binding, and alternative splicing, among others. (1,2) However, clinical development of oligonucleotide drug candidates often faces setbacks attributed to poor target engagement in vivo due to limited uptake by target organs and cells, limiting their use to a few concentrated disease settings. (3 ⁇ 5) Current strategies to improve drug potency generally focus on chemical modifications, bioconjugation with antibodies, peptides, or small molecule ligands, and polyplex carriers such as lipid nanoparticles and cationic polymers.
  • the bottlebrush polymer in our earlier system was prepared by ring-opening metathesis polymerization of norbornenyl monomers using a Grubbs third-generation catalyst. We identified three aspects where this system can be improved: (1) batch-to-batch consistency, (2) residue heavy metal content, and (3) control over the bottlebrush polymer backbone.
  • certain chemical modifiers such as lipid tails, cholesterol, etc.
  • when arranged into different patterns within the polymer give rise to variable cell uptake, plasma pharmacokinetics, and biodistribution, as described herein. Therefore, it is possible to optimize polymers as delivery vehicles to address each different disease that has specific requirements for drug delivery.
  • Nanoparticles present significant potential for advancing oligonucleotide drug delivery systems. Among these, bottlebrush polymers have emerged as a promising platform, addressing key challenges such as carrier-mediated toxicity, immunogenicity, and unintended off-target effects.
  • the present disclosure provides a polymer, comprising one or more of: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.
  • the present disclosure provides a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.
  • the present disclosure provides a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, covalently linked to the backbone.
  • the polymer further comprises at least one cholesterol, at least one spermine, or a combination thereof covalently linked to the backbone.
  • a polymer backbone is a polyphosphodiester backbone.
  • a polymer backbone is a polyphosphodiester backbone, 2-deoxyribose-based backbone, a serinol-based backbone, or a combination thereof.
  • a polymer - 10 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 backbone is a 2-deoxyribose-based backbone, a serinol-based backbone, or a combination thereof.
  • the polymer comprises from about 1 to about 100 (e.g., about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, etc.) polymer arms covalently linked to the backbone.
  • the polymer comprises from about 10 to about 40 polymer arms covalently linked to the backbone.
  • the polymer comprises about 30 polymer arms covalently linked to the backbone.
  • at least one (e.g., each) of the plurality of polymer arms is poly(ethylene glycol) (PEG), poly-L-lysine, PAMAM dendrimers, poly(2-oxazoline), polypeptides, poly(N-isopropylacrylamide), polyanhydrides, poly( ⁇ -caprolactone), chitosan, polystyrene, poly(ethyleneimine), poly(lactic-co-glycolic acid), poly(propyleneimine) dendrimers, poly( ⁇ -amino esters), poly(vinyl alcohol), poly(methyl methacrylate), polyacrylamide, polydopamine, polycarbonate, dextran, or a combination therefore.
  • each of the plurality of polymer arms is a poly(ethylene glycol).
  • each of the at least one alkyl is a C 10 -C 30 alkyl (e.g., C 10 -C 20 alkyl, C 15 -C 20 alkyl, etc.). In some embodiments, each of the at least one alkyl is a C 18 alkyl.
  • a polymer comprises from about 2 to about 30 alkyls (e.g., about 2 to about 20, about 2 to about 15, about 2 to about 10, etc.) covalently linked to the backbone. In some embodiments, a polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone.
  • a polymer comprises about 4 alkyls covalently linked to the backbone.
  • a polymer comprises 6 cholesterols covalently linked to the backbone.
  • a polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone.
  • the plurality of alkyls or the plurality of cholesterols are evenly distributed (i.e., the number of non-alkyls (or non- cholesterols) separating adjacent alkyls (or cholesterols) are the same or vary by 1 or 2 units).
  • a plurality of alkyls or a plurality of cholesterols are separated by from about 2 monomers to about 6 monomers.
  • a plurality of alkyls or a plurality of cholesterols are separated by 2, 3, 4 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 3, 4, 5 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 4, 5, 6 monomers, or a combination thereof.
  • a polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47 (Table 3).
  • the polymer has a sequence comprising 80%, 85%, 90%, 95% or 100% of a sequence disclosed herein, e.g., in Table 3.
  • Conjugates [0079]
  • a polymer is modified, e.g., conjugated to an agent (e.g. a therapeutic agent, an imaging agent, an oligonucleotide, etc.).
  • agent e.g. a therapeutic agent, an imaging agent, an oligonucleotide, etc.
  • conjugated refers to attached, via a covalent or noncovalent interaction. Conjugation can employ any of suitable linking agents; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents.
  • the present disclosure provides a conjugate comprising a polymer of the present disclosure and an agent (e.g., oligonucleotide) conjugated (e.g., covalently linked) to the polymer.
  • an agent e.g., oligonucleotide conjugated (e.g., covalently linked) to the polymer.
  • an oligonucleotide comprises deoxyribonucleotides.
  • an oligonucleotide comprises ribonucleotides.
  • Non-limiting examples of oligonucleotides include single-, double- or multistranded DNA or RNA, DNA-RNA hybrids, or - 12 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 a polymer comprising purine and pyrimidine bases, or other natural, or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of an oligonucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof.
  • an agent e.g., oligonucleotide
  • a linker e.g., a cleavable linker
  • the agent e.g., oligonucleotide
  • the agent is conjugated to the 5’ end of the polymer.
  • polymers, conjugates and compositions of the present disclosure are used to treat a disease or disorder, including those described herein.
  • polymers, conjugates and compositions of the present disclosure are used for treating Myotonic Dystrophy Type 1 (DM1).
  • a polymer has SEQ ID NO.37 or SEQ ID NO.29 (see Table 3). These sequences have a high muscle uptake.
  • Q36 (SEQ ID NO.37, Table 3): NN NNN NAN NNN NNN ANN NNN NNA NNN NNN NNN N
  • Q18 (SEQ ID NO.29, Table 3): NN NCN NNC NNN NCN NNN CNN NNC NNN CNN N
  • polymers, conjugates and compositions of the present disclosure are used for treating rheumatoid arthritis.
  • a polymer has SEQ ID NO.27 (see Table 3). This sequence has a high spleen uptake.
  • a conjugate comprises an oligonucleotide, or an analog or derivative thereof.
  • the oligonucleotide, or the analog or derivative thereof is an inhibitor of a protein.
  • the oligonucleotide, or the analog or derivative thereof is an activator of the protein.
  • the oligonucleotide, or the analog or derivative thereof decreases (e.g., reduces or neutralizes) the expression or activity of the - 13 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 protein. In other embodiments, the oligonucleotide, or the analog or derivative thereof, increases the expression or activity of the protein.
  • the oligonucleotides can have sequences containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof.
  • oligonucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., A, G, C, or T) and nucleotides comprising modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides, such as 5-methyl dCTP and 5- hydroxymethyl cytosine).
  • the oligonucleotide comprises at least one modified nucleotide.
  • Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-o- methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'- thiouridine and 2'-deoxyuridine.
  • the modification increases nuclease resistance, increases serum stability, decrease immunogenicity, or a combination of the foregoing.
  • the agent e.g., oligonucleotide
  • the agent is a DNA molecule.
  • the agent e.g., oligonucleotide
  • the oligonucleotide comprises an analog or a derivative of an oligonucleotide.
  • the analog or derivative is a peptide nucleic acid (PNA).
  • the analog or derivative is a locked nucleic acid (LNA).
  • the analog or derivative is a morpholino oligonucleotide.
  • the analog or derivative comprises one or more phosphorothioate-linkages.
  • the agent comprises a deoxyribonucleic guanidine (DNG) nucleotide.
  • the agent comprises ribonucleic guanidine (RNG) nucleotide.
  • the agent e.g., oligonucleotide
  • the agent is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamers, ribozymes, DNAzymes, antisense oligonucleotides, exon-skipping oligonucleotides, siRNA oligonucleotides, triple helix forming oligonucleotides, ribozymes, DNAzymes, chemically modified forms thereof, or a combination thereof.
  • the agent is an oligonucleotide.
  • the agent e.g., oligonucleotide
  • the agent is an antisense oligonucleotide.
  • - 14 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1
  • the administration of a conjugate or a composition may be carried out in any manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation.
  • the route of administration is determined by the tissue or tissues, or organ to which the agent or agents are targeted.
  • the tissue, tissues, or organ is the subject’s lung, ovary, immune system, skin, blood vessel, muscle, blood, brain, heart, intestine(s), pancreas, spleen, kidney, heart, bone, bone marrow, stomach, head, or any combination thereof.
  • Compositions e.g. pharmaceutical composition
  • the composition comprises a polymer or a conjugate disclosed herein, and a carrier (e.g. a pharmaceutically acceptable carrier).
  • a composition refers to a composition having pharmacological activity or other direct effect in mitigating, treating, or preventing cancer, or a finished dosage form or formulation thereof.
  • a composition e.g., pharmaceutical composition
  • Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents or tonifiers include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose or dextrins); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose or sorbitol), salt-forming counter-ions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); non-ionic surfactants (e.g., Tween), PLURONICSTM and polyethylene glycol (PEG).
  • buffers e.g., phosphate, citrate, his
  • a composition (e.g., pharmaceutical composition) is formulated for a suitable administration schedule and route.
  • administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous and topical, etc.
  • a composition e.g., pharmaceutical composition
  • a composition is stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized).
  • a composition is formulated to be administered by infusion (e.g., intravenous infusion).
  • a composition is formulated to be administered with one or more additional agents (e.g., with a second therapeutic agent) as a combination therapy.
  • a “combination therapy” or “administered in combination” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition.
  • additional agents or treatments include biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules impacting immune-mediated diseases or conditions.
  • the treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap.
  • the two or more agents are administered in a sequential manner as part of a prescribed regimen. In other embodiments, the delivery of the two or more agents is simultaneous or concurrent. In some embodiments, the two or more agents are co-formulated. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents can be administered by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the two or more therapeutic agents can be administered by the same route or by different routes.
  • Methods - 16 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00102]
  • the present disclosure provides methods of modulating or altering the expression of a gene product encoded by a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate disclosed herein.
  • the present disclosure provides methods of delivering an agent (e.g.
  • an agent e.g., an oligonucleotide
  • an agent comprises deoxyribonucleotides.
  • an agent comprises ribonucleotides.
  • Non-limiting examples of oligonucleotides include single-, double- or multistranded DNA or RNA, DNA- RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of an oligonucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof.
  • the agent e.g., oligonucleotide
  • the agent is an RNA molecule.
  • the oligonucleotide comprises an analog or a derivative of a oligonucleotide.
  • the analog or derivative is a peptide nucleic acid (PNA).
  • the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative comprises one or more phosphorothioate-linkages. In some embodiments, the agent comprises a deoxyribonucleic guanidine (DNG) nucleotide. In some embodiments, the agent comprises ribonucleic guanidine (RNG) nucleotide.
  • DNG deoxyribonucleic guanidine
  • RNG ribonucleic guanidine
  • the agent e.g., oligonucleotide
  • the agent is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamers, ribozymes, DNAzymes, antisense oligonucleotides, exon-skipping oligonucleotides, siRNA oligonucleotides, triple helix forming oligonucleotides, ribozymes, DNAzymes, chemically modified forms thereof, or a combination thereof.
  • the agent is an - 17 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 oligonucleotide.
  • the agent e.g., oligonucleotide
  • the present disclosure provides methods of delivering an agent to a cell, the method comprising: contacting the cell with a conjugate disclosed herein.
  • the present disclosure provides methods of detecting a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate disclosed herein, thereby forming a polynucleotide-conjugate hybrid; and detecting a signal (e.g., a fluorescent signal, a radioactive signal) from the polynucleotide-conjugate hybrid.
  • a conjugate disclosed herein further comprises a fluorescent dye or a radioactive dye (e.g., a radioactive isotope).
  • a conjugate disclosed herein further comprises a fluorescent dye.
  • a fluorescent dye is Cyanine 5 (Cy5), Cyanine 3 (Cy3), Cyanine 2 (Cy2), a Fluorescein (e.g., Fluorescein isothiocyanate (FITC)), an Alexa Fluor dye, Texas Red, Tetramethylrhodamine (TAMRA), 6-Carboxy-X-Rhodamine (ROX), Tetramethylrhodamine (TMR), or Boron-dipyrromethene (BODIPY).
  • a fluorescent dye is Cyanine5 (Cy5).
  • detecting a signal from a polynucleotide-conjugate hybrid comprises using imaging.
  • detecting a signal from a polynucleotide- conjugate hybrid comprises using autoradiography. In some embodiments, detecting a signal from a polynucleotide-conjugate hybrid comprises using fluorescence imaging.
  • a target polynucleotide is selected from eukaryotic, prokaryotic, and viral polynucleotides. In some embodiments, a target polynucleotide is a polynucleotide specific to a mammalian cancer cell, a mammalian non-cancer cell, a plant cell, a bacterium, or a virus. In some embodiments, a target polynucleotide is an mRNA.
  • an mRNA is KRAS mRNA or DMPK mRNA.
  • the present disclosure provides methods of treating a disease, the method comprising administering a conjugate or a composition disclosed herein to a subject in need thereof.
  • the disease is a cancer, a muscular disorder, or a central nervous system disorder.
  • muscular disorders include and are not limited to muscular dystrophy, myotonic dystrophy, myasthenia gravis, amyotrophic lateral sclerosis (ALS), dermatomyositis, polymyositis, inclusion body myositis, mitochondrial myopathy, congenital myopathy, glycogen storage disease type II (Pompe disease), spinal muscular atrophy, Becker muscular dystrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, central core disease, nemaline myopathy, distal myopathy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy.
  • the muscular disorder is myotonic dystrophy.
  • central nervous system disorders include and are not limited to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), epilepsy, stroke, migraine, meningitis, encephalitis, spinal cord injury, cerebral palsy, hydrocephalus, brain tumors, Chiari malformation, Creutzfeldt-Jakob disease (CJD), narcolepsy, schizophrenia, depression, and obsessive-compulsive disorder (OCD).
  • a wide variety of cancers are treatable according to the methods described herein.
  • the cancer comprises a solid tumor (e.g., a tumor of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testes, head and/or neck, pancreas, brain, skin).
  • a solid tumor cancer e.g., a tumor of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testes, head and/or neck, pancreas, brain, skin.
  • Solid tumor cancers that can be treated according to the methods described herein include breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, renal cancer, gastric cancer, colon cancer, rectal cancer, colorectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer and skin cancer.
  • the cancer is a hematologic cancer (e.g., leukemia, lymphoma, myeloma).
  • Hematologic cancers that can be treated according to the methods described herein include leukemias (e.g., acute leukemias, chronic leukemias), lymphomas (e.g., B-cell lymphoma, T-cell lymphoma) and multiple myeloma.
  • the cancer is a lung cancer.
  • Examples of cancers treatable according to the methods described herein include Acute Lymphoblastic Leukemia (ALL); Acute Myeloid Leukemia (AML); Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Cancer (e.g., Kaposi Sarcoma, - 19 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 AIDS-Related Lymphoma, Primary CNS Lymphoma); Anal Cancer; Appendix Cancer; Astrocytomas, Childhood; Atypical Teratoid/Rhabdoid Tumor, Childhood, Central Nervous System; Basal Cell Carcinoma of the Skin; Bile Duct Cancer; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer (including Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma); Brain Tumors/Cancer; Breast Cancer; Burkitt Lymphom
  • the administration of a polymer, conjugate, or composition of the present disclosure may be parenteral or non-parenteral, including intravenous, intra-arterial, intraperitoneal, intramuscular, intracavity, subcutaneous, intradermal, topical, inhalation, transmucosal, rectal or transdermal.
  • the administration routes is oral, rectal, subcutaneous, transdermal, ocular, and intraperitoneal.
  • the polymer may be administered ex vivo.
  • a polymer, conjugate or composition is delivered to a target site by diffusion and/or osmosis over time ranging from hours to days. In certain embodiments, the conjugate is delivered directly to the target site. In some embodiments, the procedure of delivering a conjugate to a target site is repeated several times, if needed. [00121] In some embodiments, a polymer, conjugate or composition is administered with one or more additional agents (e.g., with a second therapeutic agent) as a combination therapy.
  • additional agents e.g., with a second therapeutic agent
  • Also disclosed herein are methods of making a bottlebrush polymer comprising one or more of: providing one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof; combining the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, co
  • methods of making a bottlebrush polymer comprise: - 22 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 combining one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.
  • At least one or more of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group.
  • each of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group.
  • covalently linking a plurality of polymer arms to the backbone comprises removing the protecting group and contacting the polymer backbone with a reagent comprising the polymer arm.
  • the reagent is an amino-reactive PEG reagent (e.g., PEG NHS ester).
  • protecting groups include and are not limited to Boc (tert- butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), CBZ (carbobenzyloxy), Tos (p- toluenesulfonyl), PMB (para-methoxybenzyl), Nip (nitrophenylsulfonyl), Alloc (allyloxycarbonyl), Mtr (4-methyltrityl), and TFA (trifluoroacetyl).
  • a protecting group is fluorenylmethoxycarbonyl (Fmoc), trifluoroacetyl (TFA), or a combination thereof.
  • the polymer comprises from about 1 to about 100 (e.g., about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to - 23 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 about 50, etc.) polymer arms covalently linked to the backbone.
  • the polymer comprises from about 10 to about 40 polymer arms covalently linked to the backbone.
  • the polymer comprises about 30 polymer arms covalently linked to the backbone.
  • each of the plurality of polymer arms is poly(ethylene glycol) (PEG), poly-L-lysine, PAMAM dendrimers, poly(2-oxazoline), polypeptides, poly(N- isopropylacrylamide), polyanhydrides, poly( ⁇ -caprolactone), chitosan, polystyrene, poly(ethyleneimine), poly(lactic-co-glycolic acid), poly(propyleneimine) dendrimers, poly( ⁇ - amino esters), poly(vinyl alcohol), poly(methyl methacrylate), polyacrylamide, polydopamine, polycarbonate, dextran, or a combination therefore.
  • PEG poly(ethylene glycol)
  • PAMAM dendrimers poly(2-oxazoline)
  • polypeptides poly(N- isopropylacrylamide), polyanhydrides, poly( ⁇ -caprolactone), chitosan, polystyrene, poly(ethyleneimine), poly(lactic-co-glycolic
  • each of the plurality of polymer arms is a poly(ethylene glycol).
  • each of the one or more alkyl phosphoramidites is a C10-C30 alkyl (e.g., C 10 -C 20 alkyl, C 15 -C 20 alkyl, etc.) phosphoramidite.
  • each of the one or more alkyl phosphoramidites is a C18 alkyl phosphoramidite.
  • a polymer comprises from about 2 to about 30 alkyls (e.g., about 2 to about 20, about 2 to about 15, about 2 to about 10, etc.) covalently linked to the backbone.
  • a polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone. [00132] In some embodiments, a polymer comprises about 4 alkyls covalently linked to the backbone. [00133] In some embodiments, a polymer comprises 6 cholesterols covalently linked to the backbone. [00134] In some embodiments, a polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone. In some embodiments, the plurality of alkyls or the plurality of cholesterols are evenly distributed (i.e., the number of non-alkyls (or non- cholesterols) separating adjacent alkyls (or cholesterols) are the same or vary by 1 or 2 units).
  • a plurality of alkyls or a plurality of cholesterols are separated by from about 2 monomers to about 6 monomers.
  • a plurality of alkyls or a plurality of cholesterols are separated by 2, 3, 4 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 3, 4, 5 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 4, 5, 6 monomers, or a combination thereof.
  • a polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47 (Table 3).
  • the cholesterol phosphoramidite is . - 25 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1
  • the spermine phosphoramidite is . . is . DEFINITIONS - 26 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00141] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
  • Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”
  • “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the disclosure, claims, result or embodiment, “about” means within one standard deviation per the practice in the art, or can mean a range of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 4, ⁇ 3, ⁇ 2 or ⁇ 1% of a given value.
  • PEGs for use in the present invention will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation, or, e.g., the identity of adjacent functional groups.
  • the variable (n) - 28 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 may, for non-limiting example, range from 1 to about 5000, and the terminal groups and architecture of the overall PEG may vary.
  • treat means inhibiting or relieving a disease or disorder, including cancer, a central nervous system disorder, a muscular disorder, a wound or a tumor.
  • treatment can include a postponement of development of the symptoms associated with disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease.
  • the terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated.
  • subject refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, swine, dog, cat, rabbit, guinea pig, rat, mouse).
  • the subject is a human.
  • a “subject in need thereof” refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented).
  • the subject is a human.
  • the human is a neonate.
  • the human is a pediatric patient.
  • the human is a juvenile.
  • the human is an adult. In some embodiments, the human is less than 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.
  • the term “effective amount,” “therapeutically effective amount,” or “sufficient amount” refers to a quantity sufficient to, when administered to a subject (e.g., a mammal such as a human, e.g., a patient such as a human cancer patient), effect treatment (e.g., produce beneficial or desired results), including effects at cellular, tissue or clinical levels, etc.
  • a subject e.g., a mammal such as a human, e.g., a patient such as a human cancer patient
  • effect treatment e.g., produce beneficial or desired results
  • effects at cellular, tissue or clinical levels etc.
  • the term depends upon the context in which it is being applied. For example, in the context of treating cancer, it is an amount of an agent sufficient to achieve a response as compared to the response obtained without administration of the agent.
  • a “therapeutically effective amount” of a composition of the present disclosure is an amount that results in a beneficial or desired result in a subject (e.g., as compared to a control).
  • a therapeutically effective amount of a composition of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response.
  • EXAMPLES [00152] The entire teachings of Wei, Y. et al., Bottlebrush polymers with sequence-controlled backbones for enhanced oligonucleotide delivery, J. Am. Chem. Soc.2024, 146, 34763-34770 are incorporated herein by reference.
  • EXAMPLE 1 The poly(2-deoxyribose phosphodiester) backbone polymer is constructed using a stepwise condensation approach, employing two bespoke modified phosphoramidites: ribose NH 2 (R-NH 2 ) and ribose C 18 (R-C 18 ).
  • the modifiers were synthesized from 1-chloro-3,5-di(4- chlorbenzoyl)-2-deoxy-d-ribose in ⁇ 40% overall yields in multigram scales (FIG.1A and 6- 21; Schemes 1 and 2).
  • Scheme 1 The modifiers were synthesized from 1-chloro-3,5-di(4- chlorbenzoyl)-2-deoxy-d-ribose in ⁇ 40% overall yields in multigram scales (FIG.1A and 6- 21; Schemes 1 and 2).
  • oligonucleotide component was synthesized as an integral part of the backbone of the solid support. Due to the utilization of the same phosphoramidite chemistry, the oligonucleotide can be positioned at either the 3′ or the 5′ of the polymer backbone without requiring additional postconjugation steps.
  • antisense oligonucleotides targeting the 3′ untranslated region (UTR) of both human and mouse KRAS mRNA (ASO1 and ASO2, respectively) were selected as the payload for this ribose-based pacDNA, which were - 31 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 positioned at the 5′ of the ribose backbone (FIG.1B). (17,18) The backbone is designed to incorporate 30 repeating units of ribose NH 2 , which can be subsequently PEGylated to give the bottlebrush morphology (FIG.1C-D).
  • the partially PEGylated product was desalted, lyophilized, and subsequently reacted with another equivalent of PEG in anhydrous N,N-dimethylformamide (DMF) for full derivatization.
  • DMF N,N-dimethylformamide
  • Excess PEG and residues were removed by aqueous gel permeation chromatography (GPC), yielding highly uniform, narrowly dispersed pacDNA structures, as evidenced by aqueous and DMF GPC (FIG.2A; Table 2), dynamic light scattering (DLS) (FIG.2B and 22), and transmission electron microscopy (TEM) (FIG.2C).
  • interaction energy refers to the total energy arising from all intermolecular forces between the pacDNA molecules and surrounding water molecules. A more negative interaction energy indicates stronger interactions with water, leading to greater solubility of the pacDNA in aqueous environments.
  • Cy5-labeled pacDNAs were intravenously (i.v.) administered to C57BL/6 mice via the tail vein, and blood plasma samples were collected by submandibular puncture at predetermined time points over a 72 h period.
  • all pacDNA constructs regardless of the number of R-C18 units in the backbone, exhibited significantly prolonged circulation times (FIG.4A).
  • Analyzing the plasma PK using a two-compartment model it can be seen that the distribution half-lives (t 1/2 ⁇ ) generally decrease with increasing R-C18 number (FIG.4B).
  • the K273 cell is a KRAS G12D lung cancer line derived from a genetically engineered mouse model (GEMM).
  • mice were sacrificed 72 h after injection, and tissue - 37 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 lysates were used to determine distribution as % injected dose per gram of tissue (% ID/g, FIG.4D).
  • pacDNAs with two or four R-C 18 units exhibit stronger tumor localization compared to those with six, eight, or ten C18 modifiers.
  • pac-4 ⁇ 1C18 shows the highest tumor distribution compared with other backbone designs (FIG.4E and 27), we focused on this specific arrangement for subsequent studies.
  • pac-4 ⁇ 1C18-ASO2 which is matched to the mouse wild-type KRAS mRNA sequence at the 3′ UTR region.
  • FIG.28A Western blot shows that pac-4 ⁇ 1C 18 -ASO2 downregulated KRAS protein levels in a dose-dependent manner (FIG.28A).
  • the MTT cell viability assay showed that pac-4 ⁇ 1C18-ASO2 reduced K273 cell viability, while free ASO2 or pac-4 ⁇ 1C18-scramble showed no inhibitory effect (FIG.28B).
  • the antitumor efficacy of pac-4 ⁇ 1C 18 -ASO2 was assessed in female C57BL/6 mice bearing subcutaneous K273 allografts.
  • pac-4 ⁇ 1C 18 -ASO2 Neither pac-4 ⁇ 1C 18 -ASO2 nor free ASO2 lead to significant body weight changes (FIG.29). Immunohistostaining of tumor tissue revealed reduced KRAS protein expression after pac-4 ⁇ 1C 18 -ASO2 treatment but not with free ASO2 (FIG.5D). [00162] Next, we performed in vivo safety analysis of pac-4 ⁇ 1C18-ASO2. Hematoxylin and eosin (H & E) staining demonstrated no obvious histological changes in major internal organs (FIG. 30). Macromolecular therapeutics requiring repeated dosing may induce antidrug adaptive immunity, leading to loss of drug activity in subsequent doses.
  • pac-4 ⁇ 1C18 as overall the most favorable given in vitro cell uptake, gene silencing, and biodistribution in mice.
  • pac-PN polynorbornene bottlebrush
  • a KRAS-depleting pac-4 ⁇ 1C18 showed single-agent tumor-suppressive potency at a fraction of concentration typically used for the ASO modality.
  • Reversed-phase high- performance liquid chromatography was performed on a Waters (Waters Co., MA, USA) Breeze 2 HPLC system coupled to a Symmetry® C183.5 ⁇ m, 4.6 ⁇ 75 mm reversed-phase column and a 2998 PDA detector, using TEAA buffer (0.1 M) and HPLC-grade acetonitrile as mobile phases.
  • Aqueous GPC measurements were performed on a Waters Breeze 2 GPC system equipped with UltrahydrogelTM 1000, UltrahydrogelTM 500 and UltrahydrogelTM 250, 7.8A ⁇ 300 mm column and a 2998 PDA detector (Waters Co., MA, USA).
  • Phosphate-buffered saline PBS, pH 7.4 was used as the eluent running at a flow rate of 0.8 mL/min.
  • N, N-dimethylformamide (DMF) GPC analysis was performed on EcoSEC HLC-8320 GPC system (Tosoh Bioscience LLC, Tokyo, Japan) equipped with a TSKGel ⁇ -M 7.8 ⁇ 300 mm, 13 ⁇ m column and RI/UV-Vis detectors.
  • the mobile phase is 0.05 M lithium bromide in HPLC-grade DMF, and samples were analyzed at a flow rate of 0.4 mL/min.
  • DMF GPC calibration was based on a ReadyCal kit of polyethylene glycol standards (PSS-Polymer Standard Service-USA Inc., MA, USA). The kit covers an Mn range from 232 Da to 1015 kDa.
  • TEM transmission electron microscopy
  • HRMS High-resolution mass spectra analysis was performed on a Q Exactive Orbitrap (Thermofisher ScientificTM) using a syringe pump with direct infusion.
  • Data was acquired in positive/negative ion mode using a hybrid orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with direct infusion electrospray ion source (HESI) using a syringe pump.
  • the flow rate was 5ul/min with +4.5 kV /-3.5 kV electrospray voltages.
  • Analysis was performed in full scan mode with a mass range of 150-2000 m/z, including an automatic gain control setting of 1 ⁇ 106 and a mass resolution of 17,000. DLS and ⁇ potential measurements were performed on a Malvern Zetasizer Nano-ZSP (Malvern, UK).
  • reaction was allowed to react at room temperature (R.T.) for at least 4 h. Afterwards, the reaction was concentrated by rotary-evaporation and purified by flash chromatography on silica column using a dichloromethane with ethyl acetate (0 to 5%) eluent system. The product was then dried under high vacuum to yield a white solid.
  • Ethyl acetate was added to dilute the reaction and extracted with 0.1 M NaHCO 3 (2x) and brine (2x). The solution was dried with MgSO 4 and concentrated via rotary-evaporation. The mixture was purified by flashing chromatography on silica column with a hexane: ethyl acetate (2:1 to 1:1, v:v) eluent system supported with 0.1% triethylamine. The final product was dried under high vacuum yielding a viscous liquid.
  • the reaction was allowed to stir at R.T. for at least 4 h before dried under rotary-evaporation.
  • the product was purified by flashing chromatography on silica column with a dichloromethane (0 to 30% ethyl acetate) eluent system. The purified product was dried under high vacuum and yield a white powder.
  • the solution was extracted with 0.1 M NaHCO3 (2x) and brine (2x) and dried with MgSO4 followed by rotary-evaporation.
  • the concentrated solution was then purified by flashing chromatography on silica column with a dichloromethane added with ethyl acetate (0 to 10%) eluent system.
  • the purified product was dried with rotary-evaporation and yield a viscous liquid yellow in color.
  • the reaction was stirred for an additional 6 hours.10 ⁇ L of reacted mixture was lyophilized and then dissolved in 0.05 M lithium bromide in HPLC-grade DMF to be analyzed by DMF GPC for molecular weight and PDI measurement. Several drops of ethyl vinyl ether were added to quench the reaction, followed by 2 hours of stirring. The solution was concentrated under vacuum, and the resulting residue was precipitated into cold diethyl ether three times. The precipitate was dried under vacuum, yielding a white powder. The pac-PN brush polymer was then reacted with excess sodium azide in anhydrous DMF overnight at room temperature. The mixture was dialyzed against Nanopure water for one week and lyophilized to obtain a white powder.
  • the azide-functionalized PN bottlebrush polymer (50 nmol) was dissolved in 1 mL of 2 M sodium chloride solution and reacted with - 45 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 dibenzocyclooctyne (DBCO)-modified ASO (50 nmol) at 50 °C overnight.
  • the conjugate was purified by aqueous GPC, desalted, and lyophilized.
  • the purified PN-pacDNA was stored at -20 °C until use.
  • Oligonucleotide and ribose-based pacDNA backbone synthesis [00179] All oligonucleotide and ribose backbones were synthesized on a Dr. Oligo 48 DNA synthesizer (Biolytic Lab Performance, Inc., Fremont, CA) using dT-CPG. The time for the coupling of ribose-phosphoramidites were set at 10 min (compared to 15 s for regular phosphoramidite). Oligonucleotide strands were cleaved from the CPG support and deprotected in aqueous ammonium hydroxide solution (28-30% NH 3 basis) at room temperature for 16 h.
  • Free oligonucleotide were purified by RP-HPLC, followed by the removal of dimethoxytrityl (DMT) groups using 20% acetic acid.
  • the oligonucleotide with ribose backbones were purified with Glen-PakTM DNA purification cartridge (for use with disposable syringes) (Glen Research Co., USA.) following the manufacture’s protocol.
  • NCI-H358 cells was cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics.
  • K273 cells were cultured in DMEM media supplemented with 5% fetal bovine serum (FBS), 10 ⁇ g/mL Epidermal Growth Factor (EGF), 1mg/mL hydrocortisone, 1 mg/mL cholera toxin, 4mg/mL insulin, and 1% penicillin- streptomycin (PS). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . - 46 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00184] C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratory., USA. Animals were housed at Northeastern University animal facilities.
  • FBS fetal bovine serum
  • EGF Epidermal Growth Factor
  • PS penicillin- streptomycin
  • NCI-H358 cells were cultured in a 24-well plate with a cell seeding density of 1.0 ⁇ 10 6 cells per well in 1 mL complete RPMI medium for 24 h at 37 °C. Next, cells were washed 2 ⁇ with 1 ⁇ PBS and treated with fluorescently labeled test agents and controls, which were dissolved in serum-free RPMI culture medium at various concentrations (1 ⁇ M to 5 ⁇ M).
  • NCI-H358 cells were cultured in a 24-well glass bottom plate at 1.0 ⁇ 10 5 cells per well in 1 mL complete RPMI medium for 24 h at 37 °C.
  • Cell viability [00190] The cell viability of NCI-H358 and K273 after treatment with ribose-based pacDNA and bottlebrush polymer was analyzed by MTT (dimethylthiazol-diphenyltetrazolium bromide) colorimetric assay. Cells were seeded in 96-well plates at a density of 1 ⁇ 10 4 cells per well in 175 ⁇ L full growth media and cultured for 24 h at 37 °C with 5% CO 2 .
  • MTT dimethylthiazol-diphenyltetrazolium bromide
  • BCA bicinchoninic acid
  • Antibodies used in this study were: KRAS antibody (cat. sc-30; Santa Cruz), ⁇ - actin (cat. AM4302, Cell Signaling Technology), anti-mouse IgG, HRP-linked antibody (cat. 7076S, Cell Signaling Technology).
  • KRAS antibody cat. sc-30; Santa Cruz
  • ⁇ - actin cat. AM4302, Cell Signaling Technology
  • anti-mouse IgG HRP-linked antibody
  • HRP-linked antibody cat. 7076S, Cell Signaling Technology.
  • Blood samples (25 ⁇ L) were collected from the submandibular vein at varying time points (30 min, 2 h, 4 h, 10 h, 24 h, 48 h and 72 h) using BD VacutainerTM blood collection tubes with lithium - 48 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 heparin.
  • Heparinized plasma was obtained by centrifugation at 3000 rpm for 20 min, aliquoted into a 96-well plate, and measured for fluorescence intensity on a SynergyTM Neo2 Multi-Mode microplate reader (BioTek Instruments Inc., VT, USA).
  • PBS phosphate buffered saline
  • mice received following treatment via tail vein injection: 1) PBS, 2) pac-4 ⁇ 1C 18 -ASO2 (0.5 ⁇ mol/kg), 3) pac-4 ⁇ 1C 18 -ASO2 (0.5 ⁇ mol/kg), 4) Free ASO2 (0.5 ⁇ mol/kg).
  • Samples were injected every four days for four doses. Once the tumor volume reached 1000mm 3 , the mice were euthanized by CO2. Major organs and tumors were collected into 10% neutral buffered formalin for overnight fix and then for histological analysis carried by iHisto Inc.
  • Anti-PEG immune response [00198] Healthy C57BL/6 mice were administrated PBS, pac-4 ⁇ 1C 18 -ASO2, free ASO2 or PEG-KLH every four days for four doses.50-100 ⁇ L plasma were collected one week after the last dose. The concentration of anti-PEG IgM/IgG were assessed by ELISA kit (Cat # PEGG-1, Cat #PEGM-1, Life Diagnotics, Inc.) according to the manufacturer’s protocol. [00199] Multiplex analysis of cytokines [00200] The multiplexing analysis was performed using the LuminexTM 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta).
  • the 32-plex consisted of Eotaxin, G-CSF, GM-CSF, IFN ⁇ , IL-1 ⁇ , IL- 1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, - 49 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1 ⁇ , MIP-1 ⁇ , MIP-2, RANTES, TNF ⁇ , and VEGF.
  • Assay sensitivities of these markers range from 0.3 – 30.6 pg/mL for the 32-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol.
  • Whole-animal and ex vivo organ imaging [00202] Tumor-bearing C57BL/6 mice were i.v. injected with Cy5-labeled samples at an ASO dose of 0.5 ⁇ mol/kg. Then mice were scanned at 1, 4, 8, 24 h, 48 h, and 72 h using an IVIS Lumina II imaging system (Caliper Life Sciences, Inc. MA, USA).
  • mice were euthanized using CO 2 , and major organs, tissues and the tumor were dissected and imaged using IVIS.
  • Biodistribution [00204] The major organs, tissues and tumor were diced into small piece and weighted. Then, the flesh was homogenized in tissue protein extraction reagent (Thermo Fisher, USA), which is supported with 0.5% Triton X-100, using a BeadBlaster D2400-R refrigerated homogenizer (Benchmark scientific, NJ, USA.).
  • each pacDNA model underwent a refinement process in Maestro, during which hydrogen atoms were added to the structures.
  • the OPLS4 forcefield was used to model pacDNAs.
  • each model was solvated in a rectangular water box using the SPC water model with a minimum water shell of 10 ⁇ from any box edge to the nearest atom of the structure.
  • Sodium (Na+) and chloride (Cl-) ions were placed - 50 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 to neutralize the system.
  • each pacDNA structure underwent an extensive minimization and equilibration process, detailed as follows. Each system was first relaxed using the default relaxation protocol from Maestro. After that, the system went through an annealing process by first heating the system from 10 K to 400 K and then cooling to 300 K. Following annealing, each pacDNA model was re-solvated with a refreshed water box to accurately represent the aqueous environment. The updated simulation system was used to conduct a production run for 100 ns at a constant temperature of 300 K with 1 bar pressure (NPT ensemble).
  • the Nosé–Hoover algorithm was used for temperature control, and the Martyna– Tobias–Klein algorithm was used to control the pressure. All molecular dynamics simulations were performed using the GPU-accelerated Desmond engine in Schrodinger molecular modeling suite. (27-29) The simulation trajectories from 90 ns to 100 ns were used to calculate the potential energy for the corresponding pacDNA structure. [00208] A full-length and full-atomistic pacDNA model with the largest spacing (pac-4 ⁇ 1C18- ASO1) was built to investigate the conformational characteristics. The initial structure of pacDNA-4 ⁇ 1C 18 with truncated PEG chain was built using ChemDraw. We extended each PEG chain to 226 mer using VMD and Maestro.
  • Oligonucleotide drugs hold great potential to revolutionize disease therapeutics due to their adaptability across diverse diseases and targets. Notably, oligonucleotide-based therapies have been developed for previously untreatable rare diseases, such as Spinraza for spinal muscular atrophy and investigational drugs for DM1 (myotonic dystrophy type 1).
  • oligonucleotide drug candidates are frequently hindered by their structural and chemical properties, including their negative charge and limited membrane permeability.
  • LNPs lipid nanoparticles
  • polymer-conjugated drug carriers polymer-conjugated drug carriers
  • antibody-conjugated delivery systems designed for targeted oligonucleotide transport.
  • LNPs and polymer-based systems predominantly accumulate in the liver, limiting their applicability for targeting other organs.
  • antibody-conjugated delivery systems are typically unable to maintain a prolonged blood circulation time.
  • none of these platforms have integrated machine learning with delivery platform development to precisely modulate cellular uptake or achieve targeted delivery to specific organs.
  • the polyphosphodiester backbone polymer was assembled via a stepwise condensation process, incorporating three distinct side-chain modified phosphoramidite units in conjunction alongside an unmodified serinol phosphoramidite.
  • the C18 and cholesterol phosphoramidites served as hydrophobic units, while the spermine phosphoramidite introduced positive charge.
  • N non-modified (serinol); A: C18 alkyl; C: cholesterol; S: spermine. All monomers (N, A, C, and S) are linked to PEG (10 kDa).
  • the polyphosphodiester backbone was synthesized via standard solid-phase synthesis, incorporating both modified and unmodified phosphoramidite units to form a 30-mer structure in a stepwise manner. Following backbone assembly, 20% piperidine in DMF was used to remove the Fmoc protecting group, after which the crude product was purified using a Glen column to eliminate excess reagents and byproducts. The synthesized polymer backbone was then reacted with PEG-NHS, and after a two-step polymerization process, the final product was purified using size-exclusion chromatography to remove unreacted small molecules and achieve the desired molecular weight distribution. (FIG.31B) Our primary objective was to investigate how different arrangements of these units affect in vitro and in vivo performance.
  • the Q45 polymer featuring 8 evenly distributed C18 units and two centrally located spermine units, demonstrated the highest uptake across all cell lines, highlighting the importance of both hydrophobicity and charge distribution in optimizing uptake.
  • the inclusion of cholesterol did not significantly enhance cellular uptake, likely due to its lower affinity for cell membranes compared to C18.
  • mice were sacrificed, and their organs, including the liver, spleen, kidneys, lungs, and heart, were dissected and homogenized to quantify the distribution of the cyanine5-labeled bottlebrush polymers across different tissues. This allowed us to evaluate the biodistribution and retention of the polymers in major organs, providing key insights into their tissue-specific accumulation.
  • IVIS imaging data is presented in FIG.34A, while a quantification heatmap and a bar graph illustrating the organ-specific accumulation of different bottlebrush polymer formulations are presented in FIG.34B and FIG.34C respectively.
  • the IVIS imaging results reveal distinct biodistribution patterns, with fluorescence signals indicating polymer accumulation in different organs.
  • Microscale thermophoresis (MST) binding measurements were carried out with 10 nM Cy5-labeled samples in binding buffer (1 ⁇ PBS pH 7.4, 0.1% Triton X 100) with a range of concentration of human AXL protein (154-AL) from 2500 nM to 0.07 nM. The mixtures were transferred to Monolith NT.115 standard capillaries and analyzed on a Monolith NT.115 instrument (NanoTemper Technologies, Kunststoff, Germany) at medium MST power and 20% excitation power.
  • Samples and controls were intravenously administered via the tail vein at equal aptamer concentrations (0.5 ⁇ mol/kg equiv. of aptamer).
  • Blood samples 50 ⁇ L were collected from the submandibular vein at varying time points (30 min, 2 h, 4 h, 10 h, 24 h, 48 h, and 72 h) using BD Vacutainer blood collection tubes with sodium heparin. Heparinized plasma samples were obtained by centrifugation at 3000 rpm for 20 min, and then aliquoted into a 96-well plate. The fluorescence intensities were measured on a plate reader. The amounts of agents in the blood samples were estimated using standard curves established for each sample in freshly collected plasma.
  • the disclosed polymers, conjugates and methods improve the poor circulation of oligonucleotide drug in blood, enable oligonucleotide drug targeting at specific organs, enhance cellular uptake of oligonucleotide drugs, and/or enable disease specific optimization using polymer sequence engineering.
  • library screening can yield a large number of polymers, each having its own best match in disease.
  • the disclosed polymers conjugates and methods can be used for therapeutic development for various diseases with different drug delivery requirements, in vitro research, generation of well-defined synthetic polymers using solid phase synthesis, drug delivery, and/or a biological research tool.
  • REFERENCES [00232] (1) Moumne, L.; Marie, A.
  • Oligonucleotide Therapies The Past and the Present. Hum. Gene Ther.2015, 26 (8), 475– 485, DOI: 10.1089/hum.2015.070 [00235] (4) Lauffer, M. C.; van Roon-Mom, W.; Aartsma-Rus, A.; Collaborative, N. Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders. Commun.

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Abstract

In some embodiments disclosed herein are polymers comprising a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone.

Description

Docket No.5200.2402-001 (INV-24084) 4112432.v1 Inventors: Jiachen Lin, Ke Zhang and Yun Wei Polyphosphodiester based polymer library and combinatorial approach to identify bioactive sequences RELATED APPLICATION(S) [0001] The application claims the benefit of U.S. Provisional Application No.63/567,421, filed on March 19, 2024. The entire teachings of the above application(s) are incorporated herein by reference. GOVERNMENT SUPPORT [0002] This invention was made with government support under Grant Numbers 1R01CA251730, 1R01GM121612, and 42CA275425 awarded by the National Institutes of Health, and under Grant Number 2004947 awarded by the National Science Foundation. The Government has certain rights in the invention. INCORPORATION BY REFERENCE OF MATERIAL IN XML [0003] This application incorporates by reference the Sequence Listing contained in the following eXtensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 52002402-001_SL.xml; created: March 12, 2025; 72,394 Bytes in size. SUMMARY [0004] The clinical translation of oligonucleotide-based therapeutics continues to encounter challenges in delivery. In this disclosure, a novel class of delivery vehicles for antisense oligonucleotides (ASO), which are based on polyethylene glycol (PEG) bottlebrush polymers with sequence-defined backbones, is introduced. Using solid-phase synthesis and bespoke monomers, the ASO and polymer backbone can both be assembled on the solid support. The synthesis allows the incorporation of different units such as carbon lipids, spermine, or cholesterol into the backbone in specific patterns to modulate the cell-materials interactions. PEG side chains were grafted onto the polymer segment of the resulting polymer-oligonucleotide - 1 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 conjugate, yielding bottlebrush polymers. Several optimal patterns of modifiers which lead to improved cellular uptake, plasma pharmacokinetics, biodistribution, and antisense activity in vivo were discovered. [0005] Previously developed was a novel form of oligonucleotide therapeutic delivery platform, termed pacDNA, which is a bottlebrush polymer covalently conjugated with the oligonucleotide to the polymer backbone. pacDNA overcomes many challenges associated with oligonucleotide delivery, including carrier-mediated toxicity or immunogenicity, poor delivery to non-liver sites, and non-hybridization side effects. It is discovered that the interactions between pacDNA and cells are strongly dictated by the polymer backbone, which opens a door to explore a range of biological properties such as pharmacokinetics and biodistribution by carefully designing the backbone. In the present disclosure, a library of “digital” bottlebrush polymers, those with precise backbone arrangement of various monomers within the backbone, was designed and synthesized. The biodistribution, plasma pharmacokinetics, and cellular uptake of these digitized bottlebrush polymers were explored, and interesting structure-property relationships were seen. ln some embodiments, a novel drug delivery system can be tailored to each specific disease or disease type based upon digital polymers. [0006] One embodiment disclosed herein is a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. [0007] Another embodiment disclosed herein is a conjugate, comprising a polymer of the present disclosure and an agent conjugated to the polymer. [0008] Another embodiment disclosed herein is a composition comprising a conjugate of the present disclosure and a pharmaceutically acceptable carrier. [0009] Another embodiment disclosed herein is a method of modulating or altering the expression of a gene product encoded by a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate of the present disclosure. - 2 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0010] Another embodiment disclosed herein is a method of delivering an agent to a subject in need thereof, the method comprising: administering a conjugate or a composition of the present disclosure to the subject. [0011] Another embodiment disclosed herein is a method of delivering an agent to a cell, the method comprising: contacting the cell with a conjugate of the present disclosure. [0012] Another embodiment disclosed herein is a method of detecting a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate of the present disclosure, thereby forming a polynucleotide-conjugate hybrid; and detecting a signal from the polynucleotide-conjugate hybrid. [0013] In yet another embodiment disclosed herein is a method of treating a disease, the method comprising administering a conjugate of the present disclosure or a composition of the present disclosure to a subject in need thereof. [0014] In yet another embodiment disclosed herein is a method of making a bottlebrush polymer, the method comprising: combining one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. BRIEF DESCRIPTION OF THE DRAWINGS [0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0016] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference - 3 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. [0017] FIGs.1A-D: Design of ribose-based pacDNAs. FIG.1A: Synthetic scheme of pacDNAs. FIG.1B: Backbone designs of pacDNAs. The gray and red circles represent the R- NH2 unit and R-C18 units, respectively. ASO: antisense oligonucleotide. FIG.1C: Schematics of the PEGylation process of ribose-based pacDNA. FIG.1D: Molecular dynamics (MD) simulation of ribose-based pacDNA with four C18 modifiers (cyan: PEG; gray: backbone; red: C18; yellow: ASO). [0018] FIGs.2A-D: Characterization of ribose-based pacDNAs. FIG.2A: Aqueous GPC chromatograms of pacDNAs after two-stage PEGylation. FIG.2B: pacDNA Z-average (the intensity-weighted mean) molecular size in Nanopure water as determined by DLS. FIG.2C: Representative TEM image of pacDNA (pac-4 × 1C18-ASO1) with negative staining using 2% uranyl acetate. FIG.2D: pacDNA ζ potential in Nanopure water. [0019] FIGs.3A-G: In vitro properties of ribose-based pacDNAs. FIG.3A: Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs-ASO1 containing varying numbers and arrangement of C18 modifiers (0C18, 2C18, 4C18, 6C18, 8C18, 10C18) after 4 h incubation, as determined by flow cytometry. FIG.3B: Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs-ASO1 containing varying numbers and arrangement of C18 modifiers (1 × 6C18, 2 × 3C18, 3 × 2C18, 6 × 1C18) after 4 h incubation, as determined by flow cytometry. FIG.3C: Confocal microscopy of NCI-H358 cells treated with Cy5-labeled free ASO1 or pacDNAs- ASO1 for 8 h (additional samples: FIG.25). Cy5-labeled ASO (red), DAPI-stained nuclei (blue), and phalloidin-stained F actin (green) are presented separately. FIG.3D: Representative molecular snapshots from the simulation trajectory (top row is the structures of entire molecules, bottom row is the corresponding structures without PEG chains to show backbones more clearly; cyan: PEG; gray: backbone; red: C18; yellow: ASO). FIG.3E: Interaction energy of pacDNAs after interaction with water as determined by MD simulation. FIG.3F: KRAS depletion efficiency of 10 μM pacDNAs-ASO1 in NCI-H358 cells. The number shows the relative KRAS protein level analyzed by ImageJ software. FIG.3G: Inhibitive effect of 10 μM pacDNAs-ASO1 - 4 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 on the proliferation of NCI-H358 cells after 48 h incubation. Statistical analysis was performed using one-way ANOVA. ****P < 0.0001. [0020] FIGs.4A-E: Plasma PK and biodistribution of ribose-based pacDNAs. FIG.4A: Plasma PK of Cy5-labeled pacDNA and free DNA in C57BL/6 mice following i.v. injection. Statistical analysis was performed using two-way ANOVA. ****P < 0.0001. FIG.4B: Plasma PK parameters of ribose-based pacDNAs with variant numbers of the C18 modifier. FIG.4C: Live animal fluorescence imaging of C57BL/6 mice bearing the K273 allograft following i.v. injection of Cy5-labeled pacDNA. Areas surrounding the allograft have been shaved to facilitate imaging. FIG.4D: Quantitative biodistribution of pacDNAs in major organs/tissues of tumor- bearing C57BL/6 mice 72 h post i.v. injection. FIG.4E: Ex vivo imaging of pacDNAs in major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection. [0021] FIGs.5A-F: In vivo antitumor efficacy of ribose-based pacDNAs. FIG.5A: K273 allograft volume changes in 24 days with i.v. injection of PBS, ASO2, and pac-4 × 1C18-ASO2 at equivalent ASO doses (0.5 μmol/kg) every 4 days since day 10. Injections are indicated by black arrows. FIG.5B: Mean tumor growth curve. FIG.5C: Kaplan–Meier end point animal survival analysis. Statistical analysis was calculated by the log-rank test. FIG.5D: Immunohistostaining and H & E staining of K273 tumor after treatment. FIG.5E: Anti-PEG IgM and IgG levels in C57BL/6 mice plasma after i.v. injection of PBS, free ASO2, pac-4 × 1C18- ASO2, and PEG-KLH (positive control) at 0.5 μmol/kg every 4 days for four doses. Plasma was collected 1 week after the last dose. Statistical analysis was performed using one-way ANOVA. FIG.5F: Selected cytokine and chemokine levels in C57BL/6 mice plasma 5 h after i.v. injection of PBS, free ASO2, pac-4 × 1C18-ASO2 (2 μmol/kg), and LPS (0.5 mg/kg). Statistical analysis was performed using two-way ANOVA (B) and one-way ANOVA (E,F). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [0022] FIG.6: 1H NMR spectrum of compound 1 (Scheme 1). [0023] FIG.7: 13C NMR spectrum of compound 1. [0024] FIG.8: 1H NMR spectrum of compound 2 (Scheme 1). [0025] FIG.9: 13C NMR spectrum of compound 2. [0026] FIG.10: 1H NMR spectrum of compound 3 (Scheme 1). - 5 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0027] FIG.11: 13C NMR spectrum of compound 3. [0028] FIG.12: 1H NMR spectrum of R-NH2 (Scheme 1). [0029] FIG.13: 13C NMR spectrum of R-NH2. [0030] FIG.14: 1H NMR spectrum of compound 4 (Scheme 2). [0031] FIG.15: 13C NMR spectrum of compound 4. [0032] FIG.16: 1H NMR spectrum of compound 5 (Scheme 2). [0033] FIG.17: 13C NMR spectrum of compound 5. [0034] FIG.18: 1H NMR spectrum of compound 6 (Scheme 2). [0035] FIG.19: 13C NMR spectrum of compound 6. [0036] FIG.20: 1H NMR spectrum of R-C18 (Scheme 2). [0037] FIG.21: 13C NMR spectrum of R-C18. [0038] FIG.22: DLS measurements of ribose-based pacDNAs in Nanopure water. [0039] FIGs.23A-F: Comparison of polynorbornene bottlebrush (pac-PN) and ribose-based pac 4×1C18. FIG.23A: The molecular weight of five batches of pac-PN and pac 4×1C18 brush polymers. FIG.23B: Descriptive statistics of the molecular weight of FIG.23A. FIG.23C: Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs and free oligo. FIG.23D: Plasma pharmacokinetics of Cy5-labeled pacDNA and free DNA in C57BL/6 mice following i.v. injection. Statistical analysis was performed using two-way ANOVA. **** P <0.0001. FIG. 23E: Live animal fluorescence imaging of C57BL/6 mice bearing K273 allograft following i.v. injection of Cy5-labeled pacDNA. Areas surrounding the allograft has been shaved to facilitate imaging. FIG.23F: The ex vivo imaging of pacDNAs in major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection. [0040] FIGs.24A-B: In vitro properties of ribose-based pacDNAs without ASO. FIG.24A: Cellular uptake by NCI-H358 cells of Cy5-labeled pacDNAs containing varying numbers and arrangement of C18 modifiers after 4 h incubation, as determined by flow cytometry. MFI: mean fluorescence intensity. FIG.24B: Cytotoxicity of 10μM ribose-based pacDNAs (without ASO) on the proliferation of NCI-H358 cells after 48 h incubation. Statistical analysis was performed using one-way ANOVA. - 6 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0041] FIG.25: Confocal microscopy of NCI-H358 cells treated with Cy5-labeled free DNA or ribose-based pacDNAs for 8 h. [0042] FIG.26: Inhibitive effect of ribose-based pacDNAs-ASO1 on the proliferation of NCI-H358 cells at different concentration (from 100 nM to 5 μM) after 48 h incubation. [0043] FIG.27: Fluorescence imaging of major organs/tissues of tumor-bearing C57BL/6 mice 72 h post i.v. injection of Cy5-label ribose-based pacDNAs. [0044] FIGs.28A-B: In vitro efficacy test of ribose-based pacDNAs on K273 cell line. FIG. 28A: KRAS depletion efficacy of pac-4 × 1C18-ASO2 dose-dependently. FIG.28B: Inhibition of K273 cell proliferation 48 h after treatment of pac-4 × 1C18-ASO2, pac-4 × 1C18-scramble and free-ASO2 at different levels of dose. Statistical analysis was performed using two-way ANOVA. **** P <0.0001. [0045] FIG.29: Body weight change of C57BL/6 mice bearing K273 allograft model. See FIG.5A for the administration time frame. [0046] FIG.30: H&E staining of major organs and tissues of C57BL/6 mice after i.v. injection of PBS, free ASO2 and pac-4 × 1C18-ASO2 at doses 0.5 μmol/kg every four days, four doses at total. [0047] FIG.31A: Four different example building blocks for digital brush polymer. Example structures of cholesterol, spermine, serinol (void), and C18 alkyl phosphoramadites. [0048] FIG.31B: Synthesis of an example digital brush polymer library. Color code: spermine (R = red), cholesterol (R = yellow), C18 alkyl (R = green), and void (R = white). [0049] FIG.32A: Transmission electron microscopy (TEM) for synthesized bottle brush polymers. [0050] FIG.32B: Gel permeation chromatography (GPC) results of brushes with different backbones. [0051] FIG.32C: Dynamic light scattering (DLS) reveals size similarity of polymers with different backbones. [0052] FIG.32D: Zeta potential reveals differences in charge on different backbones. [0053] FIG.33A: Cellular uptake on NCI-H358 cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells. - 7 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0054] FIG.33B: Cellular uptake on DM1 fibroblast cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells. [0055] FIG.33C: Cellular uptake on HepG2 cell line for digital brush polymer, all synthesized polymers are labeled with cyanine 5 and under 0.5 nmol/ul for 10000 cells. [0056] FIG.34A: Biodistribution analysis of bottlebrush polymers in various organs. IVIS fluorescence imaging of excised organs, showing distinct accumulation patterns among different polymer formulations. The epi-fluorescence intensities are shown in the heatmap legend. [0057] FIG.34B: Heatmap representation of quantified fluorescence intensities, with warmer colors indicating higher accumulation levels in specific organs. [0058] FIG.34C: Quantification bar graph illustrating the comparative distribution of polymers across different organs. DETAILED DESCRIPTION [0059] A description of example embodiments follows. [0060] Oligonucleotide-based therapeutics hold immense promise for treating diseases through diverse mechanisms, such as gene regulation, receptor binding, and alternative splicing, among others. (1,2) However, clinical development of oligonucleotide drug candidates often faces setbacks attributed to poor target engagement in vivo due to limited uptake by target organs and cells, limiting their use to a few concentrated disease settings. (3−5) Current strategies to improve drug potency generally focus on chemical modifications, bioconjugation with antibodies, peptides, or small molecule ligands, and polyplex carriers such as lipid nanoparticles and cationic polymers. (6−9) However, concerns remain regarding potential toxicity and immunogenicity associated with chemical modifications and carriers, as well as suboptimal biodistribution following systemic administration. (10) Thus, the development of a delivery system that can simultaneously enhance nuclease stability, facilitate rapid intracellular delivery of oligonucleotides, and ensure sufficient distribution into target tissues holds the potential to bridge the critical development gap. (9−11) [0061] Previously, the design of a bottlebrush polymer–oligonucleotide conjugate (termed pacDNA: polymer-augmented conjugate of DNA), which effectively mitigates protein– oligonucleotide interactions while maintaining unaffected target RNA binding, was - 8 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 reported. (12,13) This unique selectivity is achieved through the more densely packed poly(ethylene glycol) (PEG) side chains compared to typical linear or slightly branched PEG, which leads to reduced protein binding and consequently, fewer side effects in vivo that derive from unwanted protein-oligonucleotide interactions. (14) Additionally, the large size of the conjugate reduces renal clearance and enhances circulation in the bloodstream, leading to significantly increased biodistribution to non-liver/kidney organs including difficult-to-target sites such as the skin, skeletal muscle, and heart. (15,16) [0062] The bottlebrush polymer in our earlier system was prepared by ring-opening metathesis polymerization of norbornenyl monomers using a Grubbs third-generation catalyst. We identified three aspects where this system can be improved: (1) batch-to-batch consistency, (2) residue heavy metal content, and (3) control over the bottlebrush polymer backbone. Herein, the design, synthesis, and biological testing of a novel deoxyribose 3′–5′ phosphodiester-derived bottlebrush– oligonucleotide conjugate, which offers unprecedented flexibility and control in the backbone chemistry, length, sequence, and composition, is reported. In some embodiments, an optimized backbone structure containing patterned C18 units for application in targeting Kirsten Rat Sarcoma Virus (KRAS) mRNA in a mouse allograft model is described herein. These findings provide valuable insights for the development of tailored vehicles to meet indication-specific delivery requirements. [0063] A method using solid-phase synthesis to construct sequence-defined polymeric materials, which allows one to modulate the interactions between materials and living systems, is developed. In some embodiments, certain chemical modifiers, such as lipid tails, cholesterol, etc., when arranged into different patterns within the polymer, give rise to variable cell uptake, plasma pharmacokinetics, and biodistribution, as described herein. Therefore, it is possible to optimize polymers as delivery vehicles to address each different disease that has specific requirements for drug delivery. [0064] Nanoparticles present significant potential for advancing oligonucleotide drug delivery systems. Among these, bottlebrush polymers have emerged as a promising platform, addressing key challenges such as carrier-mediated toxicity, immunogenicity, and unintended off-target effects. However, conventional bottlebrush polymers with uniform backbone configurations often struggle to achieve optimal cellular uptake and organ-targeting specificity, - 9 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 complicating the prediction of organ tropism and therapeutic efficacy. In this disclosure, in some embodiments, a library of sequence-defined “digital” bottlebrush polymers with precisely arranged lipids, cholesterol, and positively charged monomers along the backbone were designed and synthesized. By evaluating their biodistribution, plasma pharmacokinetics, and cellular uptake, predictable structure–property relationships were identified. This disclosure represents a significant step toward developing a refined drug delivery system, capable of being tailored to specific diseases through rational design. Polymers [0065] In some embodiments, the present disclosure provides a polymer, comprising one or more of: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. [0066] In some embodiments, the present disclosure provides a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. [0067] In some embodiments, the present disclosure provides a polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, covalently linked to the backbone. In some embodiments, the polymer further comprises at least one cholesterol, at least one spermine, or a combination thereof covalently linked to the backbone. [0068] In some embodiments, a polymer backbone is a polyphosphodiester backbone. In some embodiments, a polymer backbone is a polyphosphodiester backbone, 2-deoxyribose-based backbone, a serinol-based backbone, or a combination thereof. In some embodiments, a polymer - 10 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 backbone is a 2-deoxyribose-based backbone, a serinol-based backbone, or a combination thereof. [0069] In some embodiments, the polymer comprises from about 1 to about 100 (e.g., about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, etc.) polymer arms covalently linked to the backbone. In some embodiments, the polymer comprises from about 10 to about 40 polymer arms covalently linked to the backbone. In some embodiments, the polymer comprises about 30 polymer arms covalently linked to the backbone. [0070] In some embodiments, at least one (e.g., each) of the plurality of polymer arms is poly(ethylene glycol) (PEG), poly-L-lysine, PAMAM dendrimers, poly(2-oxazoline), polypeptides, poly(N-isopropylacrylamide), polyanhydrides, poly(ε-caprolactone), chitosan, polystyrene, poly(ethyleneimine), poly(lactic-co-glycolic acid), poly(propyleneimine) dendrimers, poly(β-amino esters), poly(vinyl alcohol), poly(methyl methacrylate), polyacrylamide, polydopamine, polycarbonate, dextran, or a combination therefore. In some embodiments, each of the plurality of polymer arms is a poly(ethylene glycol). [0071] In some embodiments, each of the at least one alkyl is a C10-C30 alkyl (e.g., C10-C20 alkyl, C15-C20 alkyl, etc.). In some embodiments, each of the at least one alkyl is a C18 alkyl. [0072] 7 In some embodiments, a polymer comprises from about 2 to about 30 alkyls (e.g., about 2 to about 20, about 2 to about 15, about 2 to about 10, etc.) covalently linked to the backbone. In some embodiments, a polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone. [0073] In some embodiments, a polymer comprises about 4 alkyls covalently linked to the backbone. [0074] In some embodiments, a polymer comprises 6 cholesterols covalently linked to the backbone. [0075] In some embodiments, a polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone. In some embodiments, the plurality of alkyls or the plurality of cholesterols are evenly distributed (i.e., the number of non-alkyls (or non- cholesterols) separating adjacent alkyls (or cholesterols) are the same or vary by 1 or 2 units). - 11 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 For non-limiting example, if there are 3, 4, and 5 non-alkyls separating any adjacent alkyls out of the plurality of alkyls linked to the polymer backbone, the plurality of alkyls are evenly distributed. For non-limiting example, if there are 2, 4, and 5 non-alkyls separating any adjacent alkyls out of the plurality of alkyls linked to the polymer backbone, the plurality of alkyls are not evenly distributed. [0076] In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by from about 2 monomers to about 6 monomers. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 2, 3, 4 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 3, 4, 5 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 4, 5, 6 monomers, or a combination thereof. [0077] In some embodiments, a polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47 (Table 3). [0078] In some embodiments, the polymer has a sequence comprising 80%, 85%, 90%, 95% or 100% of a sequence disclosed herein, e.g., in Table 3. Conjugates [0079] In some embodiments, a polymer is modified, e.g., conjugated to an agent (e.g. a therapeutic agent, an imaging agent, an oligonucleotide, etc.). The term “conjugated” refers to attached, via a covalent or noncovalent interaction. Conjugation can employ any of suitable linking agents; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents. [0080] In some embodiments, the present disclosure provides a conjugate comprising a polymer of the present disclosure and an agent (e.g., oligonucleotide) conjugated (e.g., covalently linked) to the polymer. [0081] In some embodiments, an oligonucleotide comprises deoxyribonucleotides. In certain embodiments, an oligonucleotide comprises ribonucleotides. Non-limiting examples of oligonucleotides include single-, double- or multistranded DNA or RNA, DNA-RNA hybrids, or - 12 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 a polymer comprising purine and pyrimidine bases, or other natural, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of an oligonucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof. [0082] In some embodiments, an agent (e.g., oligonucleotide) comprises a linker (e.g., a cleavable linker) used for conjugation to the polymer backbone and is otherwise unmodified (e.g., natural DNA or RNA). [0083] In some embodiments, the agent (e.g., oligonucleotide) is conjugated to the 5’ end of the polymer. [0084] In some embodiments, polymers, conjugates and compositions of the present disclosure are used to treat a disease or disorder, including those described herein. [0085] In some embodiments, polymers, conjugates and compositions of the present disclosure are used for treating Myotonic Dystrophy Type 1 (DM1). In some embodiments, a polymer has SEQ ID NO.37 or SEQ ID NO.29 (see Table 3). These sequences have a high muscle uptake. [0086] Q36 (SEQ ID NO.37, Table 3): NN NNN NAN NNN NNN ANN NNN NNA NNN NNN N [0087] Q18 (SEQ ID NO.29, Table 3): NN NCN NNC NNN NCN NNN CNN NNC NNN CNN N [0088] In some embodiments, polymers, conjugates and compositions of the present disclosure are used for treating rheumatoid arthritis. In some embodiments, a polymer has SEQ ID NO.27 (see Table 3). This sequence has a high spleen uptake. [0089] Q16 (SEQ ID NO.27, Table 3): NN NNN NNN NNN NCC CCC CNN NNN NNN NNN N [0090] In some embodiments, a conjugate comprises an oligonucleotide, or an analog or derivative thereof. In some embodiments, the oligonucleotide, or the analog or derivative thereof, is an inhibitor of a protein. In some embodiments, the oligonucleotide, or the analog or derivative thereof, is an activator of the protein. In some embodiments, the oligonucleotide, or the analog or derivative thereof, decreases (e.g., reduces or neutralizes) the expression or activity of the - 13 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 protein. In other embodiments, the oligonucleotide, or the analog or derivative thereof, increases the expression or activity of the protein. [0091] The oligonucleotides can have sequences containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Accordingly, oligonucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., A, G, C, or T) and nucleotides comprising modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides, such as 5-methyl dCTP and 5- hydroxymethyl cytosine). In some embodiments, the oligonucleotide comprises at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-o- methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'- thiouridine and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, decrease immunogenicity, or a combination of the foregoing. [0092] In some embodiments, the agent (e.g., oligonucleotide) is a DNA molecule. In some embodiments, the agent (e.g., oligonucleotide) is an RNA molecule. [0093] In some embodiments, the oligonucleotide comprises an analog or a derivative of an oligonucleotide. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative comprises one or more phosphorothioate-linkages. In some embodiments, the agent comprises a deoxyribonucleic guanidine (DNG) nucleotide. In some embodiments, the agent comprises ribonucleic guanidine (RNG) nucleotide. [0094] In some embodiments, the agent (e.g., oligonucleotide) is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamers, ribozymes, DNAzymes, antisense oligonucleotides, exon-skipping oligonucleotides, siRNA oligonucleotides, triple helix forming oligonucleotides, ribozymes, DNAzymes, chemically modified forms thereof, or a combination thereof. In some embodiments, the agent is an oligonucleotide. In some embodiments, the agent (e.g., oligonucleotide) is an antisense oligonucleotide. - 14 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0095] Determining the dosage and route of administration for a particular conjugate, patient and disease or condition is well within the abilities of one of skill in the art. In certain embodiments, the administration of a conjugate or a composition may be carried out in any manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation. [0096] In some embodiments the route of administration is determined by the tissue or tissues, or organ to which the agent or agents are targeted. In some embodiments, the tissue, tissues, or organ is the subject’s lung, ovary, immune system, skin, blood vessel, muscle, blood, brain, heart, intestine(s), pancreas, spleen, kidney, heart, bone, bone marrow, stomach, head, or any combination thereof. Compositions [0097] In some embodiments, the disclosure provides a composition (e.g. pharmaceutical composition), wherein the composition comprises a polymer or a conjugate disclosed herein, and a carrier (e.g. a pharmaceutically acceptable carrier). As used herein, the term “pharmaceutical composition” refers to a composition having pharmacological activity or other direct effect in mitigating, treating, or preventing cancer, or a finished dosage form or formulation thereof. [0098] In some embodiments, a composition (e.g., pharmaceutical composition) comprises carriers, excipients, stabilizers, diluents or tonifiers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents or tonifiers include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose or dextrins); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose or sorbitol), salt-forming counter-ions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); non-ionic surfactants (e.g., Tween), PLURONICS™ and polyethylene glycol (PEG). - 15 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [0099] In some embodiments, a composition (e.g., pharmaceutical composition) is formulated for a suitable administration schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous and topical, etc. In some embodiments, a composition (e.g., pharmaceutical composition) is stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized). In some embodiments, a composition is formulated to be administered by infusion (e.g., intravenous infusion). [00100] In some embodiments, a composition is formulated to be administered with one or more additional agents (e.g., with a second therapeutic agent) as a combination therapy. As used herein, a “combination therapy” or “administered in combination” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. Non-limiting examples of additional agents or treatments include biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules impacting immune-mediated diseases or conditions. [00101] The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the two or more agents are administered in a sequential manner as part of a prescribed regimen. In other embodiments, the delivery of the two or more agents is simultaneous or concurrent. In some embodiments, the two or more agents are co-formulated. In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents can be administered by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The two or more therapeutic agents can be administered by the same route or by different routes. Methods - 16 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00102] In some embodiments, the present disclosure provides methods of modulating or altering the expression of a gene product encoded by a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate disclosed herein. [00103] In some embodiments, the present disclosure provides methods of delivering an agent (e.g. a therapeutic agent, an imaging agent, an oligonucleotide, etc.) to a subject in need thereof, the method comprising: administering a conjugate or a composition of the present disclosure to the subject. [00104] In some embodiments, an agent (e.g., an oligonucleotide) comprises deoxyribonucleotides. In certain embodiments, an agent comprises ribonucleotides. Non-limiting examples of oligonucleotides include single-, double- or multistranded DNA or RNA, DNA- RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of an oligonucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof. [00105] In some embodiments, the agent (e.g., oligonucleotide) is a DNA molecule. In some embodiments, the agent (e.g., oligonucleotide) is an RNA molecule. [00106] In some embodiments, the oligonucleotide comprises an analog or a derivative of a oligonucleotide. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative comprises one or more phosphorothioate-linkages. In some embodiments, the agent comprises a deoxyribonucleic guanidine (DNG) nucleotide. In some embodiments, the agent comprises ribonucleic guanidine (RNG) nucleotide. [00107] In some embodiments, the agent (e.g., oligonucleotide) is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamers, ribozymes, DNAzymes, antisense oligonucleotides, exon-skipping oligonucleotides, siRNA oligonucleotides, triple helix forming oligonucleotides, ribozymes, DNAzymes, chemically modified forms thereof, or a combination thereof. In some embodiments, the agent is an - 17 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 oligonucleotide. In some embodiments, the agent (e.g., oligonucleotide) is an antisense oligonucleotide. [00108] In some embodiments, the present disclosure provides methods of delivering an agent to a cell, the method comprising: contacting the cell with a conjugate disclosed herein. [00109] In some embodiments, the present disclosure provides methods of detecting a target polynucleotide, the method comprising: contacting the target polynucleotide with a conjugate disclosed herein, thereby forming a polynucleotide-conjugate hybrid; and detecting a signal (e.g., a fluorescent signal, a radioactive signal) from the polynucleotide-conjugate hybrid. [00110] In some embodiments, a conjugate disclosed herein further comprises a fluorescent dye or a radioactive dye (e.g., a radioactive isotope). In some embodiments, a conjugate disclosed herein further comprises a fluorescent dye. [00111] In some embodiments, a fluorescent dye is Cyanine 5 (Cy5), Cyanine 3 (Cy3), Cyanine 2 (Cy2), a Fluorescein (e.g., Fluorescein isothiocyanate (FITC)), an Alexa Fluor dye, Texas Red, Tetramethylrhodamine (TAMRA), 6-Carboxy-X-Rhodamine (ROX), Tetramethylrhodamine (TMR), or Boron-dipyrromethene (BODIPY). In some embodiments, a fluorescent dye is Cyanine5 (Cy5). [00112] In some embodiments, detecting a signal from a polynucleotide-conjugate hybrid comprises using imaging. In some embodiments, detecting a signal from a polynucleotide- conjugate hybrid comprises using autoradiography. In some embodiments, detecting a signal from a polynucleotide-conjugate hybrid comprises using fluorescence imaging. [00113] In some embodiments, a target polynucleotide is selected from eukaryotic, prokaryotic, and viral polynucleotides. In some embodiments, a target polynucleotide is a polynucleotide specific to a mammalian cancer cell, a mammalian non-cancer cell, a plant cell, a bacterium, or a virus. In some embodiments, a target polynucleotide is an mRNA. In some embodiments, an mRNA is KRAS mRNA or DMPK mRNA. [00114] In some embodiments, the present disclosure provides methods of treating a disease, the method comprising administering a conjugate or a composition disclosed herein to a subject in need thereof. In some embodiments, the disease is a cancer, a muscular disorder, or a central nervous system disorder. - 18 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00115] Examples of muscular disorders include and are not limited to muscular dystrophy, myotonic dystrophy, myasthenia gravis, amyotrophic lateral sclerosis (ALS), dermatomyositis, polymyositis, inclusion body myositis, mitochondrial myopathy, congenital myopathy, glycogen storage disease type II (Pompe disease), spinal muscular atrophy, Becker muscular dystrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, central core disease, nemaline myopathy, distal myopathy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy. In some embodiments, the muscular disorder is myotonic dystrophy. [00116] Examples of central nervous system disorders include and are not limited to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), epilepsy, stroke, migraine, meningitis, encephalitis, spinal cord injury, cerebral palsy, hydrocephalus, brain tumors, Chiari malformation, Creutzfeldt-Jakob disease (CJD), narcolepsy, schizophrenia, depression, and obsessive-compulsive disorder (OCD). [00117] A wide variety of cancers are treatable according to the methods described herein. In some embodiments, the cancer comprises a solid tumor (e.g., a tumor of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testes, head and/or neck, pancreas, brain, skin). Accordingly, in some embodiments, the cancer is a solid tumor cancer. Solid tumor cancers that can be treated according to the methods described herein include breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, renal cancer, gastric cancer, colon cancer, rectal cancer, colorectal cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer and skin cancer. In some embodiments, the cancer is a hematologic cancer (e.g., leukemia, lymphoma, myeloma). Hematologic cancers that can be treated according to the methods described herein include leukemias (e.g., acute leukemias, chronic leukemias), lymphomas (e.g., B-cell lymphoma, T-cell lymphoma) and multiple myeloma. In some embodiments, the cancer is a lung cancer. [00118] Examples of cancers treatable according to the methods described herein include Acute Lymphoblastic Leukemia (ALL); Acute Myeloid Leukemia (AML); Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Cancer (e.g., Kaposi Sarcoma, - 19 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 AIDS-Related Lymphoma, Primary CNS Lymphoma); Anal Cancer; Appendix Cancer; Astrocytomas, Childhood; Atypical Teratoid/Rhabdoid Tumor, Childhood, Central Nervous System; Basal Cell Carcinoma of the Skin; Bile Duct Cancer; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer (including Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma); Brain Tumors/Cancer; Breast Cancer; Burkitt Lymphoma; Carcinoid Tumor (Gastrointestinal); Carcinoid Tumor, Childhood; Cardiac (Heart) Tumors, Childhood; Embryonal Tumors, Childhood; Germ Cell Tumor, Childhood; Primary CNS Lymphoma; Cervical Cancer; Childhood Cervical Cancer; Cholangiocarcinoma; Chordoma, Childhood; Chronic Lymphocytic Leukemia (CLL); Chronic Myelogenous Leukemia (CML); Chronic Myeloproliferative Neoplasms; Colorectal Cancer; Childhood Colorectal Cancer; Craniopharyngioma, Childhood; Cutaneous T-Cell Lymphoma (e.g., Mycosis Fungoides and Sézary Syndrome); Ductal Carcinoma In Situ (DCIS); Embryonal Tumors, Central Nervous System, Childhood; Endometrial Cancer (Uterine Cancer); Ependymoma, Childhood; Esophageal Cancer; Childhood Esophageal Cancer; Esthesioneuroblastoma; Ewing Sarcoma; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Eye (Ocular) Cancer; Childhood Intraocular Melanoma; Intraocular Melanoma; Retinoblastoma; Fallopian Tube Cancer; Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Childhood Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumors (GIST); Childhood Gastrointestinal Stromal Tumors; Germ Cell Tumors; Childhood Central Nervous System Germ Cell Tumors (e.g., Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer); Gestational Trophoblastic Disease; Hairy Cell Leukemia; Head and Neck Cancer; Heart Tumors, Childhood; Hepatocellular (Liver) Cancer; Histiocytosis, Langerhans Cell; Hodgkin Lymphoma; Hypopharyngeal Cancer; Intraocular Melanoma; Childhood Intraocular Melanoma; Islet Cell Tumors, Pancreatic Neuroendocrine Tumors; Kaposi Sarcoma; Kidney (Renal Cell) Cancer; Langerhans Cell Histiocytosis; Laryngeal Cancer; Leukemia; Lip and Oral Cavity Cancer; Liver Cancer; Lung Cancer (Non-Small Cell and Small Cell); Childhood Lung Cancer; Lymphoma; Male Breast Cancer; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma; Melanoma; Childhood Melanoma; Melanoma, Intraocular (Eye); - 20 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Childhood Intraocular Melanoma; Merkel Cell Carcinoma; Mesothelioma, Malignant; Childhood Mesothelioma; Metastatic Cancer; Metastatic Squamous Neck Cancer with Occult Primary; Midline Tract Carcinoma With NUT Gene Changes; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes; Multiple Myeloma/Plasma Cell Neoplasms; Mycosis Fungoides; Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms; Myelogenous Leukemia, Chronic (CML); Myeloid Leukemia, Acute (AML); Myeloproliferative Neoplasms, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin Lymphoma; Non-Small Cell Lung Cancer; Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer; Childhood Ovarian Cancer; Pancreatic Cancer; Childhood Pancreatic Cancer; Pancreatic Neuroendocrine Tumors; Papillomatosis (Childhood Laryngeal); Paraganglioma; Childhood Paraganglioma; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer; Pheochromocytoma; Childhood Pheochromocytoma; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Primary Central Nervous System (CNS) Lymphoma; Primary Peritoneal Cancer; Prostate Cancer; Rectal Cancer; Recurrent Cancer; Renal Cell (Kidney) Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Sarcoma (e.g., Childhood Rhabdomyosarcoma, Childhood Vascular Tumors, Ewing Sarcoma, Kaposi Sarcoma, Osteosarcoma (Bone Cancer), Soft Tissue Sarcoma, Uterine Sarcoma); Sézary Syndrome; Skin Cancer; Childhood Skin Cancer; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Squamous Cell Carcinoma of the Skin; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Childhood Stomach (Gastric) Cancer; T-Cell Lymphoma, Cutaneous (e.g., Mycosis Fungoides and Sèzary Syndrome); Testicular Cancer; Childhood Testicular Cancer; Throat Cancer (e.g., Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer); Thymoma and Thymic Carcinoma; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Childhood Vaginal Cancer; Vascular Tumors; Vulvar Cancer; and Wilms Tumor and Other Childhood Kidney Tumors. - 21 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00119] The administration of a polymer, conjugate, or composition of the present disclosure may be parenteral or non-parenteral, including intravenous, intra-arterial, intraperitoneal, intramuscular, intracavity, subcutaneous, intradermal, topical, inhalation, transmucosal, rectal or transdermal. In some embodiments, the administration routes is oral, rectal, subcutaneous, transdermal, ocular, and intraperitoneal. In other embodiments, the polymer may be administered ex vivo. [00120] In some embodiments, a polymer, conjugate or composition is delivered to a target site by diffusion and/or osmosis over time ranging from hours to days. In certain embodiments, the conjugate is delivered directly to the target site. In some embodiments, the procedure of delivering a conjugate to a target site is repeated several times, if needed. [00121] In some embodiments, a polymer, conjugate or composition is administered with one or more additional agents (e.g., with a second therapeutic agent) as a combination therapy. [00122] Also disclosed herein are methods of making a bottlebrush polymer, the methods comprising one or more of: providing one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof; combining the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. [00123] In some embodiments, methods of making a bottlebrush polymer comprise: - 22 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 combining one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. [00124] In some embodiments, at least one or more of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group. [00125] In some embodiments, each of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group. [00126] In some embodiments, covalently linking a plurality of polymer arms to the backbone comprises removing the protecting group and contacting the polymer backbone with a reagent comprising the polymer arm. In some embodiments, the reagent is an amino-reactive PEG reagent (e.g., PEG NHS ester). [00127] Examples of protecting groups include and are not limited to Boc (tert- butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), CBZ (carbobenzyloxy), Tos (p- toluenesulfonyl), PMB (para-methoxybenzyl), Nip (nitrophenylsulfonyl), Alloc (allyloxycarbonyl), Mtr (4-methyltrityl), and TFA (trifluoroacetyl). In some embodiments, a protecting group is fluorenylmethoxycarbonyl (Fmoc), trifluoroacetyl (TFA), or a combination thereof. [00128] In some embodiments, the polymer comprises from about 1 to about 100 (e.g., about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to - 23 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 about 50, etc.) polymer arms covalently linked to the backbone. In some embodiments, the polymer comprises from about 10 to about 40 polymer arms covalently linked to the backbone. In some embodiments, the polymer comprises about 30 polymer arms covalently linked to the backbone. [00129] In some embodiments, each of the plurality of polymer arms is poly(ethylene glycol) (PEG), poly-L-lysine, PAMAM dendrimers, poly(2-oxazoline), polypeptides, poly(N- isopropylacrylamide), polyanhydrides, poly(ε-caprolactone), chitosan, polystyrene, poly(ethyleneimine), poly(lactic-co-glycolic acid), poly(propyleneimine) dendrimers, poly(β- amino esters), poly(vinyl alcohol), poly(methyl methacrylate), polyacrylamide, polydopamine, polycarbonate, dextran, or a combination therefore. In some embodiments, each of the plurality of polymer arms is a poly(ethylene glycol). [00130] In some embodiments, each of the one or more alkyl phosphoramidites is a C10-C30 alkyl (e.g., C10-C20 alkyl, C15-C20 alkyl, etc.) phosphoramidite. In some embodiments, each of the one or more alkyl phosphoramidites is a C18 alkyl phosphoramidite. [00131] 7 In some embodiments, a polymer comprises from about 2 to about 30 alkyls (e.g., about 2 to about 20, about 2 to about 15, about 2 to about 10, etc.) covalently linked to the backbone. In some embodiments, a polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone. [00132] In some embodiments, a polymer comprises about 4 alkyls covalently linked to the backbone. [00133] In some embodiments, a polymer comprises 6 cholesterols covalently linked to the backbone. [00134] In some embodiments, a polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone. In some embodiments, the plurality of alkyls or the plurality of cholesterols are evenly distributed (i.e., the number of non-alkyls (or non- cholesterols) separating adjacent alkyls (or cholesterols) are the same or vary by 1 or 2 units). For non-limiting example, if there are 3, 4, and 5 non-alkyls separating any adjacent alkyls out of the plurality of alkyls linked to the polymer backbone, the plurality of alkyls are evenly distributed. For non-limiting example, if there are 2, 4, and 5 non-alkyls separating any adjacent - 24 - 4112432.v1 Docket No. 5200.2402-001 (INV-24084) 4112432.v1 alkyls out of the plurality of alkyls linked to the polymer backbone, the plurality of alkyls are not evenly distributed. [00135] In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by from about 2 monomers to about 6 monomers. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 2, 3, 4 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 3, 4, 5 monomers, or a combination thereof. In some embodiments, a plurality of alkyls or a plurality of cholesterols are separated by 4, 5, 6 monomers, or a combination thereof. [00136] In some embodiments, a polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47 (Table 3). [00137] In some embodiments, the cholesterol phosphoramidite is . - 25 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00138] In some embodiments, the spermine phosphoramidite is . . is . DEFINITIONS - 26 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00141] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. [00142] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, example materials and methods are described herein. [00143] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” [00144] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [00145] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include”, as well as variations thereof, such as “comprises” and “comprising”, are to be construed in an open, inclusive sense, e.g., “including, but not limited to.” The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element - 27 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 or step not specified in the claim; and (iii) “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention and disclosure. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.” [00146] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the disclosure, claims, result or embodiment, “about” means within one standard deviation per the practice in the art, or can mean a range of ± 20%, ± 10%, ± 5%, ±4, ±3, ±2 or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Examples. [00147] All percents are intended to be weight percent unless otherwise specified. The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, other various embodiments of and modifications to the present disclosure, in addition to those described herein, will be apparent to those of ordinary skill in the art from the foregoing description and accompanying drawings. Thus, such other embodiments and modifications are intended to fall within the scope of the present disclosure. Further, although the present disclosure has been described herein in the context of a particular implementation in a particular environment for a particular purpose, those of ordinary skill in the art will recognize that its usefulness is not limited thereto and that the present disclosure may be beneficially implemented in any number of environments for any number of purposes. Accordingly, the claims set forth below should be construed in view of the full breadth and spirit of the present disclosure as described herein. [00148] “PEG” or “polyethylene glycol” as used herein, is meant to encompass any water- soluble polyethylene oxide. Typically, PEGs for use in the present invention will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation, or, e.g., the identity of adjacent functional groups. The variable (n) - 28 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 may, for non-limiting example, range from 1 to about 5000, and the terminal groups and architecture of the overall PEG may vary. [00149] As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder, including cancer, a central nervous system disorder, a muscular disorder, a wound or a tumor. For example, treatment can include a postponement of development of the symptoms associated with disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. [00150] As used herein, “subject” refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, swine, dog, cat, rabbit, guinea pig, rat, mouse). In a particular embodiment, the subject is a human. A “subject in need thereof” refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented). In some embodiments, the subject is a human. In some embodiments, the human is a neonate. In some embodiments, the human is a pediatric patient. In some embodiments, the human is a juvenile. In some embodiments, the human is an adult. In some embodiments, the human is less than 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. [00151] As used herein, the term “effective amount,” “therapeutically effective amount,” or “sufficient amount” refers to a quantity sufficient to, when administered to a subject (e.g., a mammal such as a human, e.g., a patient such as a human cancer patient), effect treatment (e.g., produce beneficial or desired results), including effects at cellular, tissue or clinical levels, etc. As such, the term depends upon the context in which it is being applied. For example, in the context of treating cancer, it is an amount of an agent sufficient to achieve a response as compared to the response obtained without administration of the agent. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of - 29 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. In some embodiments, a “therapeutically effective amount” of a composition of the present disclosure is an amount that results in a beneficial or desired result in a subject (e.g., as compared to a control). A therapeutically effective amount of a composition of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response. EXAMPLES [00152] The entire teachings of Wei, Y. et al., Bottlebrush polymers with sequence-controlled backbones for enhanced oligonucleotide delivery, J. Am. Chem. Soc.2024, 146, 34763-34770 are incorporated herein by reference. EXAMPLE 1 [00153] The poly(2-deoxyribose phosphodiester) backbone polymer is constructed using a stepwise condensation approach, employing two bespoke modified phosphoramidites: ribose NH2 (R-NH2) and ribose C18 (R-C18). The modifiers were synthesized from 1-chloro-3,5-di(4- chlorbenzoyl)-2-deoxy-d-ribose in ∼40% overall yields in multigram scales (FIG.1A and 6- 21; Schemes 1 and 2). Scheme 1. Synthesis of ribose-amine phosphoramidite (R-NH2) - 30 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00154] The oligonucleotide component was synthesized as an integral part of the backbone of the solid support. Due to the utilization of the same phosphoramidite chemistry, the oligonucleotide can be positioned at either the 3′ or the 5′ of the polymer backbone without requiring additional postconjugation steps. For the purposes of this proof-of-concept study, antisense oligonucleotides (ASOs) targeting the 3′ untranslated region (UTR) of both human and mouse KRAS mRNA (ASO1 and ASO2, respectively) were selected as the payload for this ribose-based pacDNA, which were - 31 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 positioned at the 5′ of the ribose backbone (FIG.1B). (17,18) The backbone is designed to incorporate 30 repeating units of ribose NH2, which can be subsequently PEGylated to give the bottlebrush morphology (FIG.1C-D). (16,19) Because the phosphodiester ribose backbone is significantly more hydrophilic than the polynorbornene (PN)-based pacDNA, which may limit material–cell membrane interactions, leading to reduced cell uptake, we investigated how hydrophobic C18 modifiers introduced into the backbone can affect the cellular uptake and biodistribution in vivo. We varied the numbers of C18 modifiers (ranging from 0 to 10) as well as their distribution patterns (for backbones containing six C18 modifiers) in order to probe the structure–property relationship (FIG.1B). [00155] Following the completion of solid-phase backbone/ASO synthesis, the trifluoroacetyl protecting groups were removed, and the hybrid strand was cleaved from the solid support. The successful strand was isolated using a dimethoxytrityl (DMT)-affinity column. To construct the bottlebrush structure, the amine groups on the backbone were derivatized with a heterodifunctional N-hydroxysuccinimide (NHS)- and methyl-terminated 10 kDa PEG, using a two- stage process. (19) The purified strand was PEGylated initially in 1× phosphate buffered saline (PBS) at 4 °C overnight. The partially PEGylated product was desalted, lyophilized, and subsequently reacted with another equivalent of PEG in anhydrous N,N-dimethylformamide (DMF) for full derivatization. Excess PEG and residues were removed by aqueous gel permeation chromatography (GPC), yielding highly uniform, narrowly dispersed pacDNA structures, as evidenced by aqueous and DMF GPC (FIG.2A; Table 2), dynamic light scattering (DLS) (FIG.2B and 22), and transmission electron microscopy (TEM) (FIG.2C). The uniformity of pacDNA particles was consistently observed across all samples irrespective of the quantity or arrangement of R-C18 units, suggesting that the C18 modifiers do not cause aggregation in solution. ζ potential measurement revealed that all pacDNA samples are slightly anionic (−3.26 to −11.71 mV) in Nanopure water compared with free oligonucleotides (−33.6 mV) (FIG.2D). Collectively, these results demonstrate that ribose-based pacDNAs can be robustly synthesized, allowing for fine-tuning of the size/backbone sequence of the pacDNAs and the potential to modulate their bioactivities. A comparison between ribose- and PN-based pacDNAs can be found in FIG.23A-F. - 32 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Table 1. All sequences with poly(2-deoxyribose phosphodiester) backbone used in this study. N: PEG (10kDa PEG); S: C18 alkyl. Sample ID SEQ ID NO. Sequence pac-0×C18 1 NN NNN NNN NNN NNN NNN NNN NNN NNN N N - 33 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 pac-10×C18- 13 5'-GCT ATT AGG AGT CTTTNN SNN SNN NSN ASO1 NNS NNN SNN NSN NNS NNN SNN NSN NNS NNT- - N - - 34 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Free ASO2 17 5' -CAT GTA AAT ATA GCC CT-3' pac-4×C18- 18 5' -CAT GTA AAT ATA GCC CTN NNN NNS NNN Sample ID Mn(kDa) Mw(kDa) PDI ac-0C -ASO1 3625 4386 121 [00156] Next, we assessed the intracellular delivery efficacy of the pacDNA panel in NCI-H358 cells, a nonsmall lung carcinoma line harboring KRASG12C mutation. Flow cytometry analysis revealed that as the number of R-C18 units increased within the bottlebrush backbone, cellular uptake also increased (FIG.3A), suggesting the structurally more hydrophobic pacDNA constructs may exhibit stronger material–cell interactions. The trend does not change when bottlebrush polymers lacking the ASO component were tested (FIG.24A). Interestingly, when the same number of C18 units was incorporated into the bottlebrush backbone, the evenly distributed patterns demonstrated higher cellular uptake efficiencies (FIG.3B), while the clustering of C18 units reduced cell uptake. In fact, when all six C18 units were clustered together (pac-1 × 6C18-ASO1), the cellular uptake was comparable to that of the pacDNA without any C18 units (pac-0C18-ASO1). The flow cytometry measurements were further supported by confocal microscopy. Cy5-labeled pacDNAs - 35 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 were incubated with NCI-H358 cells for 8 h before imaging. Again, pacDNAs with a higher number of C18 units showed greater cell uptake (FIG.3C and 25). Without being bound to a theory, we postulate that when R-C18 units are positioned adjacent to one another, the self-interaction among C18 decreases their tendency to interact with the cellular membrane. In contrast, separating the C18 units spatially reduces such self-interactions, leading to a higher potential energy state and stronger tendency to bind with the cell membrane upon contact. Increased cellular uptake of ASO by lipid conjugation has been reported. (20,21) However, lipid conjugates often exhibit increased cytotoxicity, possibly due to membrane lytic activity of the amphiphilic conjugate. (22,23) In contrast, the pacDNAs do not cause cytotoxicity (FIG.24B) even though they contain multiple C18 units. One interpretation is that the sterics of the pacDNA make it difficult for the lipid chains to aggregate and exhibit surfactant-like properties, limiting the potential membrane lytic activity. [00157] To gain molecular level insights, we carried out full atomistic MD simulations to quantitatively assess the energy of interaction between water molecules and pacDNAs with six C18 units arranged in different distribution patterns. In this context, interaction energy refers to the total energy arising from all intermolecular forces between the pacDNA molecules and surrounding water molecules. A more negative interaction energy indicates stronger interactions with water, leading to greater solubility of the pacDNA in aqueous environments. We hypothesize that the pacDNA with the least amount of lipid self-interactions will be the most prone to membrane binding. This hypothesis can be tested by measuring pacDNA interaction energy in water─structures with the least interaction energy with water (most difficult to dissolve) should exhibit the highest cell uptake. To make the simulation computationally feasible, all PEG side chains were truncated to 20 mer, which was found to be sufficiently large to inhibit backbone coiling under simulation conditions. The simulation trajectory confirms more extensive lipid–lipid interactions for clustered vs spaced arrangement of C18 units (FIG.3D). The pacDNA with the smallest spacing (pac-1 × 6C18) exhibited the most interaction energy (most negative). This result suggests that when a constant number of C18 units are present in the backbone, their clustering increases solubility in water and therefore reduces C18 interactions with cell membranes, leading to lower cell uptake (FIG.3E). Conversely, the pacDNA with the largest spacing (pac-6 × 1C18) exhibited the lowest interaction energy (least negative), indicating that pac-6 × 1C18 has the least tendency to be dissolved in water, and by extension, an enhanced propensity for interacting with the cell membrane and cellular uptake. These - 36 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 observations underscore the pivotal role of the digital backbone design, which allows for precise tuning of the biophysical behavior of pacDNAs. [00158] Next, we assessed the efficacy of the pacDNAs to engage with cytosolic mRNA targets by using pacDNAs bearing ASO1 (vide supra) and varying numbers of evenly distributed C18 units. Western blot analysis showed the downregulation of KRAS protein levels in all pacDNA-treated groups (FIG.3F). Interestingly, when pacDNA has at least two C18 units, KRAS depletion levels did not vary significantly. We also analyzed cell viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. All pacDNAs significantly inhibited tumor cell growth, with inhibition levels ranging from 40 to 60% compared to nontreated cells, while the free ASO1 showed no inhibitory activity (FIG.3G and 26). [00159] Prior to nominating a candidate for efficacy studies, it is important to understand how the pacDNA backbone series affects plasma pharmacokinetics (PK) and biodistribution. Cy5-labeled pacDNAs were intravenously (i.v.) administered to C57BL/6 mice via the tail vein, and blood plasma samples were collected by submandibular puncture at predetermined time points over a 72 h period. Compared to free ASO, all pacDNA constructs, regardless of the number of R-C18 units in the backbone, exhibited significantly prolonged circulation times (FIG.4A). Analyzing the plasma PK using a two-compartment model, it can be seen that the distribution half-lives (t1/2α) generally decrease with increasing R-C18 number (FIG.4B). Interestingly, a straightforward correlation was not observed for the elimination half-life (t1/2β), which increases first and then decreases with C18 numbers, peaking at pac-4 × 1C18. The insertion of two or four R-C18 units may represent optimized structures because these specific arrangements are capable of significantly enhancing the bioavailability of the ASO as characterized by area under the curve (AUC∞), while being sufficiently strong for cellular uptake. [00160] We next carried out a biodistribution study comparing the different pacDNA constructs using C57BL/6 mice bearing the K273 tumor allograft. The K273 cell is a KRASG12D lung cancer line derived from a genetically engineered mouse model (GEMM). The pacDNAs were administered i.v., and fluorescence images were captured at predetermined time points (FIG.4C). The fluorescence signal at the tumor site first increases and then decreases with increasing C18 numbers, following a similar trend to the plasma elimination half-life. To quantitatively analyze pacDNA biodistribution among major organs and tissues, mice were sacrificed 72 h after injection, and tissue - 37 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 lysates were used to determine distribution as % injected dose per gram of tissue (% ID/g, FIG.4D). Again, pacDNAs with two or four R-C18 units exhibit stronger tumor localization compared to those with six, eight, or ten C18 modifiers. Because pac-4 × 1C18 shows the highest tumor distribution compared with other backbone designs (FIG.4E and 27), we focused on this specific arrangement for subsequent studies. [00161] To investigate the antitumor efficacy of pac-4 × 1C18 using the K273 allograft model, we prepared pac-4 × 1C18-ASO2, which is matched to the mouse wild-type KRAS mRNA sequence at the 3′ UTR region. First, we validated the potency of pac-4 × 1C18-ASO2 using the K273 cell line in vitro. Western blot shows that pac-4 × 1C18-ASO2 downregulated KRAS protein levels in a dose- dependent manner (FIG.28A). The MTT cell viability assay showed that pac-4 × 1C18-ASO2 reduced K273 cell viability, while free ASO2 or pac-4 × 1C18-scramble showed no inhibitory effect (FIG.28B). The antitumor efficacy of pac-4 × 1C18-ASO2 was assessed in female C57BL/6 mice bearing subcutaneous K273 allografts. When the allografts reached a volume of 100 mm3, 0.5 μmol/kg of pac-4 × 1C18-ASO2, free ASO2, or vehicle (PBS) were administrated i.v. once every 4 days for a total of four doses (FIG.5A). By day 24 after tumor inoculation, the average tumor volume of the vehicle group reached 864 mm3, while pac-4 × 1C18-ASO2 significantly inhibited tumor growth and extended mice survival with the average tumor volume at 299 mm3 (FIG.5B,C). In contrast, free ASO2 elicited no survival benefit. Neither pac-4 × 1C18-ASO2 nor free ASO2 lead to significant body weight changes (FIG.29). Immunohistostaining of tumor tissue revealed reduced KRAS protein expression after pac-4 × 1C18-ASO2 treatment but not with free ASO2 (FIG.5D). [00162] Next, we performed in vivo safety analysis of pac-4 × 1C18-ASO2. Hematoxylin and eosin (H & E) staining demonstrated no obvious histological changes in major internal organs (FIG. 30). Macromolecular therapeutics requiring repeated dosing may induce antidrug adaptive immunity, leading to loss of drug activity in subsequent doses. To evaluate the anti-PEG immunoglobulin IgM/IgG response, healthy C57BL/6 mice were administrated pac-4 × 1C18-ASO2 or free ASO2 every 4 days for four doses. Mouse plasma was collected 1 week after the last dose. Enzyme-linked immunosorbent assays (ELISA) revealed that pac-4 × 1C18-ASO2 induced very limited levels of anti- PEG IgM/IgG (FIG.5E). Both responses were minor compared to the positive control (PEG-keyhole limpet hemocyanin [KLH] conjugate). To further investigate possible unintended activation of the immune system, C57BL/6 mouse plasma was collected 3 h after i.v. injection of pac-4 × 1C18-ASO2 - 38 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 or free ASO2, and cytokines and chemokines related to innate and adaptive immune responses were measured. The assay detected no apparent cytokines or chemokines for either pac-4 × 1C18-ASO2 or free ASO2 treatment groups, while lipopolysaccharide (LPS) i.v. injection (positive control) generated high levels of cytokines/chemokines (FIG.5F). [00163] In conclusion, our study presents a robust approach to construct bottlebrush polymers with arbitrary control of the backbone size, composition, and monomer sequence. These capabilities allow us to take advantage of C18 modifiers and optimize biological properties such as cell uptake and tumor localization. Experimental and computational studies suggest that the different arrangements of C18 affect their self-interaction, which, in turn, modifies their readiness to engage with the cell membrane. Through optimization, we identified pac-4 × 1C18 as overall the most favorable given in vitro cell uptake, gene silencing, and biodistribution in mice. A comparison between pac-4 × 1C18 and the previously published polynorbornene bottlebrush (pac-PN) indicates that pac-4 × 1C18 exhibits superior batch-to-batch consistency, improved structural control, and enhanced tumor distribution. In a KRASG12D lung cancer allograft, a KRAS-depleting pac-4 × 1C18 showed single-agent tumor-suppressive potency at a fraction of concentration typically used for the ASO modality. Taken together, our results provide valuable insights into the pacDNA structure-property relationship, for example, for tuning the polymer backbone to meet the delivery requirements of various diseases. [00164] Materials and reagents [00165] 1-Chloro-3,5-di(4-chlorbenzoyl)-2-deoxy-D-ribose, 6-(Trifluoroacetamido) hexanol, 4,4′-Dimethoxytrityl chloride, 1-Octadecanol, and 2-Cyanoethyl N,N- diisopropylchlorophosphoramidite were purchased from Sigma-Aldrich Co., USA. Methoxypolyethylene glycol (PEG) glutaramide succinimidyl ester (Mn=10 kDa) was purchased from Creative PEGWorks (Chapel hill, NC, USA). Phosphoramidites and supplies for DNA synthesis were obtained from Glen Research Co., USA. All other materials were obtained from Fisher Scientific Inc., USA, or VWR International LLC., USA, and were used as received unless otherwise indicated. [00166] Instrumentations - 39 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00167] 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 700 MHz NMR spectrometer (Bruker Scientific LLC, MA, USA). Reversed-phase high- performance liquid chromatography (RP-HPLC) was performed on a Waters (Waters Co., MA, USA) Breeze 2 HPLC system coupled to a Symmetry® C183.5 μm, 4.6×75 mm reversed-phase column and a 2998 PDA detector, using TEAA buffer (0.1 M) and HPLC-grade acetonitrile as mobile phases. Aqueous GPC measurements were performed on a Waters Breeze 2 GPC system equipped with Ultrahydrogel™ 1000, Ultrahydrogel™ 500 and Ultrahydrogel™ 250, 7.8A×300 mm column and a 2998 PDA detector (Waters Co., MA, USA). Phosphate-buffered saline (PBS, pH 7.4) was used as the eluent running at a flow rate of 0.8 mL/min. N, N-dimethylformamide (DMF) GPC analysis was performed on EcoSEC HLC-8320 GPC system (Tosoh Bioscience LLC, Tokyo, Japan) equipped with a TSKGel α-M 7.8×300 mm, 13 μm column and RI/UV-Vis detectors. The mobile phase is 0.05 M lithium bromide in HPLC-grade DMF, and samples were analyzed at a flow rate of 0.4 mL/min. DMF GPC calibration was based on a ReadyCal kit of polyethylene glycol standards (PSS-Polymer Standard Service-USA Inc., MA, USA). The kit covers an Mn range from 232 Da to 1015 kDa. For transmission electron microscopy (TEM) measurements, samples were imaged on a JEOL JEM 1010 electron microscope with an accelerating voltage of 80 kV. High-resolution mass spectra (HRMS) analysis was performed on a Q Exactive Orbitrap (Thermofisher ScientificTM) using a syringe pump with direct infusion. Data was acquired in positive/negative ion mode using a hybrid orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with direct infusion electrospray ion source (HESI) using a syringe pump. The flow rate was 5ul/min with +4.5 kV /-3.5 kV electrospray voltages. Analysis was performed in full scan mode with a mass range of 150-2000 m/z, including an automatic gain control setting of 1×106 and a mass resolution of 17,000. DLS and ζ potential measurements were performed on a Malvern Zetasizer Nano-ZSP (Malvern, UK). Samples were dissolved in Nanopure water at a concentration of 1 μM and filtered through a 0.2 μm PTFE filter before measurement. Histopathology (H&E) and immunohistochemistry studies were carried out at iHisto Inc. [00168] Synthesis of TFA-amine-ribose (1, Scheme 1).1-Chloro-3,5-di(4-chlorbenzoyl)-2- deoxy-D-ribose (1.04g, 2.42 mmol, 1 eq.) was dissolved with 10 mL of anhydrous DCM in a - 40 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 round bottom flask.6-(Trifluoroacetamido) hexanol (0.62 g, 2.90 mmol, 1.2 eq.) and 4- Dimethylaminopyridine (DMAP) (87.90 mg, 0.72 mmol, 0.3 eq.) were dissolved in 5 mL of anhydrous DCM, separately, and the solutions were added into the flask. The reaction was allowed to react at room temperature (R.T.) for at least 4 h. Afterwards, the reaction was concentrated by rotary-evaporation and purified by flash chromatography on silica column using a dichloromethane with ethyl acetate (0 to 5%) eluent system. The product was then dried under high vacuum to yield a white solid.1H NMR (700 MHz, CDCl3) δ 7.93 – 7.86 (m, 4H), 7.37 – 7.31 (m, 4H), 6.24 (s, 1H), 5.33 (ddd, J = 8.0, 3.7, 2.2 Hz, 1H), 5.22 – 5.19 (m, 1H), 4.53 (dd, J = 11.7, 4.0 Hz, 1H), 4.48 – 4.40 (m, 2H), 3.66 (dt, J = 9.6, 6.7 Hz, 1H), 3.37 (dt, J = 9.6, 6.3 Hz, 1H), 3.25 (q, J = 6.9 Hz, 2H), 2.42 (ddd, J = 14.6, 8.0, 5.3 Hz, 1H), 2.13 (ddd, J = 14.4, 2.2, 1.0 Hz, 1H), 1.56 – 1.42 (m, 5H), 1.38 – 1.23 (m, 4H).13C NMR (176 MHz, CDCl3) δ 165.46, 165.40, 157.27, 157.06, 139.81, 139.70, 131.16, 131.13, 131.04, 128.82, 128.77, 128.21, 116.68, 115.05, 103.79, 80.75, 75.09, 67.40, 64.59, 39.88, 39.11, 29.52, 28.96, 28.85, 26.42, 25.75. HRMS: calculated for C27H28Cl2F3NO7 [M-H]-: 604.12, found: 604.11. [00169] Synthesis of deprotected TFA-amine-ribose (2, Scheme 1). Compound 1 (1.01 g, 1.66 mmol, 1 eq.) was dissolved in 9 mL 0.5 M NaOMe in methanol (4.16 mmol, 2.5 eq.) and allowed to stir at R.T. for 2 h. The solution was dried loaded on silica column and purified using a 5% ethyl acetate in dichloromethane added with methanol (0 to 5%) eluent system. The deprotected compound 1 was then dried under high vacuum to yield a viscous transparent liquid (Compound 2).1H NMR (700 MHz, CDCl3) δ 7.42 (t, J = 5.9 Hz, 1H), 5.19 – 5.13 (m, 1H), 4.43 (td, J = 6.2, 3.6 Hz, 1H), 4.13 – 4.03 (m, 1H), 3.99 (q, J = 3.9 Hz, 1H), 3.73 – 3.65 (m, 2H), 3.65 – 3.55 (m, 2H), 3.38 (tt, J = 9.7, 5.7 Hz, 2H), 3.34 – 3.25 (m, 2H), 2.21 (ddd, J = 13.8, 7.0, 2.1 Hz, 1H), 2.15 – 2.04 (m, 1H), 1.54 (dtd, J = 15.7, 12.4, 7.2 Hz, 4H), 1.32 (tq, J = 14.6, 7.3 Hz, 4H).13C NMR (176 MHz, CDCl3) δ 157.81, 157.60, 157.58, 157.39, 157.37, 157.18, 116.75, 115.11, 104.41, 87.22, 71.93, 68.00, 63.53, 42.23, 39.77, 29.27, 28.61, 26.24, 26.20, 25.56. HRMS: calculated for C13H22F3NO5 [M-H]-: 328.15, found: 328.14. [00170] Synthesis of TFA-amine-ribose-DMT (3, Scheme 1). Compound 2 (0.55 g, 1.65 mmol, 1 eq.) was dissolved in 10 mL anhydrous pyridine and the solution was allowed to cool with an ice-bath. Dimethoxytrityl chloride (0.62 g, 1.82 mmol, 1.1 eq.) was dissolved in 5 mL - 41 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 anhydrous pyridine and added dropwise into the reaction under positive nitrogen flow. The reaction was allowed to gradually reach to R.T. and stirred overnight. Afterwards, the solution was quenched with methanol and concentrated by rotary evaporation to remove pyridine using toluene to co-evaporate. The concentrated reaction was then dissolved in ethyl acetate and extracted with 0.1 M NaHCO3 (2x) and brine (2x). The final solution was dried with MgSO4, concentrated by rotary evaporation, and purified by flashing chromatography on silica column with a hexane: ethyl acetate (3:1 to 1:1, v:v) eluent system supported with 0.1% triethylamine. The product was then dried under high vacuum to yield viscous liquid, orange in color.1H NMR (700 MHz, CDCl3) δ 7.34 – 7.10 (m, 9H), 6.73 (d, J = 8.7 Hz, 4H), 5.14 (d, J = 4.5 Hz, 1H), 4.13 (dt, J = 5.4, 2.8 Hz, 1H), 4.09 (dd, J = 9.4, 5.8 Hz, 1H), 3.67 (s, 7H), 3.35 – 3.29 (m, 1H), 3.27 – 3.18 (m, J = 6.7 Hz, 2H), 3.08 – 2.95 (m, 3H), 2.10 (ddd, J = 13.6, 6.0, 4.6 Hz, 1H), 1.95 – 1.90 (m, 1H), 1.54 – 1.44 (m, 4H), 1.27 (qd, J = 9.5, 5.7 Hz, 4H).13C NMR (176 MHz, CDCl3) δ 158.49, 157.39, 157.19, 144.83, 136.03, 135.93, 130.07, 128.16, 127.84, 126.80, 116.77, 115.14, 113.14, 104.35, 86.80, 86.12, 73.54, 67.22, 64.05, 55.21, 40.89, 39.82, 29.42, 28.83, 26.33, 25.77. HRMS: calculated for C34H40F3NO7 [M-H]-: 630.28, found: 630.27. [00171] Synthesis of ribose-amine (R-NH2, Scheme 1). Compound 3 (0.42 g, 0.65 mmol, 1 eq.) was dissolved in 5 mL anhydrous dichloromethane and 452.9 μL of triethylamine (0.33 g, 3.25 mmol, 5 eq.) was added to the solution. The solution was cooled with an ice-bath and undergo continuous nitrogen flow. The 2-Cyanoethyl N, N-diisopropylchlorophosphoramidite was dissolved in 2 mL anhydrous dichloromethane and added into the previous reaction mixture. The reaction was allowed to stir on ice for 30 min and at R.T. for 2.5 h. Ethyl acetate was added to dilute the reaction and extracted with 0.1 M NaHCO3 (2x) and brine (2x). The solution was dried with MgSO4 and concentrated via rotary-evaporation. The mixture was purified by flashing chromatography on silica column with a hexane: ethyl acetate (2:1 to 1:1, v:v) eluent system supported with 0.1% triethylamine. The final product was dried under high vacuum yielding a viscous liquid.1H NMR (700 MHz, CDCl3) δ 7.39 – 7.18 (m, 9H), 6.78 – 6.72 (m, 4H), 5.14 (dd, J = 5.4, 1.6 Hz, 1H), 4.28 (ddt, J = 11.4, 7.7, 3.6 Hz, 1H), 4.13 (q, J = 4.2 Hz, 1H), 3.75 – 3.63 (m, 7H), 3.50 (ddt, J = 17.0, 13.5, 6.9 Hz, 4H), 3.37 (dt, J = 9.5, 6.2 Hz, 1H), 3.29 – 3.25 (m, 2H), 3.25 – 3.20 (m, 1H), 3.04 (dd, J = 10.1, 4.6 Hz, 1H), 2.34 (t, J = 6.5 Hz, 2H), 2.28 (ddd, J = - 42 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 13.6, 8.0, 5.5 Hz, 1H), 1.92 (ddd, J = 13.7, 3.1, 1.6 Hz, 1H), 1.43 – 1.11 (m, 8H), 1.06 (dd, J = 19.3, 6.8 Hz, 12H).13C NMR (176 MHz, CDCl3) δ 158.37, 157.22, 157.02, 145.04, 136.30, 136.23, 130.17, 128.27, 127.72, 126.65, 117.57, 116.71, 113.04, 104.21, 85.89, 84.40, 84.36, 74.43, 74.33, 67.68, 64.62, 60.41, 58.14, 58.03, 55.22, 40.76, 40.74, 39.83, 29.33, 28.69, 26.34, 25.66, 24.59, 24.55, 24.50, 24.46, 20.34, 20.30. HRMS: calculated for C43H57F3N3O8P [M+H] +: 832.38, found: 832.40. [00172] Synthesis of C18-ribose (4, Scheme 2).1-Chloro-3,5-di(4-chlorbenzoyl)-2-deoxy-D- ribose (1.00 g, 2.33 mmol, 1 eq.) and DMAP (85.3 mg, 0.70 mmol, 0.3 eq.) was dissolved in 15 mL anhydrous dichloromethane.1-Octadecanol (0.75 g, 2.80 mmol, 1.2 eq.) was dissolved separately in 5 ml anhydrous tetrahydrofuran and added into the previous mixture. The reaction was allowed to stir at R.T. for at least 4 h before dried under rotary-evaporation. The product was purified by flashing chromatography on silica column with a dichloromethane (0 to 30% ethyl acetate) eluent system. The purified product was dried under high vacuum and yield a white powder.1H NMR (700 MHz, CDCl3) δ 7.96 – 7.84 (m, 4H), 7.36 – 7.29 (m, 4H), 5.52 (ddd, J = 7.5, 4.7, 2.9 Hz, 1H), 5.26 (dd, J = 5.6, 2.4 Hz, 1H), 4.55 – 4.37 (m, 3H), 3.65 (ddt, J = 19.9, 9.4, 6.8 Hz, 1H), 3.30 (dt, J = 9.6, 6.8 Hz, 1H), 2.47 (ddd, J = 14.2, 7.2, 2.4 Hz, 1H), 2.28 (dt, J = 14.2, 5.2 Hz, 1H), 1.47 – 1.11 (m, 32H), 0.80 (t, J = 7.0 Hz, 3H).13C NMR (176 MHz, CDCl3) δ 165.44, 165.29, 139.88, 139.60, 131.22, 131.19, 131.12, 131.09, 128.86, 128.80, 128.78, 128.25, 127.92, 104.67, 81.67, 76.03, 68.41, 65.51, 39.19, 39.17, 32.00, 29.80, 29.79, 29.76, 29.72, 29.70, 29.66, 29.60, 29.57, 29.51, 29.47, 26.35, 26.20, 22.79, 14.27. HRMS: calculated for C37H52Cl2O6 [M+H] +: 663.31, found: 663.46. [00173] Synthesis of deprotected C18-ribose (5, Scheme 2). Compound 4 (2.44 g, 3.68 mmol, 1 eq.) was dissolved in 18.4 mL 0.5 M NaOMe in methanol solution (9.21 mmol, 2.5 eq.) and stirred at R.T. for 2 h. The reaction mixture was dried by rotary-evaporation and purified by flashing chromatography on silica column with a dichloromethane:ethyl acetate (7:3, v:v) added with methanol (0 to 5%). The purified product was dried under high vacuum and yield a viscous liquid.1H NMR (700 MHz, CDCl3) δ 5.21 – 5.14 (m, 1H), 4.13 – 4.07 (m, 1H), 3.67 (dddd, J = 15.8, 9.2, 6.6, 4.6 Hz, 2H), 3.57 (ddd, J = 32.5, 12.0, 4.2 Hz, 1H), 3.34 (ddt, J = 15.8, 9.5, 6.7 Hz, 1H), 2.05 – 1.95 (m, 1H), 1.49 (p, J = 5.6 Hz, 2H), 1.18 (d, J = 3.3 Hz, 28H), 0.81 (td, J = - 43 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 7.1, 3.7 Hz, 3H).13C NMR (176 MHz, CDCl3) δ 104.48, 104.45, 104.38, 104.34, 87.77, 87.60, 73.06, 72.36, 68.68, 67.72, 67.70, 63.56, 63.20, 42.89, 41.50, 32.00, 29.79, 29.77, 29.75, 29.70, 29.66, 29.63, 29.46, 26.25, 26.14, 22.79, 14.27. HRMS: calculated for C23H46O4 [M-H]-: 385.34, found: 385.33. [00174] Synthesis of C18-ribose-DMT (6, Scheme 2). Compound 5 (1.03 g, 2.66 mmol, 1 eq.) was dissolved in 10 mL anhydrous pyridine and cooled with an ice-bath. Dimethoxytrityl chloride (2.02 g, 2.93 mmol, 1.1 eq.) was dissolved in 5 mL anhydrous pyridine and added dropwise into the previous mixture. The reaction was allowed to gradually come back to R.T. and stirred overnight. Pyridine was then removed from the reaction mixture via rotary evaporation (with the addition of toluene), and the mixture was diluted with ethyl acetate. The solution was extracted with 0.1 M NaHCO3 (2x) and brine (2x) and dried with MgSO4 followed by rotary-evaporation. The concentrated solution was then purified by flashing chromatography on silica column with a dichloromethane added with ethyl acetate (0 to 10%) eluent system. The purified product was dried with rotary-evaporation and yield a viscous liquid yellow in color.1H NMR (700 MHz, CDCl3) δ 7.38 – 7.10 (m, 9H), 6.77 – 6.71 (m, 4H), 5.05 (dd, J = 5.4, 2.1 Hz, 1H), 4.33 (tt, J = 6.8, 3.4 Hz, 1H), 3.86 (dt, J = 6.9, 5.0 Hz, 1H), 3.70 (s, 6H), 3.50 (dt, J = 9.4, 6.8 Hz, 1H), 3.25 – 3.17 (m, 2H), 3.07 (dd, J = 9.4, 6.9 Hz, 1H), 2.09 (ddd, J = 13.2, 6.8, 2.1 Hz, 1H), 1.94 (ddd, J = 12.7, 6.6, 5.4 Hz, 1H), 1.74 (d, J = 3.7 Hz, 1H), 1.38 – 1.05 (m, 32H), 0.80 (t, J = 7.0 Hz, 3H).13C NMR (176 MHz, CDCl3) δ 158.48, 144.87, 136.13, 136.10, 130.06, 130.04, 129.15, 128.19, 127.87, 127.83, 127.78, 126.79, 113.12, 103.82, 86.20, 84.53, 73.63, 67.89, 65.28, 55.26, 55.20, 41.19, 31.95, 29.73, 29.70, 29.68, 29.64, 29.62, 29.60, 29.57, 29.48, 29.38, 22.71, 14.14. HRMS: calculated for C44H64O6 [M+H] +: 689.47, found: 688.47. [00175] Synthesis of R-C18 (R-C18, Scheme 2). Compound 6 (0.39 g, 0.57 mmol, 1 eq.) and triethylamine (276.1 μL, 1.98 mmol, 3.5 eq.) were dissolved in 8 mL anhydrous dichloromethane and cooled in an ice-bath.2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (315.6 μL, 1.42 mmol, 2.5 eq.) was dissolved in 2 mL anhydrous dichloromethane and added into the reaction mixture. The reaction was reacted on ice for 30 min and R.T. for 2.5 h. Ethyl acetate was added to dilute the reaction and extracted with 0.1 M NaHCO3 (2x) and brine (2x). The solution was dried with MgSO4 and concentrated via rotary-evaporation. The mixture was purified by flashing - 44 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 chromatography on silica column with a hexane added with ethyl acetate (0 to 20%) eluent system. The final product is dried under high vacuum and yield a viscous liquid.1H NMR (700 MHz, CDCl3) δ 7.53 – 7.28 (m, 7H), 7.28 – 7.19 (m, 2H), 6.86 – 6.80 (m, 4H), 5.23 (dd, J = 5.2, 2.8 Hz, 1H), 4.46 (dtd, J = 10.2, 6.1, 4.2 Hz, 1H), 4.17 (q, J = 5.0 Hz, 1H), 3.80 (s, 7H), 3.75 (ddt, J = 10.2, 8.0, 6.7 Hz, 1H), 3.68 (dt, J = 9.4, 6.9 Hz, 1H), 3.55 (dh, J = 10.3, 6.7 Hz, 2H), 3.35 (dt, J = 9.4, 6.8 Hz, 1H), 3.21 (dd, J = 9.8, 4.9 Hz, 1H), 3.16 (dd, J = 9.8, 5.8 Hz, 1H), 2.61 (td, J = 6.5, 2.2 Hz, 2H), 2.24 – 2.14 (m, 2H), 1.47 (qt, J = 9.5, 4.3 Hz, 2H), 1.35 – 1.29 (m, 3H), 1.28 (s, 14H), 1.25 (d, J = 14.3 Hz, 13H), 1.16 (d, J = 6.8 Hz, 7H), 1.07 (d, J = 6.8 Hz, 6H), 0.90 (t, J = 7.0 Hz, 3H).13C NMR (176 MHz, CDCl3) δ 158.38, 144.99, 136.26, 136.24, 130.15, 130.14, 128.30, 127.71, 126.63, 117.52, 113.01, 104.17, 85.93, 84.40, 84.37, 74.60, 74.50, 68.05, 64.91, 58.24, 58.13, 55.17, 43.20, 43.13, 40.76, 40.74, 31.94, 29.73, 29.68, 29.64, 29.53, 29.38, 26.16, 24.60, 24.56, 24.50, 24.46, 22.71, 20.32, 20.28, 14.14. HRMS: calculated for C53H81N2O7P [M+H] +: 889.58, found: 889.59. [00176] Synthesis of polynorbornene bottlebrush (pac-PN) [00177] Norbornenyl bromide and norbornenyl PEG were synthesized following previously reported procedures. (16) Norbornenyl bromide (5 eq.) was dissolved in deoxygenated DCM under a nitrogen atmosphere. The 2nd generation Grubbs catalyst (1 eq.), dissolved in deoxygenated DCM, was then added to the norbornenyl bromide solution. The mixture was stirred vigorously for 30 minutes, then norbornenyl PEG (50 eq.) in deoxygenated DCM was added. The reaction was stirred for an additional 6 hours.10 µL of reacted mixture was lyophilized and then dissolved in 0.05 M lithium bromide in HPLC-grade DMF to be analyzed by DMF GPC for molecular weight and PDI measurement. Several drops of ethyl vinyl ether were added to quench the reaction, followed by 2 hours of stirring. The solution was concentrated under vacuum, and the resulting residue was precipitated into cold diethyl ether three times. The precipitate was dried under vacuum, yielding a white powder. The pac-PN brush polymer was then reacted with excess sodium azide in anhydrous DMF overnight at room temperature. The mixture was dialyzed against Nanopure water for one week and lyophilized to obtain a white powder. To synthesize pac-PN-ASO, the azide-functionalized PN bottlebrush polymer (50 nmol) was dissolved in 1 mL of 2 M sodium chloride solution and reacted with - 45 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 dibenzocyclooctyne (DBCO)-modified ASO (50 nmol) at 50 °C overnight. The conjugate was purified by aqueous GPC, desalted, and lyophilized. The purified PN-pacDNA was stored at -20 °C until use. [00178] Oligonucleotide and ribose-based pacDNA backbone synthesis [00179] All oligonucleotide and ribose backbones were synthesized on a Dr. Oligo 48 DNA synthesizer (Biolytic Lab Performance, Inc., Fremont, CA) using dT-CPG. The time for the coupling of ribose-phosphoramidites were set at 10 min (compared to 15 s for regular phosphoramidite). Oligonucleotide strands were cleaved from the CPG support and deprotected in aqueous ammonium hydroxide solution (28-30% NH3 basis) at room temperature for 16 h. Free oligonucleotide were purified by RP-HPLC, followed by the removal of dimethoxytrityl (DMT) groups using 20% acetic acid. The oligonucleotide with ribose backbones were purified with Glen-Pak™ DNA purification cartridge (for use with disposable syringes) (Glen Research Co., USA.) following the manufacture’s protocol. [00180] PEGylation of ribose-based pacDNAs [00181] The purified backbone (50 nmol, 1 eq.) and NHS-terminated 10kDa mPEG (50 nmol, 1 eq.) were dissolved in 800 μL of phosphate buffered saline (PBS, pH 7.4). The mixture was shaken overnight at 4 °C and then desalted using Nap-Column.50 equivalents of tetrabutylammonium nitrate were added to the desalted product. The desalted product was vacuum dried and then dissolved in 1 mL anhydrous N, N-Dimethylformamide (DMF) containing 42 μL triethylamine. To this mixture, 1 equiv of NHS-terminated PEG (dissolved in 1 mL DMF) was added in 5 aliquots (12 h between each aliquot), and the mixture was gently shaken at room temperature. The mixture was dried in vacuo and purified by aqueous GPC. [00182] Cell culture and animals [00183] NCI-H358 cells was cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. K273 cells were cultured in DMEM media supplemented with 5% fetal bovine serum (FBS), 10μg/mL Epidermal Growth Factor (EGF), 1mg/mL hydrocortisone, 1 mg/mL cholera toxin, 4mg/mL insulin, and 1% penicillin- streptomycin (PS). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. - 46 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00184] C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratory., USA. Animals were housed at Northeastern University animal facilities. Animal protocols (protocol number: 22-0309R) were approved by the Institutional Animal Care and Use Committee of Northeastern University and carried out in accordance with the approved guidelines. [00185] Cellular uptake [00186] NCI-H358 cells were cultured in a 24-well plate with a cell seeding density of 1.0´106 cells per well in 1 mL complete RPMI medium for 24 h at 37 °C. Next, cells were washed 2´ with 1´ PBS and treated with fluorescently labeled test agents and controls, which were dissolved in serum-free RPMI culture medium at various concentrations (1 μM to 5 μM). Cells were further incubated with the samples for 4 h at 37 °C, before being washed with 1´ PBS 3´ and treated with 100 μL of 0.25% trypsin/EDTA solution (Gibco, USA) followed by 700 μL of cold 1´ PBS. Detached cells were collected for flow cytometry analysis (CytoFLEX Flow Cytometer, Beckman Coulter, USA.). [00187] Confocal microscopy [00188] NCI-H358 cells were cultured in a 24-well glass bottom plate at 1.0×105 cells per well in 1 mL complete RPMI medium for 24 h at 37 °C. On the following day, cells were washed with 1´ PBS 3´, and fluorescently labeled test agents and controls dissolved in serum- free RPMI culture medium at were added to give 5 µM of total DNA. Cells were further incubated at 37 °C for 8 h. Next, cells were washed with 1´ PBS 3´ and fixed with 4% paraformaldehyde for 30 min at R.T., followed by another 3´ wash with 1´ PBS. Finally, the cells were stained with DAPI for 10 min before imaged on an LSM-880 confocal laser scanning microscope (Carl Zeiss Ltd., Cambridge, UK). All imaging settings were kept identical in each study. [00189] Cell viability [00190] The cell viability of NCI-H358 and K273 after treatment with ribose-based pacDNA and bottlebrush polymer was analyzed by MTT (dimethylthiazol-diphenyltetrazolium bromide) colorimetric assay. Cells were seeded in 96-well plates at a density of 1×104 cells per well in 175 µL full growth media and cultured for 24 h at 37 °C with 5% CO2. Then cells were treated with - 47 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 pacDNA and bottlebrush polymer in the concentration range of 0.1 – 10 μM (equiv. of DNA). Cells treated with vehicle served as a control. After 48 h of incubation, 20 μL of 5 mg/mL MTT stock solution in PBS was added to each well. After incubation for another 4 h, the media was carefully removed. The resulting blue formazan crystals were dissolved in DMSO (200 μL per well) and measured at 490 nm on a BioTek® Synergy™ Neo2 Multi-Mode microplate reader (BioTek Inc., VT, USA). [00191] Western blot analysis [00192] Cells were seeded in 24-well plates at a density of 2.0×105 cells per well in 1 mL full growth media and cultured for 24 h at 37 °C with 5% CO2. After washing by PBS 1×, pacDNA and bottlebrush polymer (1 μM – 10 μM equiv. of DNA) dissolved in full media (1 mL) was added, and cells were further incubated at 37 °C for 72 h. Next, cells were harvested and whole cell lysates were collected in 100 μL of RIPA cell lysis buffer supplemented with 1% phosphate inhibitor and 1% phosphatase inhibitor. Total proteins in cell lysate were quantified using a bicinchoninic acid (BCA) protein assay kit. Equal amounts of total proteins were separated on a 10% SDS-PAGE gel and electro-transferred to nitrocellulose membrane. The membrane was then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline supplemented with 0.05% Tween-20 (TBST). After blocking, the membrane was incubated with appropriate primary antibodies overnight at 4 °C. After washing with TBST for three times (10 min per time), the membrane was incubated with secondary antibodies at room temperature for 1 h. The detected proteins were visualized by chemiluminescence using the ECL Western Blotting Substrate (Bio- rad, MA, USA). Antibodies used in this study were: KRAS antibody (cat. sc-30; Santa Cruz), β- actin (cat. AM4302, Cell Signaling Technology), anti-mouse IgG, HRP-linked antibody (cat. 7076S, Cell Signaling Technology). [00193] Plasma pharmacokinetics (PK) studies [00194] Immunocompetent C57BL/6 mice were used to examine the plasma PK of free ASO, pacDNAs. Mice were randomly divided into seven groups (n = 5). Cy5-labeled samples were intravenously (i.v.) administrated via the tail vein at equal ASO dosage (0.5 μmol/kg). Blood samples (25 μL) were collected from the submandibular vein at varying time points (30 min, 2 h, 4 h, 10 h, 24 h, 48 h and 72 h) using BD Vacutainer™ blood collection tubes with lithium - 48 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 heparin. Heparinized plasma was obtained by centrifugation at 3000 rpm for 20 min, aliquoted into a 96-well plate, and measured for fluorescence intensity on a Synergy™ Neo2 Multi-Mode microplate reader (BioTek Instruments Inc., VT, USA). The amounts of ASO in the blood samples were estimated using standard curves established for each sample. To establish the standard curves, samples of known quantities were incubated with freshly collected plasma for 1 h at room temperature before fluorescence was measured. [00195] Allograft tumor model [00196] To establish allograft tumor model, approximately 5×105 K273 cells in 100 μL phosphate buffered saline (PBS) were implanted subcutaneously on the right flank of 6-week-old C57BL/6 (female, n = 5). Mice were monitored for tumor growth and body weight every other day. Once tumor volume reached 100mm3, mice were divided into groups to keep initial average tumor volumes of each group very close. The mice received following treatment via tail vein injection: 1) PBS, 2) pac-4×1C18-ASO2 (0.5 µmol/kg), 3) pac-4×1C18-ASO2 (0.5 µmol/kg), 4) Free ASO2 (0.5 µmol/kg). Samples were injected every four days for four doses. Once the tumor volume reached 1000mm3, the mice were euthanized by CO2. Major organs and tumors were collected into 10% neutral buffered formalin for overnight fix and then for histological analysis carried by iHisto Inc. [00197] Anti-PEG immune response [00198] Healthy C57BL/6 mice were administrated PBS, pac-4×1C18-ASO2, free ASO2 or PEG-KLH every four days for four doses.50-100µL plasma were collected one week after the last dose. The concentration of anti-PEG IgM/IgG were assessed by ELISA kit (Cat # PEGG-1, Cat #PEGM-1, Life Diagnotics, Inc.) according to the manufacturer’s protocol. [00199] Multiplex analysis of cytokines [00200] The multiplexing analysis was performed using the Luminex™ 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Thirty-two markers were simultaneously measured in the samples using Eve Technologies' Mouse Cytokine 32-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 32-plex consisted of Eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL- 1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, - 49 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. Assay sensitivities of these markers range from 0.3 – 30.6 pg/mL for the 32-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol. [00201] Whole-animal and ex vivo organ imaging [00202] Tumor-bearing C57BL/6 mice were i.v. injected with Cy5-labeled samples at an ASO dose of 0.5 μmol/kg. Then mice were scanned at 1, 4, 8, 24 h, 48 h, and 72 h using an IVIS Lumina II imaging system (Caliper Life Sciences, Inc. MA, USA). To evaluate the biodistribution of pacDNAs and the bottlebrush polymer, mice were euthanized using CO2, and major organs, tissues and the tumor were dissected and imaged using IVIS. [00203] Biodistribution [00204] The major organs, tissues and tumor were diced into small piece and weighted. Then, the flesh was homogenized in tissue protein extraction reagent (Thermo Fisher, USA), which is supported with 0.5% Triton X-100, using a BeadBlaster D2400-R refrigerated homogenizer (Benchmark scientific, NJ, USA.). After centrifugation, the supernatants were aliquoted into a 96-well plate and measured for fluorescence intensity on a Synergy™ Neo2 Multi-Mode microplate reader (BioTek Instruments Inc., VT, USA). The amounts of ASO in the supernatant were estimated using standard curves established for each sample. [00205] All-atom molecular dynamics (MD) simulation [00206] All-atom molecular dynamics (MD) simulations of pacDNAs were conducted to understand the molecular mechanism of how the lipids distribution affects the energy state of pacDNAs. The four initial pacDNAs with six C18 units arranged in different distributions were built using ChemDraw. For these four pacDNA models, all PEG side chains were truncated to a 20 mer. Subsequently, each pacDNA model underwent a refinement process in Maestro, during which hydrogen atoms were added to the structures. (25) The OPLS4 forcefield was used to model pacDNAs. (26) To mimic the aqueous environment, each model was solvated in a rectangular water box using the SPC water model with a minimum water shell of 10 Å from any box edge to the nearest atom of the structure. Sodium (Na+) and chloride (Cl-) ions were placed - 50 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 to neutralize the system. Furthermore, the salt (NaCl) concentration was set to 0.15 M to mimic physiological conditions. [00207] Prior to the production run, each pacDNA structure underwent an extensive minimization and equilibration process, detailed as follows. Each system was first relaxed using the default relaxation protocol from Maestro. After that, the system went through an annealing process by first heating the system from 10 K to 400 K and then cooling to 300 K. Following annealing, each pacDNA model was re-solvated with a refreshed water box to accurately represent the aqueous environment. The updated simulation system was used to conduct a production run for 100 ns at a constant temperature of 300 K with 1 bar pressure (NPT ensemble). The Nosé–Hoover algorithm was used for temperature control, and the Martyna– Tobias–Klein algorithm was used to control the pressure. All molecular dynamics simulations were performed using the GPU-accelerated Desmond engine in Schrodinger molecular modeling suite. (27-29) The simulation trajectories from 90 ns to 100 ns were used to calculate the potential energy for the corresponding pacDNA structure. [00208] A full-length and full-atomistic pacDNA model with the largest spacing (pac-4×1C18- ASO1) was built to investigate the conformational characteristics. The initial structure of pacDNA-4×1C18 with truncated PEG chain was built using ChemDraw. We extended each PEG chain to 226 mer using VMD and Maestro. (25,30) After that, we followed the same process as described above to minimize and equilibrate the structure. The same annealing protocol was used to optimize the structure. The simulation system with an optimized structure (~1.6 million atoms) was used to run the production run for 60 ns in an NPT ensemble (300 K, 1 bar). EXAMPLE 2 [00209] Oligonucleotide drugs hold great potential to revolutionize disease therapeutics due to their adaptability across diverse diseases and targets. Notably, oligonucleotide-based therapies have been developed for previously untreatable rare diseases, such as Spinraza for spinal muscular atrophy and investigational drugs for DM1 (myotonic dystrophy type 1). However, the clinical advancement of oligonucleotide drug candidates is frequently hindered by their structural and chemical properties, including their negative charge and limited membrane permeability. - 51 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Those properties present significant barriers to effective targeting and uptake, requiring more precise delivery strategies. To enhance delivery efficiency, recent studies have developed various nanoparticle-based delivery systems, including lipid nanoparticles (LNPs), polymer-conjugated drug carriers, and antibody-conjugated delivery systems designed for targeted oligonucleotide transport. Although these delivery systems have demonstrated efficacy, they come with inherent limitations. LNPs and polymer-based systems predominantly accumulate in the liver, limiting their applicability for targeting other organs. Similarly, antibody-conjugated delivery systems are typically unable to maintain a prolonged blood circulation time. Furthermore, none of these platforms have integrated machine learning with delivery platform development to precisely modulate cellular uptake or achieve targeted delivery to specific organs. [00210] The polyphosphodiester backbone polymer was assembled via a stepwise condensation process, incorporating three distinct side-chain modified phosphoramidite units in conjunction alongside an unmodified serinol phosphoramidite. The C18 and cholesterol phosphoramidites served as hydrophobic units, while the spermine phosphoramidite introduced positive charge. The unmodified serinol units acted as spacers, allowing precise control over the polymer backbone and facilitating the construction of a 30-mer polymer. (FIG.31A) The C18 phosphoramidite was synthesized from octadecylsuccinic anhydride, which was subsequently grafted with (9H-fluoren-9-yl)methyl (5-aminopentyl)carbamate to incorporate a side-chain amine group (see Scheme 3). Scheme 3. Synthesis of C18 phosphoramidite - 52 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 , introduced, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite was employed to construct the C18 phosphoramidite 16. Similarly, starting with cholesterol amine, a two-step reaction was performed to yield cholesterol-NHS (compound 17) (Scheme 4), which was reacted with Fmoc- Lys-OH to graft an amine group, producing compound 18 (cholesterol-Lys-OH). Compound 18 was then reacted with serinol-DMT and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to afford the cholesterol phosphoramidite 11. Scheme 4. Synthesis of cholesterol phosphoramidite - 53 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 spermine. The unmodified units, as reported in our previous research, were synthesized from Fmoc-beta-alanine through a four-step reaction (Scheme 5). Scheme 5. Synthesis of spermine phosphoramidite Table 3. Sequences with polyphosphodiester backbone used in this study. N: non-modified (serinol); A: C18 alkyl; C: cholesterol; S: spermine. All monomers (N, A, C, and S) are linked to PEG (10 kDa). Sample ID SEQ ID NO. Sequence - 54 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Q1 20 NN NNN NNN NNN NNN NNN NNN NNN NNN NNN N N N N C N A A - 55 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Q32 34 NA NNA NNS NNA NNA NNA NNA NNS NNA NNA N N N N N N A - 56 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00213] Following the successful synthesis of the three side-chain modified phosphoramidite units, we proceeded with the design of polymer sequences based on various combinations of these modifications. The polyphosphodiester backbone was synthesized via standard solid-phase synthesis, incorporating both modified and unmodified phosphoramidite units to form a 30-mer structure in a stepwise manner. Following backbone assembly, 20% piperidine in DMF was used to remove the Fmoc protecting group, after which the crude product was purified using a Glen column to eliminate excess reagents and byproducts. The synthesized polymer backbone was then reacted with PEG-NHS, and after a two-step polymerization process, the final product was purified using size-exclusion chromatography to remove unreacted small molecules and achieve the desired molecular weight distribution. (FIG.31B) Our primary objective was to investigate how different arrangements of these units affect in vitro and in vivo performance. For the C18 units, we systematically increased the number from 1 to 8 to evaluate the impact of increasing hydrophobicity. In the case of the 6 C18 configuration, we examined various spatial arrangements, including clustering, pairing, and even distribution along the backbone (FIG.31B, Table 3). Similarly, for cholesterol, we incorporated 6 units into the polymer backbone and compared the effects of clustering versus even distribution on polymer performance (FIG.31B). To introduce positive charge, spermine units were integrated into the C18 sequences, allowing us to assess whether the proximity of these charges influences structural or functional outcomes (FIG.31B). Through this process, we successfully synthesized a library consisting of 31 distinct combinations of these units (FIG.31B). [00214] Modulation of bottlebrush polymer architecture by C18, cholesterol, and spermine units [00215] Following the successful synthesis of the bottlebrush polymer library, Gel Permeation Chromatography (GPC) (FIG.32A) was utilized to confirm the molecular weight distribution, a 25 min retention time showing consistent molecular weights (300kDa) and low polydispersity across all samples. To further explore the structural organization, Transmission Electron Microscopy (TEM) provided visual evidence of uniform, spherical nanostructures with an average core diameter of 20 nm, confirming the controlled assembly achieved through the synthetic route (FIG.32B). Complementary Dynamic Light Scattering (DLS) measurements - 57 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 showed a slightly larger hydrodynamic diameter of around 25 nm (FIG.32C), which can be attributed to the hydration shell present in solution, indicating good stability and dispersion in aqueous environments. This consistency across different techniques reflects the robustness and reproducibility of the synthetic process. Zeta potential measurements revealed that polymers with higher spermine content displayed increasingly positive surface charges, suggesting that electrostatic interactions (FIG.32D), driven by the positively charged spermine units, play a crucial role in influencing polymer compaction and size. To further elucidate these structural effects, molecular dynamics simulations demonstrated that increased lipid and spermine content resulted in more compact polymer conformations, driven by enhanced hydrophobic and electrostatic forces. Together, these findings underscore the critical impact of polymer composition on structural and physicochemical properties, offering valuable insights for the rational design of bottlebrush polymers tailored for specific biomedical applications. [00216] Enhanced cellular uptake by hydrophobic and positively charged units [00217] To investigate how hydrophobic and positively charged units influence cellular uptake, we applied 0.5 µM of the synthesized bottlebrush polymer library to three cell lines (NCIH358, HEPG2, and DM1 fibroblasts) (FIGs.33A-C). Our results demonstrated that cellular uptake consistently increased as the number of hydrophobic units (i.e. C18 and cholesterol units) was increased, with an optimal uptake observed between 6 and 10 C18 units. Notably, polymers with 6 evenly distributed C18 units exhibited the highest uptake, outperforming both clustered configurations and those with 8 C18 units. This suggests that the spatial arrangement of hydrophobic units significantly impacts polymer-cell interactions. Due to synthesis limitations, only two spermine units were incorporated into the polymers, but even with this constraint, the addition of positive charges led to a remarkable increase in cellular uptake, likely driven by electrostatic interactions with the negatively charged cell membranes. The Q45 polymer, featuring 8 evenly distributed C18 units and two centrally located spermine units, demonstrated the highest uptake across all cell lines, highlighting the importance of both hydrophobicity and charge distribution in optimizing uptake. In contrast, the inclusion of cholesterol did not significantly enhance cellular uptake, likely due to its lower affinity for cell membranes compared to C18. Molecular dynamics simulations aligned with the experimental results, - 58 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 showing that Q45 had the strongest membrane binding affinity. These findings provide a mechanistic explanation for the role of both hydrophobic and charged units in enhancing cellular uptake and underscore the importance of unit arrangement in optimizing polymer performance. [00218] Pharmacokinetics, biodistribution, and predictive modeling of digital bottlebrush polymers in vivo performance [00219] To assess the in vivo performance of the bottlebrush polymer library, we conducted intravenous (IV) injections in C57BL/6 mice. Each mouse was administered 10nmol per mice of cyanine5-labeled bottlebrush polymers via tail vein injection. Over the course of three days, we collected blood samples daily to analyze the pharmacokinetics and determine how the polymers circulated in the bloodstream over time. After the third day, the mice were sacrificed, and their organs, including the liver, spleen, kidneys, lungs, and heart, were dissected and homogenized to quantify the distribution of the cyanine5-labeled bottlebrush polymers across different tissues. This allowed us to evaluate the biodistribution and retention of the polymers in major organs, providing key insights into their tissue-specific accumulation. [00220] IVIS imaging data is presented in FIG.34A, while a quantification heatmap and a bar graph illustrating the organ-specific accumulation of different bottlebrush polymer formulations are presented in FIG.34B and FIG.34C respectively. The IVIS imaging results reveal distinct biodistribution patterns, with fluorescence signals indicating polymer accumulation in different organs. Notably, Q36 and Q18 exhibit strong fluorescence in muscle tissue, suggesting a preferential accumulation, while Q19 shows a pronounced signal in the spleen, as confirmed by the heatmap analysis. The heatmap (FIG.34B), derived from quantification data, further highlights the relative distribution intensity of each polymer across various organs, with warmer colors indicating higher accumulation. The bar graph (FIG.34C) provides a comparative visualization of the biodistribution trends, emphasizing the variations in organ targeting among different formulations. These findings indicate that polymer composition significantly influences biodistribution, which could be strategically applied for targeted drug delivery in disease- relevant animal models. [00221] Materials and Methods [00222] Microscale thermophoresis - 59 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00223] Microscale thermophoresis (MST) binding measurements were carried out with 10 nM Cy5-labeled samples in binding buffer (1× PBS pH 7.4, 0.1% Triton X 100) with a range of concentration of human AXL protein (154-AL) from 2500 nM to 0.07 nM. The mixtures were transferred to Monolith NT.115 standard capillaries and analyzed on a Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) at medium MST power and 20% excitation power. Data was analyzed using the MO. Affinity Analysis software (version 2.3, NanoTemper Technologies) and MST-on time was set at 1.5 s. [00224] Flow cytometry [00225] Cells were seeded in 24-well plates at a density of 1.0×105 cells per well in 1 mL full growth media and cultured overnight at 37 °C with 5% CO2. After incubation, cells were inhibited by 0.1% sodium azide in serum-free culture media for 1 h. After washing by PBS 1×, Cy5-labeled samples and controls (1 μM equiv. of aptamer) dissolved in serum-free culture media (400 µL) were added, and cells were further incubated at 37 °C for 4 h. Next, cells were washed with PBS 2× and treated with trypsin (60 μL per well). Thereafter, 1 mL of PBS was added to each culture well to suspend the cells. Cells were then analyzed on a Attune™ NxT flow cytometer (Invitrogen, MA). Data for 1.0×104 gated events were collected. [00226] Pharmacokinetics [00227] Animal protocols were approved by the Institutional Animal Care and Use Committee of Northeastern University. Animal experiments and operations were conducted following the approved guidelines.8~12-week-old female C57BL/6 mice (Charles River, MA, USA) were randomly divided into 5 groups (n=5). Samples and controls were intravenously administered via the tail vein at equal aptamer concentrations (0.5 μmol/kg equiv. of aptamer). Blood samples (50 μL) were collected from the submandibular vein at varying time points (30 min, 2 h, 4 h, 10 h, 24 h, 48 h, and 72 h) using BD Vacutainer blood collection tubes with sodium heparin. Heparinized plasma samples were obtained by centrifugation at 3000 rpm for 20 min, and then aliquoted into a 96-well plate. The fluorescence intensities were measured on a plate reader. The amounts of agents in the blood samples were estimated using standard curves established for each sample in freshly collected plasma. [00228] Conclusion - 60 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00229] In summary, we have developed a library of bottle-brush polymers with diverse backbone structures. Through the combination of phosphoramidite monomers with modifications, we meticulously designed and synthesized digital bottlebrush polymers featuring distinct backbones. Furthermore, through in vitro and in vivo testing, we identified sequences with optimal targeting efficiency. [00230] In some example embodiments, the disclosed polymers, conjugates and methods improve the poor circulation of oligonucleotide drug in blood, enable oligonucleotide drug targeting at specific organs, enhance cellular uptake of oligonucleotide drugs, and/or enable disease specific optimization using polymer sequence engineering. In addition, library screening can yield a large number of polymers, each having its own best match in disease. [00231] In some example embodiments, the disclosed polymers conjugates and methods can be used for therapeutic development for various diseases with different drug delivery requirements, in vitro research, generation of well-defined synthetic polymers using solid phase synthesis, drug delivery, and/or a biological research tool. REFERENCES [00232] (1) Moumne, L.; Marie, A. C.; Crouvezier, N. Oligonucleotide Therapeutics: From Discovery and Development to Patentability. Pharmaceutics 2022, 14 (2), 260, DOI: 10.3390/pharmaceutics14020260 [00233] (2) Zhang, Q.; Yang, L.; Liu, Y. H.; Wilkinson, J. E.; Krainer, A. R. Antisense oligonucleotide therapy for H3.3K27M diffuse midline glioma. Sci. Transl. Med.2023, 15, 691, DOI: 10.1126/scitranslmed.add8280 [00234] (3) Lundin, K. E.; Gissberg, O.; Smith, C. I. E. Oligonucleotide Therapies: The Past and the Present. Hum. Gene Ther.2015, 26 (8), 475– 485, DOI: 10.1089/hum.2015.070 [00235] (4) Lauffer, M. 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H.; Jia, F.; Tan, X.; Davis, S.; Krishnan, S.; Amiji, M. M.; Zhang, K. Providing Oligonucleotides with Steric Selectivity by Brush-Polymer-Assisted Compaction. J. Am. Chem. Soc.2015, 137 (39), 12466– 12469, DOI: 10.1021/jacs.5b08069 [00244] (13) Lu, X.; Zhang, K. PEGylation of therapeutic oligonucletides: From linear to highly branched PEG architectures. Nano Res.2018, 11 (10), 5519– 5534, DOI: 10.1007/s12274-018-2131-8 [00245] (14) Jia, F.; Lu, X.; Tan, X.; Wang, D.; Cao, X.; Zhang, K. Effect of PEG Architecture on the Hybridization Thermodynamics and Protein Accessibility of PEGylated - 62 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 Oligonucleotides. Angew. Chem., Int. Ed. Engl.2017, 56 (5), 1239– 1243, DOI: 10.1002/anie.201610753 [00246] (15) Wang, D.; Lin, J.; Jia, F.; Tan, X.; Wang, Y.; Sun, X.; Cao, X.; Che, F.; Lu, H.; Gao, X.Bottlebrush-architectured poly (ethylene glycol) as an efficient vector for RNA interference in vivo. Sci. 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Utilizing endosomal capture for tumor therapy via membrane-lytic mechanism-based Pickering emulsion. J. Controlled Release 2023, 354, 523– 537, DOI: 10.1016/j.jconrel.2023.01.035 - 63 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00254] (23) Lee, Y.; Thompson, D. H. Stimuli-responsive liposomes for drug delivery. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol.2017, 9, e1450, DOI: 10.1002/wnan.1450 [00255] (24) Wei, Y.; Chen, P.; Ren, M.; Li, D.; Lin, J.; Sun, T.; Wang, Y.; Yang, S.; Nenopoulos, C.; Oetheimer, C.; Li, Y. Bottlebrush polymers with sequence-controlled backbones for enhanced oligonucleotide delivery. J. Am. Chem. Soc.2024, 146, 34763-34770. [00256] (25) Schrödinger, L., New York, NY. Schrödinger Release 2023-2: Maestro.2023. [00257] (26) Lu, C.; Wu, C.; Ghoreishi, D.; Chen, W.; Wang, L.; Damm, W.; Ross, G. A.; Dahlgren, M. K.; Russell, E.; Von Bargen, C. D.; et al. OPLS4: Improving Force Field Accuracy on Challenging Regimes of Chemical Space. J Chem Theory Comput 2021, 17 (7), 4291-4300. DOI: 10.1021/acs.jctc.1c00302 From NLM PubMed-not-MEDLINE. [00258] (27) Bowers, K. J.; Chow, D. E.; Xu, H.; Dror, R. O.; Eastwood, M. P.; Gregersen, B. A.; Klepeis, J. L.; Kolossvary, I.; Moraes, M. A.; Sacerdoti, F. D.; et al. Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters. In SC '06: Proceedings of the 2006 ACM/IEEE Conference on Supercomputing, 11-17 Nov.2006, 2006; pp 43-43. DOI: 10.1109/SC.2006.54. [00259] (28) D. E. Shaw Research, N. Y., NY. Schrödinger Release 2024-1: Desmond Molecular Dynamics System.2024. [00260] (29) Schrödinger, N. Y., NY. Maestro-Desmond Interoperability Tools.2024. [00261] (30) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. Journal of Molecular Graphics & Modelling 1996, 14 (1), 33-38. DOI: Doi 10.1016/0263- 7855(96)00018-5. [00262] (31) Zhang, Ke; Wang, Yuyan. International App. Pub. No. WO 2023/115031 A1. [00263] (32) Zhang, Ke; Wang, Dali. International App. Pub. No. WO 2023/023662 A1. [00264] (33) Zhang, Ke. International App. Pub. No. WO 2020/210207 A2. [00265] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. - 64 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 [00266] The teachings of all patents, published applications and references cited herein and in the attached documents are incorporated herein by reference in their entirety. - 65 - 4112432.v1

Claims

Docket No.5200.2402-001 (INV-24084) 4112432.v1 CLAIMS What is claimed is: 1. A polymer, comprising: a backbone comprising phosphodiester; a plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. 2. The polymer of claim 1, wherein the backbone is a 2-deoxyribose-based backbone, a serinol-based backbone, or a combination thereof. 3. The polymer of claim 1 or 2, wherein the polymer comprises from about 10 to about 40 polymer arms covalently linked to the backbone. 4. The polymer of any one of claims 1-3, wherein each of the plurality of polymer arms is a poly(ethylene glycol). 5. The polymer of any one of claims 1-4, wherein each of the at least one alkyl is a C10-C30 alkyl. 6. The polymer of any one of claims 1-5, wherein each of the at least one alkyl is a C18 alkyl. 7. The polymer of any one of claims 1-6, wherein the polymer comprises from about 2 to about 10 alkyls covalently linked to the backbone. 8. The polymer of any one of claims 1-7, wherein the polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone. - 66 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 9. The polymer of any one of claims 1-8, wherein the polymer comprises about 4 alkyls covalently linked to the backbone. 10. The polymer of any one of claims 1-9, wherein the polymer comprises 6 cholesterols covalently linked to the backbone. 11. The polymer of any one of claims 7-10, wherein the polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone, and wherein the plurality of alkyls or the plurality of cholesterols are evenly distributed. 12. The polymer of claim 11, wherein the plurality of alkyls or the plurality of cholesterols are separated by from about 2 monomers to about 6 monomers. 13. The polymer of any one of claims 1-12, wherein the polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47. 14. A conjugate, comprising the polymer of any one of claims 1-13 and an agent conjugated to the polymer. 15. The conjugate of claim 14, wherein the agent is conjugated to the 5’ end of the polymer. 16. The conjugate of claim 14 or 15, wherein the agent is an oligonucleotide. 17. The conjugate of any one of claims 14-16, wherein the agent is single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamers, ribozymes, DNAzymes, antisense oligonucleotides, exon-skipping oligonucleotides, siRNA oligonucleotides, triple helix forming oligonucleotides, ribozymes, DNAzymes, chemically modified forms thereof, or a combination thereof. 18. The conjugate of any one of claims 14-17, wherein the agent is an antisense oligonucleotide. - 67 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 19. A composition comprising the conjugate of any one of claims 14-18 and a pharmaceutically acceptable carrier. 20. A method of delivering an agent to a subject in need thereof, the method comprising: administering the conjugate of any one of claims 14-18 or the composition of claim 19 to the subject. 21. A method of delivering an agent to a cell, the method comprising: contacting the cell with the conjugate of any one of claims 14-18. 22. A method of modulating or altering the expression of a gene product encoded by a target polynucleotide, the method comprising: contacting the target polynucleotide with the conjugate of any one of claims 16-18. 23. A method of detecting a target polynucleotide, the method comprising: contacting the target polynucleotide with the conjugate of any one of claims 16-18, thereby forming a polynucleotide-conjugate hybrid; and detecting a signal from the polynucleotide-conjugate hybrid. 24. The method of claim 23, wherein the conjugate further comprises a fluorescent dye. 25. The method of claim 24, wherein the fluorescent dye is Cyanine5 (Cy5). 26. The method of any one of claims 23-25, wherein detecting a signal from the polynucleotide-conjugate hybrid comprises using fluorescence imaging. 27. The method of any one of claims 22-26, wherein the target polynucleotide is selected from eukaryotic, prokaryotic, and viral oligonucleotides. 28. The method of claim 27, wherein the target polynucleotide is an oligonucleotide specific to a mammalian cancer cell, a mammalian non-cancer cell, a plant cell, a bacterium, or a virus. 29. The method of claim 28, wherein the target polynucleotide is an mRNA. - 68 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 30. The method of claim 29, wherein the mRNA is KRAS mRNA or DMPK mRNA. 31. A method of treating a disease, the method comprising administering the conjugate of any one of claims 14-18 or the composition of claim 19 to a subject in need thereof. 32. The method of claim 31, wherein the disease is a cancer, a muscular disorder, or a central nervous system disorder. 33. The method of claim 32, wherein the cancer is a lung cancer. 34. The method of claim 32, wherein the muscular disorder is myotonic dystrophy. 35. A method of making a bottlebrush polymer, the method comprising: combining one or more alkyl phosphoramidites, one or more cholesterol phosphoramidites, one or more spermine phosphoramidites, one or more serinol phosphoramidites, or a combination thereof to form a polymer backbone comprising phosphodiester; and covalently linking a plurality of polymer arms to the polymer backbone, thereby making the bottlebrush polymer, wherein the bottlebrush polymer comprises: the backbone comprising phosphodiester; the plurality of polymer arms covalently linked to the backbone; and at least one alkyl, at least one cholesterol, at least one spermine, or a combination thereof, covalently linked to the backbone. 36. The method of claim 35, wherein at least one of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group. - 69 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 37. The method of claim 35 or 36, wherein each of the one or more alkyl phosphoramidites, the one or more cholesterol phosphoramidites, the one or more spermine phosphoramidites, the one or more serinol phosphoramidites, or the combination thereof comprises a protecting group. 38. The method of claim 36 or 37, wherein covalently linking a plurality of polymer arms to the backbone comprises removing the protecting group and contacting the polymer backbone with a reagent comprising the polymer arm. 39. The method of any one of claims 36-38, wherein the protecting group is fluorenylmethoxycarbonyl (Fmoc), trifluoroacetyl (TFA), or a combination thereof. 40. The method of claims 38 or 39, wherein the reagent is an amino-reactive PEG reagent. 41. The method of any one of claims 35-40, wherein each of the one or more alkyl phosphoramidites is a C10-C30 alkyl phosphoramidite. 42. The method of any one of claims 35-41, wherein each of the one or more alkyl phosphoramidites is a C18 alkyl phosphoramidite. 43. The method of any one of claims 35-42, wherein the polymer comprises from about 2 to about 10 alkyls covalently linked to the backbone. 44. The method of any one of claims 35-43, wherein the polymer comprises from about 4 to about 10 alkyls covalently linked to the backbone. 45. The method of any one of claims 35-44, wherein the polymer comprises about 4 alkyls covalently linked to the backbone. 46. The method of any one of claims 35-45, wherein the polymer comprises 6 cholesterols covalently linked to the backbone. - 70 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 47. The method of any one of claims 35-46, wherein the polymer comprises a plurality of alkyls or a plurality of cholesterols covalently linked to the backbone, and wherein the plurality of alkyls or the plurality of cholesterols are evenly distributed. 48. The method of any one of claims 35-47, wherein the plurality of alkyls or the plurality of cholesterols are separated by from about 2 monomers to about 6 monomers. 49. The method of any one of claims 35-48, wherein the polymer has a sequence comprising SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 46 or SEQ ID NO: 47. 50. The method of any one of claims 35-49, wherein the cholesterol phosphoramidite is . 51. The method of any one of claims 35-50, wherein the spermine phosphoramidite is - 71 - 4112432.v1 Docket No.5200.2402-001 (INV-24084) 4112432.v1 . phosphoramidite is . wherein the serinol phosphoramidite is - 72 - 4112432.v1
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